Transcript 组织学与胚胎学HISTOLOGY&EMBRYOLOGY
组织学与胚胎学
HISTOLOGY & EMBRYOLOGY
霍冠华
Huo Guanhua
滨州医学院组织学与胚胎学教研室
Department of Histology & Embryology Binzhou Medical University September, 2013
第
1
章 绪论
Chapter 1 Introduction Histology & Embryology is composed of 2 related sciences.
一、组织学与胚胎学的研究内容及意义
The contents and importance of H&E
组织学（
histology
）
:
是研究机体微细结构及 其相关功能的科学。 在组织、细胞、亚细胞和分子水平对机体进 行研究
A science: study normal micro-structure & its related function of human body.
光镜结构
:Microstructure
电镜结构
: Ultrastructure
描述组织学与描述胚胎学descriptive histology & embryology ：用显微镜观察人体组织结构和胚胎发生过 程形态演变的科学。
Descriptive histology & embryology
：
Observes human tissue structures and the process of embryogenesis and the development.
比较组织学与比较胚胎学comparative histology and embryology:比较不同种系动物的组织结构功能和胚胎发 育过程的科学。
Comparative histology and embryology: Compares tissue structure and the process of embryogenesis and the development in different species.
实验组织学与实验胚胎学
experimental histology and embryology
：应用实验方法研究细胞与组织之间的 关系，以及理化因子或生物因素对组织结构功能和 生长发育的影响、作用机理及其防治的科学。
Experimental histology
：
Studies the relationship between cells and tissues in vivo.
Experimental embryology: Studies the mechanisms controlling the individual development of animals, even human being, by means of experiments in vivo, using such methods as marking, removal, transplantation, and isolation of body parts and organs. It also studies the action of various external factors (physical, chemical or biological factors) on embryonic development, and explores control
or
prevention methods.
Experimental embryology identifies the stages of the determination of the material of rudimentary organs and tissues, the sources of formative or inductive influences, the role of synthesis of macromolecules in the processes of determination and differentiation, and the factors responsible for morphogenesis. By removing, inactivating, or transplanting cell nuclei, experimental embryologists investigate the interaction of the nucleus and cytoplasm during gametogenesis and embryonic development, as well as the stages and factors of differential activation of genes in the course of development.
-
致病、致畸、致突变、致癌
test Pathogenicity test, teratology test, mutagenic test and carcinogenicity
分子生物学和分子胚胎学
molecular biology and molecular embryology:
从分子水平研究生命活动的物质基础及其异常 变化的科学。
Molecular biology and embryology: Studies molecules responsible for structure, morphogenesis and functions in human body and embryo.
Molecular biology is the study of biology at a molecular level. The field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry. Molecular biology chiefly concerns itself with understanding the interactions between the various systems of a cell, including the interactions between DNA, RNA and protein biosynthesis as well as learning how these interactions are regulated. Molecular embryology studies molecules responsible for morphogenesis and functions during gametogenesis
配子形成
and embryonic development.
组织（
tissue
）： 由细胞和 细胞外基质 extracellular matrix 构成。 人体
(
基本
)
组织：上皮组织、结缔组织、肌组织和神经组织
---
胚胎发生来源、 细胞构成、形态特点和功能等方面，具有明显的特性。
Tissue is composed of cells and extracellular matrix;collections of cells with similar morphological characteristics: Original 4 types of (basic, primary, or fundamental) tissues: Epithelial tissues – surface coverage Muscular tissues – contractile property Nervous tissues – cells forming brain, spinal cord, and nerves Connective tissues – to link or support other specialized tissues Tissues are different from each other in their origin, cellulosity
细胞构成
, morphological characteristics and functions.
器官organ：四种基本组织以不同的种类、数量 和方式组合形成器官。
系统system：若干功能相关的器官构成系统。
Each of the fundamental tissues is formed by several types of cells and typically by specific associations of cells and extracellular matrix. These characteristic associations facilitate the recognition of the many subtypes of tissues. Most organs are formed by an orderly combination of several tissues, except the central nervous system, which is formed almost solely by nervous tissue. The precise combination of these tissues allows the functioning of each organ and of the organism as a whole. A system is a group of organs that work together and perform one or more functions.
细胞外基质 extracellular matrix:
The extracellular matrix (ECM) is the noncellular component present within all tissues and organs, and provides not only essential physical scaffolding for the cellular constituents but also initiates crucial biochemical and biomechanical cues that are required for tissue morphogenesis, differentiation and homeostasis. The importance of the ECM is vividly illustrated by the wide range of syndromes, which can be anything from minor to severe, that arise from genetic abnormalities in ECM proteins. Although, fundamentally, the ECM is composed of water, proteins and polysaccharides, each tissue has an ECM with a unique composition and topology that is generated during tissue development through a dynamic and reciprocal, biochemical and biophysical dialogue between the various cellular components (e.g. epithelial, fibroblast, adipocyte, endothelial elements) and the evolving cellular and protein microenvironment.
细胞 Cell
构成：细胞膜、细胞质和细胞核，不同的细胞有不同的亚细胞结构特点。
The cell is the basic structural, functional and biological unit of all known living organisms. Cells are the smallest unit of life that is classified as a living thing, and are often called the "building blocks of life".
Cells consist of a protoplasm enclosed within a membrane, the cell membrane or plasma membrane about 100 trillion (10 14 , which contains many biomolecules such as proteins and nucleic acids. Organisms can be classified as unicellular (consisting of a single cell; including most bacteria) or multicellular (including plants and animals). While the number of cells in plants and animals varies from species to species, humans contain microscope. ) cells. Most plant and animal cells are between 1 and 100 micrometres and therefore are visible only under the The cytoplasm comprises cytosol (or intracellular fluid (ICF) or cytoplasmic matrix) – the gel-like substance enclosed within the cell membrane – and the organelles – the cell's internal sub-structures. Within the cells of eukaryote organisms the contents of the cell nucleus are separated from the cytoplasm, and are then called the nucleoplasm. The cytoplasm is about 70% to 90% water and usually colorless.
亚细胞结构：
A subcellular structure is simply structures within a cell. They are smaller than a cell and commonly means within a cell. They are individual components of a cell that when put together forms a complete cell. Subcellular structures can only be seen by an electron microscope, either SEM or TEM microscopy. Examples of subcellular structures are organelles Golgi apparatus, smooth and rough endoplasmic reticulum, nucleus and mitochondria.
各种分子，其中生物大分子，特别是核酸、蛋白质是决 定细胞形态和功能的关键因素。
Molecules, among them the biomacromolecules, especially nucleic acid and proteins, determine forms and functions of cells.
胚胎学
embryology:
研究个体发生、发育及其机制的学科,包括：胚胎早期发育和 各器官系统发育，先天性畸形及其成因。 与组织学互相联系，但相互独立。
Embryology is a science which is about the development of an embryo from the fertilization of the ovum to the fetus stage, mainly concerning the developmental processes and mechanism of a human being, is divided into general embryology that studies the formations and development of germ cells, fertilization and early development of human embryo, and special embryology that studies the formation and malformations of different organs. It also studies the causes inducing malformations. Embryology and Histology are connected with each other, and are separated from each other.
Conception 5weeks
畸形学
Teratology
：研究致畸因子作用和先天性畸形的成因及预防的科学
Teratology is the study of abnormalities of physiological development. It is often thought of as the study of human birth defects, but it is much broader than that, taking in other non-birth developmental stages, including puberty; and other non-human life forms, including plants. A newer term developmental toxicity includes all manifestations of abnormal development, not only frank terata. These may include growth retardation or delayed mental development without any structural malformations.
Anencephaly
[ænən'sefəlɪ]
Cyclopia
学习组织学与胚胎学的意义 学习其他相关学科的前提： 病理学
Histology and Pathology:
Histology: observe the conditions of cells and tissues under disease-free condition.
Pathology: observe the changes of cells and tissues by the disease processes.
生理学 其他学科：儿科学、内科学、外科学、 妇产科学 科学研究：
Research The subject no longer merely deals with the structure of the body; it also concerns itself with the body’s function. In fact, histology has a direct relationship to other disciplines and is essential for their understanding.
You will recognize the importance of this subject as they refer to the text later in your careers. An excellent example of this relationship will be evident when the reader learns about the histology of the kidney and realizes it is the intricate and almost sublime structure of that organ (down to the molecular level) that is responsible for the kidney’s ability to perform its function. Alterations of the kidney’s structure are responsible for a great number of life-threatening conditions.
This program includes lectures and laboratory practices. The fast developing Histology & Embryology is a main and fundamental course for different specialty of medicine, and makes the students know the basic knowledge and techniques, and acquire the ability to analyze and solve problems in further studies in other medical courses and clinic management, even in your future careers. The remainder of this chapter will discusses the methods used by histologists and embryologists.
二、当代组织学与胚胎学
Modern H & E
电子显微镜（
electron microscope
，简称电镜 ） 的发明 和超微结构的发现
The German physicist Ernst Ruska and the electrical engineer Max Knoll constructed the prototype electron microscope in 1931, capable of four-hundred-power magnification; the apparatus was the first demonstration of the principles of electron microscopy. Two years later, in 1933, Ruska built an electron microscope that exceeded the resolution attainable with an optical (light) microscope. Moreover, Reinhold Rudenberg, the scientific director of
Siemens-Schuckertwerke
, obtained the patent for the electron microscope in May 1931.
分辨率达 的
0.2nm
超微结构（ 。
20
年后发展了超薄切片术；扫描电镜出 现。可以观察细胞膜、细胞器、染色体、细胞间纤维成分
ultrastructure
） ，发现了组织、器官中的大 量新的细胞种类、各种细胞间的连接和空间配置关系
---
达 到亚细胞水平。
The maximizing resolution power of an electron microscope may reach 0.2nm. Microtomy was developed, after that 20 years, and then, scanning electron microscope was invented. Subcellular structures, such as plasma membrane and organelles, were seen with the aid of electron microscopy.
The structures visible under the electron microscope is termed ultrastructure.
普通光学显微镜和电子显微镜观察常用长度单位
:
毫米 mm, millimeter；微米μm, micrometer；纳米nm, nanometer
Length units commonly used in microscopy and EM are
μm (1/1000mm)and nm (1/1000 μ m).
mm,
近代组织学与胚胎学进展： 改进、发展各种技术，大量使用新仪器、技术： 图像分析仪
image analyzer
镜
Confocal Laser Scanning Microscope
胞术
flow cytometry, FCM Immunocytochemistry
。 、共焦激光扫描显微 、流式细 ，免疫组织化学
With development and advance in sciences and the advent of new technology, many new instruments and technique were applied by histologists and embryologists.
Confocal Laser Scanning Microscope Image analyzer Flow cytometry, FCM Immunocytochemistry, and so on
三、组织学与胚胎学的研究方法
Research methods used in Histology and Embryology:
组织学技术种类繁多，其原理涉及物理、化学、生物化学、免 疫学、分子生物学等知识
The small size of cells and matrix components makes histology dependent on the use of microscopes. Advances in physics, chemistry, biochemistry, physiology, immunology, molecular biology and pathology—and the interactions among these fields—are essential for a better knowledge of tissue biology. Familiarity with the tools and methods of any branch of science is essential for a proper understanding of the subject. This chapter reviews some of the more common methods used to study cells and tissues and the principles involved in these methods.
（一）一般光学显微镜技术
Light (Optical) microscopy
常用光镜标本制备 石蜡切片（
paraffin sectioning
）： ①取材和固定
Tissue Specimen collection and fixation
： 固定 剂
—
蛋白质凝固剂：常用甲醛
formaldehyde--
在很大程度上保存组织的 原本结构。组织块一般不超
1.0cm
大小。
To avoid tissue digestion by enzymes present within the cells (autolysis) or by bacteria and to preserve the structure and molecular composition, pieces of organs should be promptly and adequately treated before or as soon as possible after removal from the body. This treatment—fixation — can be done by chemical or, less frequently, physical methods. In chemical fixation, the tissues are usually immersed in solutions of stabilizing or cross-linking agents called fixatives. Because the fixative needs some time to fully diffuse into the tissues, the tissues are usually cut into small fragments (about 1.0cm in length, width and thickness ) before fixation to facilitate the penetration of the fixative and to guarantee preservation of the tissue. The most common fixative for light microscopy is 10% neutral buffered formalin ( 4% formaldehyde in phosphate buffered saline).
Tissue preparation
A. paraffin section preparation
Specimen: as fresh as possible
l
Fixation: fixative: formalin solution; purpose: to preserve the structural organisation Dehydration: replace the water in the tissue
by alcohol Clearing: replace the alcohol by xylene Embedding: replace the xylene w/ melted paraffin Sectioning & mounting
The fixative preserves tissues or cells mainly by irreversibly cross-linking proteins. The main action of these aldehyde fixatives is to cross-link amino groups in proteins through the formation of methylene bridges (-CH2-), in the case of formaldehyde. This process, while preserving the structural integrity of the cells and tissue can damage the biological functionality of proteins, particularly enzymes , and can also denature them to a certain extent. This can be detrimental to certain histological techniques. Further fixatives are often used for electron microscopy such as osmium tetroxide or uranyl acetate.
Formalin fixation leads to degradation of mRNA, miRNA and DNA in tissues. However, extraction, amplification and analysis of these nucleic acids from formalin-fixed, paraffin embedded tissues is possible using appropriate protocols.
② 脱 水 dehydration 和 包 埋 embedding ： 梯 度 酒 精 ascending graded ethanol 脱去固定好的组织块中 的水分；二甲苯 dimethylbenzene; xylene; xylol 置换组织中的酒精—不溶于石蜡；浸蜡—将组织块 置 于 融 化 的 石 蜡 paraffin; paraffin wax; paraffine 中 ， 让 石 蜡 浸 入 组 织 细 胞 ， 冷 却 — 蜡 块—具有石蜡的硬度。
Biological specimens often need to be solidified to allow fine sectioning.
Embedding and electron microscopy.
materials include paraffin and plastic resins. Paraffin is used routinely for light microscopy; resins are used for both light
Tissue blocks are first stabilized by fixatives. Then:
Dehydration:
ethanol Blocks are dehydrated in ascending graded series ( usually from 70% to 100% ethanol ).
Clearing: The ethanol is then replaced with a solvent miscible with the embedding medium. In paraffin embedding, the solvent used is usually xylene . As the tissues are infiltrated with the ( clearing ).
solvent, they generally become transparent
Embedding:
The blocks are embedded in paraffin wax the typical embedding medium for routine histology.
which is
Once the tissue is impregnated with the solvent, it is placed in melted paraffin ( paraffin wax being taken out of the oven. ) in the oven, typically at 58– 60℃. The heat causes the solvent to evaporate, and the spaces within the tissues become filled with paraffin. The tissue together with its impregnating paraffin hardens after
Tissue processing Embedding moulds: (A) paper boat; (B) metal boat mould; (C) Dimmock embedding mould; (D) Peel-a-way disposable mould; (E) base mould used with embedding ring ( F) or cassette bases (G)
③切片
Sectioning
和染色
staining
：用切片机将组织切 为
5-10μm
薄片，贴在载玻片上，脱蜡，水化（由 高到低浓度酒精
descending grades of alcohol
，到 水），染色。
The hard blocks containing the tissues are then taken to a microtome and are sectioned by the microtome’s steel or glass blade to a thickness of 1 10μm. The sections are floated on water and transferred to glass slides to be stained.
Because many tissue constituents have approximately the same optical densities, they must be stained for light microscopy, usually with watersoluble stains. Therefore, the paraffin must first be removed from the section, after which the tissue is rehydrated and stained.
After staining, the section is again dehydrated so that the coverslip may be permanently affixed by the use of a suitable mounting medium. The coverslip not only protects the tissue from damage but also is necessary for viewing the section with the microscope.
Various types of stains have been developed for visualization of the many components of cells and tissues; they may be grouped into three classes:
Stains that differentiate between acidic and basic components of the cell Specialized stains that differentiate the fibrous components of the extracellular matrix
Metallic salts that precipitate on tissues, forming metal deposits on them The most commonly used stains in histology are hematoxylin and eosin (H&E).
④封片
Mounting
：切片经脱水
—
梯度酒精（由低浓度到高浓 度），滴加树胶，盖玻片密封保存。
The final step in this procedure is to permanently mount the sections under a coverslip. This is accomplished by covering the section in a medium that will harden and produce a clear binder between the slide and coverslip. The ideal mounting medium should not distort the stain color, or yellow and become brittle with age.
Rinsing, Dehydration & Mounting Prep
water 70 % ethanol 2-3 minutes 80 % ethanol 2-3 minutes 95% ethanol 2-3 minutes 100% ethanol 2-3 minutes Clearing Agent 10 minute Clearing Agent 10 minute I used mounting resin called Permount (Fisher Scientific) and Neutral balsam.
How to get tissues for study?
Steps in tissue preparation
Fresh tissues from the body 1. fixation Formalin ( 10% formaldehyde)
Osmium tetroxide for EM
Mechanism - Forms cross links with proteins (Lysine) 2. Embedding – gives support for tissue slicing Paraffin or plastic resin 3. Washing & dehydration (dehydration by graded alcohols in ascending order) 4. clearing – to remove paraffin & alcohol By xylol 5. block making
6. section cutting – 5-10μ thick sections with microtome 7. mounting – on glass slide ( adhesive – albumin) 8. clearing – xylol 9. rehydrate – alcohols in descending order staining nuclear stain – Hematoxylin ( basic stain & water soluble) counter stain – Eosin ( less water soluble but soluble in alcohol) – dehydrate in ascending order
10. dehydrate and Clearing – alcohols in ascending order – xylol 11.Mounting medium – cover glass
Staining
Purpose: To make tissue section pigment
for observation.
H-E Staining: Hematoxylin
:
basic dye, purple-blue Eosin
:
acid dye, pink color
B
asophilic: components bonded by basic dye (H); pruple-blue
（
nuclear chromatin & basophilic substance in cytoplasm
）
Acidophilic: components bonded by acidic dye (E); pink (cytoplasm & collagenous fiber)
Neutrophilic: do not stain w/ both basic and acid dyes Metachromasia: a dye stains tissue a different color from that of dye solution, e.g. toluidine blue stains mast cells in purple color
With few exceptions, most tissues are colorless, so observing them unstained in the light microscope is useless. Methods of staining tissues have therefore been devised that not only make the various tissue components conspicuous but also permit distinctions to be made between them. The dyes stain tissue components more or less selectively. Most of these dyes behave like acidic or basic compounds and have a tendency to form electrostatic (salt) linkages with ionizable radicals of the tissues. Tissue components, such as ribosomes that stain more readily with basic dyes are termed basophilic , so basophilia is the affinity of cellular structures for basic dyes, such as methylene blue ; those with an affinity for acid dyes are termed acidophilic, acidophilia is the affinity of cellular structures for acidic dyes, such as eosin . Those with an weak affinity for both basic dyes and acid dyes are termed neutrophilic, neutrophilia is the a ffinity of cellular structures for neutral dyes Hematoxylin behaves like a basic dye, that is, it stains the basophilic tissue components. The main tissue components that ionize and react with basic dyes do so because of acids in their composition (nucleic acids and acid glycoproteins). Acid dyes (eg, orange G, eosin, acid fuchsin) stain the acidophilic components of tissues such as mitochondria, secretory granules, and collagen.
苏木精
—
伊红染色法
(HE
染色法
) (hematoxylin-eosin staining)
二甲苯 1 二甲苯 2 100% 酒精 在染色缸中 二甲苯 2 二甲苯 1 100% 酒精 100% 酒精 95% 精 酒 95% 精 酒 85% 精 酒 75% 精 酒 苏木精 染色液 盐酸酒 精 水 洗 水 洗 100% 酒精 95% 酒 精 95% 酒 精 85% 酒 精 75% 酒 精 伊红染 色液
Hematoxylin and Eosin (H & E) Staining H & E is a charge-based, general purpose stain. Hematoxylin stains acidic molecules shades of blue. Eosin stains basic materials shades of red, pink and orange. H & E stains are universally used for routine histological examination of tissue sections. Fixation
Any well fixed tissue.
Staining Procedure
1- Deparaffinize and hydrate to water 2- If sections are Zenker-fixed, remove the mercuric chloride crystals with iodine and clear with sodium thiosulphate (hypo)
3- Hematoxylin for 15 minutes 4- Wash in running tap water for 20 minutes 5- Counterstain with eosin from 15 seconds to 2 minutes depending on the age of the eosin, and the depth of the counterstain desired. For even staining results dip slides several times before allowing them to set in the eosin for the desired time 6- Dehydrate in 95% and absolute alcohols, two changes of 2 minutes each or until excess eosin is removed. Check under microscope 7- Clear in xylene, two changes of 2 minutes each 8- Mount in Permount or Histoclad Results
Nuclei - blue - with some metachromasia Cytoplasm - various shades of pink-identifying components different tissue
切片及染色设备 自动染色机 切片机及其它设备
Microtome for sectioning resin- and paraffin-embedded tissues for light microscopy. Rotation of the drive wheel moves the tissue-block holder up and down. Each turn of the drive wheel advances the specimen holder a controlled distance, generally between 1 and 10μ m. After each forward move, the tissue block passes over the knife edge, which cuts the sections.
其他： 火棉胶包埋celloidin embedding，火棉胶切片机切片：制 作大组织块切片。
Nitrocellulose, otherwise known as cellulose nitrate, goes by several names. The commonest, in the histological world, are celloidin and collodion. Tissues embedded in celloidin are usually sectioned with stroke, and is quite long, a sliding microtome.
In this instrument the block is mounted on a platform facing upwards and is fixed. The knife is held at a significant slant so that most of the blade edge is used during the cutting often in excess of 25 cm.
冰冻切片
frozen section
： 新鲜组织，液氮 liquid nitrogen （-196 0 C）冷冻，恒冷 箱切片机 cryostat microtome —检查组织化学、酶等
Cryostat are used in medicine to cut histological slides. They are usually used in a process called frozen section histology. The cryostat is essentially an ultrafine "deli-slicer", called a microtome, placed in a freezer. The cryostat is usually a stationary upright freezer, with an external wheel for rotating the microtome. The temperature can be varied, depending on the tissue being cut - usually from minus 20 to minus 30 degree Celsius. The freezer is either powered by electricity, or by a refrigerant like liquid nitrogen.
1. Freeze a fresh, unfixed tissue sample, up to 2.0 cm in diameter, in OCT in a suitable tissue mold. Freeze the OCT containing the tissue onto the specialized metal grids that fit onto the cryostat. OCT is viscous at room temperature and miscible with H2O, but freezes into a solid support at −20
°
C.
Certain soft tissues, such as brain, are optimally frozen in M-1 medium at −3
°
C.
2. Cut sections 5-15 μm thick in the cryostat at −20
°
C. If necessary, adjust the temperature of the cutting chamber
±
5
°
C, according to the tissue under study. OCT :Optimal Cutting Temperature Compound, the formulation of water-soluble glycols and resins, is used to embed tissue samples prior to frozen sectioning on a microtome-cryostat. This process is undertaken so as to mount slices (sections) of a sample onto slides for analysis.
特殊染色
Histology Special Stains
硝 酸 银 （ 镀 银 ） 染 色 Silver impregnation,
staining
，染神经细胞，纤维成分等。
silver
某些结构经硝酸银处理使硝酸银还原，形成银微粒附 在 组 织 结 构 上
-
棕 黑 色 ， 此 性 质 称 亲 银 性 （
argentaffin)
。有些组织结构不能使硝酸银还原， 需 加 还 原 剂 使 硝 酸 银 还 原 ， 称 为 嗜 银 性 （
argyrophilia
）。
Silver (Silver nitrate) staining(
Silver impregnation
aids the visualisation of targets of interest, namely intracellular and extracellular cellular components such as DNA and proteins, such as type III collagen and reticulin fibres by the deposition of metallic silver particles on the targets of interest.
)
Argentaffin: Of or relating to cells or tissue elements that reduce silver ions in solution, thereby staining brown or black. In the argentaffin reaction, the tissue contains reducing groups that are sufficiently strong and present in sufficient quantity to give a visible deposit without added reducing agents.
These groups are often aldehyde groups and silver solutions can be used to replace the Schiff ’s reagent in the PAS technique to give periodic acid–silver.
Argyrophilia : Many tissue groups are able to adsorb silver, possibly by ionic mechanisms as for dyeing. The silver is mainly adsorbed as silver ions but small amounts are reduced to silver atoms. These silver atoms are deposited at the site of reduction. The initial reduction reaction with silver only deposits submicroscopic atoms of silver at particularly reactive sites. In this case the developer does the main reduction and the tissue simply provides places where there are silver atoms to catalyse the reduction. This type of reaction where an external reducer or developer is added is called an argyrophilic reaction.
异染性
metachromasia:
用甲苯胺蓝等碱性染料染色呈紫红色。
The term metachromasia is used when a dye stains a tissue component a different color to the dye solution. For example, toluidine blue is a strong basic blue dye that stains nuclei a deep blue color; however, it will also stain mast cell granules a pink color. This color shift that occurs with mast cells is called metachromasia, whilst the usual blue staining is called orthochromasia.Many dyes can show metachromasia but the thiazine group dyes are especially good for this type of staining.
Silver staining of the neuron and the bile canaliculi HO (Hoechst 33258) PI staining shows the apoptosis in human HL-60 cells
涂片
smear
：直接将细胞（如血细胞）涂于玻片上， 制作血涂片、细胞涂片等。
Smear
：
A small quantity of cells (e.g. blood cells, cells collected by a swab or cultured ) spread thinly on a slide for microscopic examination.
铺片：将组织撕成薄片铺在载玻片上，制作结缔 组 织 铺 片 、 肠 系 膜 铺 片 等 。
Whole-mounts, stretched preparation , where an entire organism or structure is small enough or thin enough to be placed directly onto a microscope slide (e.g. a small unicellular or multicellular organism or a membrane that can be stretched thinly on to a slide)
磨片
ground section
：将硬组织磨成薄片，制作骨 磨片、牙磨片等。
A ground section, (e.g. bone), refers to a microscope slide of bone that is prepared by taking a larger piece of the bone and placing it between two pieces of abrasive material--such as carbide paper. These are rotated and "grind" the section down until the bone is adequately thin to transmit incident light in a light microscope, allowing for observation of the bone structure. This section is then transferred to a glass slide, mounted, and cover slipped.
显微镜：
Microscopy Light microscopy Conventional light, phase contrast, differential interference, polarizing, confocal, and fluorescence microscopy are all based on the interaction of light and tissue components.
With the light microscope, stained preparations are usually examined by means of light that passes through the specimen. The microscope is composed of mechanical and optical parts. The optical components consist of 3 systems: condenser, objective, and eyepiece. The condenser collects and focuses light, producing a cone of light that illuminates the object to be observed. The objective lenses enlarge and project the illuminated image of the object in the direction of the microscope. The microscope further magnifies this image and projects it onto the viewer’s retina, a photographic plate, or ( to obtained by digital image) a detector such as a charged coupled device (CCD
电荷耦合设 备
) camera. The total magnification is obtained by multiplying the magnifying power of the objective and ocular lenses.
3
、 荧光显微镜（
fluorescence microscope
）： 使用荧光染料染色或作为标记物，以紫外线为 光源，激发染料发出荧光。
Fluorescence Microscopy When certain substances are irradiated by light of a certain wavelength, they emit light with a longer wavelength. This phenomenon is called fluorescence. In fluorescence microscopy, tissue sections are irradiated with either ultraviolet (UV) light or laser, and the emission is in the visible portion of the spectrum. The fluorescent substances appear brilliant or colored on a dark background. For this method, the microscope has a strong UV light source and special filters that select rays of different wavelengths emitted by the substances.
Fluorescent compounds that have an affinity for cell macromolecules may be used as fluorescent stains. Acridine orange, which can combine with DNA and RNA, is an example. When observed in the fluorescence microscope, the DNA–acridine orange complex emits a yellowish-green light, and the RNA–acridine orange complex emits a reddish-orange light. It is thus possible to identify and localize nucleic acids in the cells . Another important application of fluorescence microscopy is achieved by coupling fluorescent substances (such as fluorescein isothiocyanate[ɪsəʊθɪəʊ'sɪəneɪt] , FITC) to marker molecules that will specifically bind to components of the tissues and will thus allow the identification of these components under the microscope.
（二）电镜术
Electron microscopy
用电子束代替可见光，电磁透镜代替光 学透镜，用荧光屏将肉眼不可见的电子 束呈像。
Transmission and scanning electron microscopes are based on the interaction of electrons and tissue components.
透射电镜（
transmission electron microscope, TEM
）：
Transmission Electron Microscope (TEM) Using a beam of electrons (short wave-lengths) instead of visible light.
Section preparation: similar to those for L.M mainly, glutaraldehyde, osmium tetroxide
10% formaldehyde)
instead of
Formalin (
plastic,
resin ,
instead of paraffin, 50-70nm thick,
ultramicrotome
heavy metal salts uranyl acetate, lead citrate, instead of HE.
Resolution: 0.1-0.5
nm
(0.2nm) Ultrastructrue: The structure in EM Electron-dense/ electron lucent: Dark areas of an electron micrograph are usually called electron dense, whereas light areas are called electron lucent
Schematic view
示意图
of a transmission electron microscope with its lenses and the pathway of the electrons. CCD, charged coupled device.
Diagrams of TEM
The transmission electron microscope functions on the principle that a beam of electrons can be deflected by electromagnetic fields in a manner similar to light deflection in glass lenses. In the electron microscope, electrons are released by heating a very thin metallic (usually tungsten) filament ( the cathode
阴极，负极
) in a vacuum. The electrons released are then submitted to a voltage difference of 60–120 kV between the cathode and the anode
阳极
, which is a metallic plate with a hole in its center. Electrons are thus attracted to the anode and accelerated to high speeds . They pass through the central opening in the anode, forming a constant stream (or beam) of electrons that penetrates the tube of the microscope. The beam passes inside electric coils and is deflected in a way roughly analogous to what occurs in optical lenses, because electrons change their path when submitted to electromagnetic fields. For this reason, the electric coils of electron microscopes are called electromagnetic lenses.
The configuration of the electron microscope is very similar to that of the optical microscope, although the optics of the electron microscope are usually placed upside down. The first lens is a condenser that focuses the beam of electrons on the section. Some electrons interact with atoms of the section and continue their course, whereas others simply cross the specimen without interacting. Most electrons reach the objective lens, which forms a magnified image that is then projected through other magnifying lenses. Because the human eye is not sensitive to electrons, the image is finally projected on a image is always fluorescent screen or is registered by photographic plates or a charged coupled device camera in black and white . . Because most of the image in the TEM is produced by the balance between the electrons that hit the fluorescent screen (or the photographic plate) and the electrons that are retained in the tube of the microscope, the resulting To provide a good interaction between the specimen and the electrons, electron microscopy requires very thin sections ( 40–90 nm ); therefore, embedding is performed with a resin that becomes very hard. The blocks thus obtained are so hard that analyzed.
glass or diamond knives are usually necessary to section them. The extremely thin sections are collected on small metal grids and transferred to the interior of the microscope to be
电镜细胞化学技术（
electron microscope cytochemistry
）
:
组织化学与电镜技术结合 免疫电镜术（
immuno-electron microscope
）： 免疫细胞化学与电镜结合 电镜放射自显影术（
electron microscope autoradiography
）： 电镜技术与显微放射 自显影术结合 冷冻蚀刻法（
freeze etching method
）： 用 电镜观察细胞断裂面形态结构的一种冷冻 干燥技术。
冷冻蚀刻法
freeze etching method or Freeze fracture A method of specimen preparation for the electron microscope in which rapidly frozen tissue is cracked so as to produce a fracture plane through the specimen. The surface of the fracture plane is then shadowed by heavy Metal vapour, strengthened by a carbon film and the underlying specimen is digested away, leaving a replica that can be picked up on a grid and examined in the transmission electron microscope. The great advantage of the method is that the fracture plane tends to pass along the centre of lipid bilayers and it is therefore possible to get en face views of membranes that reveal the pattern of integral membrane proteins. The e face is the outer lamella of the plasma membrane viewed as if from within the cell, the p face the inner lamella viewed from outside the cell. Fracture planes also often pass along lines of weakness such as the interface between cytoplasm and membrane, so that outer and inner membrane surfaces can be viewed. Further information about the structure can be revealed by freeze etching. Extremely rapid freezing followed by deep etching has allowed the structure of the cytoplasm to be studied without the artefacts that might be introduced by fixation.
It is a technique used to look at membranes that reveal the pattern of integral membrane proteins. General outline of technique: 1. Cells are quickly frozen in liquid nitrogen (196C), which immobilizes cell components instantly.
2. Block of frozen cells is fractured. This fracture is irregular and occures along lines of weakness like the plasma membrane or surfaces of organelles.
3. Surface ice is removed by a vacuum (freeze etching) 4. A thin layer of carbon is evaporated vertically onto the surface to produce a carbon replica.
5. Surface is shadowed with a platinum vapor.
6. Organic material is digested away by acid, leaving a replica.
7. Carbon-metal replica is put on a grid and examined by a transmission electron microscope.
（三）组织化学和细胞化学术
Histochemistry and cytochemistry
The terms histochemistry and cytochemistry are used to indicate methods for localizing substances in tissue sections. Several procedures are used to obtain this type of information, most of them based on specific chemical reactions or on high-affinity interactions between macromolecules . These methods usually produce insoluble colored or electron-dense compounds be quantified by that enable the localization of specific substances by means of light or electron microscopy.The specific substances can microspectrophotometry or image analyzer.
A + B C↓
tissue or cell chromogenic reagent insoluble coloured compund to be tested electron-dense compund
It is referred to as cytochemistry to detect some substances in free cell samples using techniques of histochemistry.
For instance
:
PAS
(P
eriodic Acid Schiff)
reaction:
for manifesting polysaccharide and proteoglycan (e.g. glycogen).
polysaccharide + periodic acid ( HIO4)
(hydroxyl group) (oxidise)
Aldehyde group + Shiff’s reagent
(colorless)
Purplish red depositor
Lipids Lipids are best revealed with dyes that are soluble in lipids. Frozen sections are immersed in alcohol solutions saturated with the dye. Sudan IV and Sudan black are the most commonly used dyes. The dye dissolves in the cellular lipid droplets, which become stained in red or black. Additional methods used for the localization of cholesterol and its esters, phospholipids, and glycolipids are useful in diagnosing metabolic diseases in which there are intracellular accumulations of different kinds of lipids.
（
3
）核酸：福尔根反应（
Feulgen reaction
）：稀 盐酸水解
DNA
，用希夫试剂处理
——
紫红色产物。 同时显示
DNA
和
RNA
，用甲基绿
methyl green -
派 若宁
pyronine
反应
——
甲基绿与细胞核
DNA
结合呈 蓝绿色，派若宁与核仁、胞质内的
RNA
结合呈红 色。 Nucleic Acids DNA can be identified and quantified in cell nuclei using the Feulgen reaction, which produces a red color in DNA. DNA and RNA can also be analyzed by staining cells or tissue sections with a basic stain, such as
methyl green –pyronine reaction, at the same time
.
（
4
） 酶类
enzymes
：通过显示酶的催化活性表明酶的存 在。切片用含特异性底物的溶液孵育，底物经酶的作用形 成初级反应产物，它再与某种捕捉剂结合
——
形成镜下可 见的沉淀物。 酶多为蛋白质
—
免疫组化显示
Enzymes Examples of enzymes that can be detected include the following: Phosphatases or lead sulfide.
are enzymes widely found in the body. They split the bond between a phosphate group and an alcohol residue of phosphorylated molecules. The colored insoluble reaction product of phosphatases is usually lead phosphate Alkaline phosphatases, which have their maximum activity at an alkaline pH, can be detected .
Acid phosphatases are frequently used to demonstrate lysosomes, cytoplasmic organelles that contain acid phosphatase
Proteins Although there are general methods to detect proteins in tissue sections, the histochemical methods usually do not permit identification of specific proteins in cells and tissues. Immunocytochemistry, presented later in this chapter, can do so.
荧光染色： Fluorescent staining 敏感性高 以紫外线照射荧光物质，激发荧光 自发荧光：组织中的某些成分
Vitamin A—
自发绿色荧光 生物胺类
:
多巴胺、肾上腺素、
5-
羟色胺
—
甲醛处理
,
不同 荧光 与荧光素结合发荧光： 荧光染料：吖啶橙
—
与
DNA
结合呈黄或黄绿色荧光，与
RNA
结合呈橘黄、橘红色荧光
Tissues or cells + fluorescein fluorescence Autofluorescence: Vitamin A-green; dopamine, adrenalin, 5-hydroxytryptamine (5-HT, serotonin ) after treating with formaldehyde- different fluorescence Fluorescent dyes: acridine orange, Hoechst 33258 and 33342, PI (Propidium iodide )
，
irradiated with ultraviolet rays
Immunohistochemistry and
Immunocytochemistry
Based on antigen binds to specific antibody.
Tissue section w/ Antigen
+
labeled
a
ntibody labeled Ag-Ab complex
Fluorescein labeling enzyme labeling colloidal gold labeling
常用标记物：荧光素 过氧化物酶
luciferin —
用荧光显微镜观察；辣根
horseradish peroxidase, HRP —
经酶的组织化 学处理后用光镜或电镜观察；胶体金
colloidal gold —
电镜 观察。
Labeling with
luciferin, horseradish peroxidase, HRP , and colloidal gold (for EM)
HO (Hoechst 33258) PI staining shows the apoptosis in human HL-60 cells FITC staining shows the translocation of TFAR19 protein in HeLa cells induced by staurosporin
Photomicrograph of a section of small intestine in which an antibody against the enzyme lysozyme was applied to demonstrate lysosomes in macrophages and Paneth cells. The brown color, indicating the presence of lysozyme, results from the reaction done to show peroxidase, which was linked to the secondary antibody. Nuclei were counterstained with hematoxylin. Medium magnification.
Photomicrograph of kidney cells in culture, stained with acridine orange. Under a fluorescence microscope, DNA (within the nuclei) emits yellow light, and the RNA-rich cytoplasm appears reddish or orange. (Courtesy of A Geraldes and JMV Costa.)
4
、原位杂交术
in situ hybridization
即核酸分子杂交组织化学术。 检测
DNA
、
mRNA
片段有无及 活性
—
特异性 的核酸组织化学术。 用带标记的已知碱基顺序的核酸
(DNA
，
RNA)
探针
probe
与细胞内待测核酸按碱基配对 原则，进行特异性原位结合
-
杂交，检测、显示 标记物，获得待测核酸的有无、相对量。
Hybridization Techniques The central challenge in modern cell biology is to understand the workings of the cell in molecular detail. This goal requires techniques that permit analysis of the molecules involved in the process of information flow from DNA to protein. Many techniques are based on hybridization. Hybridization is the binding between two single strands of nucleic acids (DNA with DNA, RNA with RNA, or RNA with DNA) that recognize each other if the strands are complementary. The greater the similarities of the sequences, the more readily complementary strands form "hybrid" double-stranded molecules. Hybridization thus allows the specific identification of sequences of DNA or RNA.
In Situ Hybridization (ISH)
—nucleic acid molecular hybridization
Use nucleotide probe to check target fragment of intracellular DNA or mRNA in situ, in order to study the gene expression.
Tissue section of a benign epithelial tumor (condyloma) submitted to in situ hybridization. The brown areas are places where DNA of human papillomavirus type 2 is present. The counterstain was hematoxylin. Medium magnification. (Courtesy of JE Levi.)
Hybridization can also be performed with purified DNA or RNA in solid supports. Mixtures of DNA or RNA are separated by electrophoresis in an agarose gel or a polyacrylamide gel. After electrophoresis, the fragments of nucleic acids are transferred to a nylon or nitrocellulose sheet by solvent drag: a buffer flows through the gel and membrane by capillarity, carrying the nucleic acid molecules that bind strongly to the nylon or nitrocellulose sheet, where the nucleic acids can be further analyzed. This technique of DNA identification is called Southern blotting . When electrophoresis of RNA is performed, the technique is called Northern blotting .
Hybridization techniques are highly specific and are routinely used in research, clinical diagnosis, and forensic medicine.
Probs
核酸探针的种类：
cDNA prob cRNA prob
常用标记物：放射性核素
—
35
S
、
32
P
、
3
H—
经放射自显影术处理后观察； 荧光素、生物 素、地高辛
—
小分子药物，经免疫组织化学 处理后观察
Markers commonly used for
hybridization include 35
S,
32
P,
3
H, fluorescein
, biotin, and
digoxin, which should be treated with IHC for revewing.
原位杂交
:
端粒酶在卵巢癌中的表达（地高辛标记）
Expression of telomerase in ovarian carcinoma (labeled by digoxin)
组织培养或细胞培养术
Cell & Tissue Culture
Live cells and tissues can be maintained and studied outside the body. In a complex organism, tissues and organs are formed by several kinds of cells. These cells are bathed in blood plasma, which contains hundreds of different molecules. Cell and tissue culture has been very helpful in isolating the effect of a single molecule on one type of cell or tissue. It also allows the direct observation of the behavior of living cells under a microscope. Several experiments that cannot be performed in the living animal can be reproduced in vitro.
The cells and tissues are grown in complex solutions of known composition primary cell cultures (salts, amino acids, vitamins) to which serum components are frequently added. In preparing cultures from a tissue or organ, cells must be initially dispersed either mechanically or by treating the tissue with enzymes. Once isolated, the cells can be cultivated in a suspension or spread out on a Petri dish or glass slide, to which they adhere, usually as a single layer of cells . Cultures of cells that are isolated in this way are called . Many cell types were once isolated in this way from normal or pathological tissue and have been maintained in vitro ever since because they have been immortalized and now constitute a permanent cell line .
Most cells obtained from normal tissues have a finite, genetically programmed life span. Certain changes, however (mainly related to oncogenes), can promote cell immortality, a process called transformation, which may be a first step in transforming a normal cell into a cancer cell. Because of transformation and other improvements in culture technology, most cell types can now be maintained in the laboratory indefinitely. All procedures with living cells and tissues must be performed in a sterile area, using sterile solutions and equipment.
培养室及设备
细胞或组织化学定量术
Quantitative cyto- or histochemistry
显微分光光度定量术microspectrophotometry 显微镜技术+分光光度技术---定量检测细胞化学 成分：DNA、RNA、蛋白质、酶、脂类、糖类等
Microscopy + spectrophotometry Quantitative analysis of the chemical composition in cells, e.g.
DNA,RNA, proteins,
enzyme
s, lipids and
carbohydrates.
A microscopic thin beam of light enters the outersegment of the receptor. Some of the light is absorbed by the photopigment and the rest emerges to be captured by the very sensitive detector. The difference between that which enters the outersegment and that which emerges is the amount that is absorbed. The technique measures the light absorbed, reflected, or emitted by a microscopic specimen at different wavelengths. Such a technique used to characterize and quantify nucleoproteins in single cells or cell organelles on the basis of their natural ultraviolet absorption spectra or following stoichiometric binding in selective cytochemical staining reactions.
流式细胞术
flow cytometry
：定量分析、分选流体单个 细胞及其它生物微粒
—
细胞大小、形态、细胞内颗 粒多少，荧光染色测
DNA
、
RNA
蛋白质、细胞膜 受体和抗原等
Flow cytometry (FCM) is a technique for counting and examining microscopic particles, such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of up to thousands of particles per second. Flow cytometry is routinely used in the diagnosis of health disorders, especially blood cancers, but has many other applications in both research and clinical practice. A common variation is to physically sort particles based on their properties , so as to purify populations of interest .
The technology has applications in a number of fields, including molecular biology, pathology, immunology, plant biology and marine biology. It has broad application in medicine (especially in transplantation, hematology, tumor immunology and chemotherapy, genetics and sperm sorting for sex preselection) and so forth.
How to study Histology and Embryology
Describe the structures of major organ what tissues formation Describe the structures of cells distribute-where, how morphological features-microstructure,
ultrastructure
s Understand the functions of the cells, organs and the sysytems Understand some molecules, especially biomacromolecules, and their functions Combine the 2 dimentional structure with 3 dimentional Combine the theory with practice Combine the structure with functions
主要参考书：
References
全国高等学校教材：组织学与胚胎学
,
第
7
版，邹仲 之 李继承主编，人民卫生出版社，
2008
年
6
月， 北京
Textbook of Histology and Embryology, 2d edition, Gao Yingmao, Science Press, Beijing.
Basic Histology, Tenth Edition, Luiz Carlos Junqueira, Jose Carneiro, McGraw-Hill Companies, Inc., 2003 Basic Concepts in Embryology, Lauren J. Sweeney, McGraw-Hill Companies, Inc., 1998
第
2
章
相关知识导读
上皮组织
Epithelial Tissue
细胞与细胞是怎样连接在一起的？ 胃肠的消化吸收功能由那种细胞参与的？ 细胞间可以通过什么样的结构进行信息交 流？ 头皮屑是怎么形成的？ 痰液是如何形成及如何从气管中排出的？
上 皮 组 织 （
epithelial tissue
） 简 称 上 皮 （
epithelium
）： 由大量形态规则、排列密集的细 胞组成。上皮组织的特性： 细胞多，排列密集。 上皮细胞具有明显的极性（
polarity
）
—
细胞不同 表面在结构和功能上有明显差别。 有游离面： 朝 向身体的表面或有腔器官的腔面。 基底面： 细胞 朝向深部结缔组织的面。 侧面： 上皮细胞之间的 连接面。 上皮基底面附着在基膜上，并借此与结 缔组织相连。上皮内大多无血管
—
靠结缔组织内 的血管提供营养。 分类：主要为被覆上皮和腺上皮 功能：保护、吸收、分泌、排泄等。
一、被覆上皮（
covering epithelium
）
覆盖于身体表面，衬贴在体腔和有腔器官 内表面。 分类：根据构成 细胞的层数 和在 垂直切面 上的形状 分类。
被覆上皮的类型和主要分布 上皮类型 主要分布 ————————————————————————— 单层 单层扁平上皮 内皮：心、血管、淋巴管腔面 上皮 间皮：胸膜、腹膜、心包膜 单层立方上皮 其它：肺泡、肾小囊壁层等 甲状腺滤泡、肾小管等 单层柱状上皮 胃、肠、胆囊、子宫等 假复层纤毛柱状上皮 呼吸管道等 复层 复层扁平上皮 未角化的：口腔、食道、阴道 上皮 复层柱状上皮 角化的：皮肤表皮等 眼睑结膜、男性尿道等 变移上皮 肾盂、肾盏、输尿管、膀胱等 —————————————————————————
1
、单层扁平上皮（
simple squamous epithelium
）： 也称单层鳞状上皮。
1
、一层扁平细胞组成。 表面观：细胞不规则或多边形，核椭圆形，位 中央。细胞边缘呈锯齿或波浪状，互相嵌合。
单层扁平上皮垂切面观察：细胞扁薄，胞质很少， 含核部厚。分类： 内皮
endothelium
）：衬在心血管和淋巴管腔面； 间皮（
mesothelium
）：在胸膜、腹膜和心包膜表面。 微动脉纵切面
其它：肺泡、肾小囊 功能：主要是保持器官表面光滑，利于血液或 淋巴的流动，减少器官间的磨擦。
2
、单层立方上皮（
simple cuboidal epithelium
）： 由一层立方形细胞组成。 表面观：细胞呈六角形或 多角形。 垂直面观：细胞呈立方形， 核圆居中。
3、单层柱状上皮 simple columnar epithelium：
由一层棱柱状细胞组成。 表面观：细胞六角形或多边形。
垂直面：细胞为柱状，核长圆，常位细胞近 基底部，长轴与细胞长轴一致。 分布：胃肠、胆囊、子宫等。 有吸收、分泌功能
肠道的单层柱状上皮除 柱状细胞，还有散在的 杯状细胞（ 满分泌颗粒
goblet cell
）： 高脚酒杯状，底部狭窄， 核深染，顶部膨大，充
—
含有粘蛋 白
—PAS
反应阳性称粘 原颗粒
mucinogen granule —
分泌后与水结合成粘 液，润滑、保护上皮。 在子宫、输卵管等腔面 的，细胞游离面有纤 毛
—
单层纤毛柱状上皮
simple ciliated columnar epithelium
4
、假复层纤毛柱状上皮（
pseudostratified ciliated columnar epithelium
）： 主要在呼吸管道。 柱状细胞：最多，表面有纤毛 梭形细胞，锥形细胞，杯状细胞 细胞形态不同， 高矮不一，核 位置高低不齐， 细胞基部均附 于基膜
5
、复层扁平上皮（
stratified squamous epithelium
）： 表层细胞 呈扁平鳞片状，又称复层鳞状上皮。 基底细胞 ：矮柱状，幼稚，有增殖分化能力。 中间 ：数层多边形细胞。 表层 ：几层梭形或扁平细胞。 上皮与
CT
连接凹凸不平
—
增加连接面，保证供应，连接牢固。
分类： 角化的复层扁平上皮 位于皮肤表皮。浅层细胞的 核消失，胞质充满角蛋 白，细胞干硬，不断脱 落。 未角化的复层扁平上皮 位于：口腔、食管等腔面， 浅层细胞有核，角蛋白 少。 复层扁平上皮功能： 耐磨擦
—
保护，阻止异物 侵入，受伤后再生修复 能力强。
6
、复层柱状上皮（
stratified columnar epithelium
）： 浅层：为一层排列整齐的柱状细胞 深层：为一层或多层多边形细胞。
变移上皮 （
transitional epithelium
） 分布于排尿管道。 细胞分层： 表层细胞
——
大，厚， 称盖细胞。 中间层：细胞多边形 基底层：细胞较小。 特点：细胞形状、层次 随器官收缩、扩张状态 而变化
——
收缩时：上 皮变厚，细胞层数增多， 细胞大立方形；扩张时： 上皮变薄，细胞层数减 少，细胞呈梭形。
二、腺上皮和腺 腺上皮（
glandular epithelium
） ： 由腺细胞 组成，以分泌功能为主的上皮。 以腺上皮为主要成分构成的器官称为腺 。 腺细胞分泌物：酶类、糖
(
粘
)
蛋白、激素等。 分类： 外分泌腺（
exocrine gland
）
—
有导管 ， 分泌物经导管排至体表或器官腔内。如汗 腺、唾液腺等。 内分泌腺（ 上腺等。
endocrine gland
）
—
无导管 ， 分 泌物（激素）释放入血液。如甲状腺、肾
外分泌腺的结构和分类
按细胞数目：单细胞腺 多细胞腺： 一般由分泌部和导管组成
外分泌腺腺泡与导管
外分泌腺： 导管部：无分支
—
单腺 有分支
—
复腺 分泌部： 管状、泡状、管泡状 单管状腺 单泡状腺
按形态
复管状腺 复泡状腺 复管泡状腺
1
、分泌部
secretory protion
一般由一层腺细胞组成，中央有腔。 形状：管状、泡状和管泡状 泡状和管泡状的分泌部称为 腺泡
(acinus)
。 根据分泌部形状分：管状腺
tubular gland
、泡 状 腺
acinar gland
和 管 泡 状 腺
tubuloacinar gland
。 腺细胞多呈锥形 ，由于分泌物不同形态各异 腺泡： Acinus 由腺细胞组成。消化、呼吸系统一般分为 浆液性和黏液性细胞。
1
）蛋白质分泌细胞
protien secreting cell
浆液性细胞（
serous cell
） ：
锥体形，核圆， 靠近细胞基底部。 基部胞质强嗜碱性， 顶部胞质有许多嗜 酸性的分泌颗粒
—
酶原颗粒
zymogen granule
电镜： 胞质（尤其基部胞质） 有密集的粗面内质 网，核上区有发达 的高尔基复合体和 数量不等的分泌颗 粒
-
蛋白质分泌细胞 的超微结构特点 合成、分泌蛋白质 。 分泌物：稀薄，含 有消化酶
2
）糖蛋白分泌细胞
glycoprotein secreting cell,
黏 液性细胞
mucous cell:
分泌糖蛋白（黏蛋白）
细胞锥体形， 核扁圆位细胞基部。 胞质色浅，含粘原颗粒，
HE
片呈泡沫或空泡状。 核周少量胞质，嗜碱性。
电镜： 基部胞质有较 丰富的粗面内质网， 核上高尔基复合体发达 有丰富、粗大的黏原颗 粒。分泌粘液。 杯状细胞： 散在的粘液性细胞 分泌过程：高尔基复合 体内合成多糖，并与 粗面内质网合成的蛋白 质结合
—
糖蛋白
—
分泌 颗粒
—
胞吐释放
杯 状 细 胞 电 镜 图
腺体：
Glands
浆液性腺泡
——
由浆液性腺细胞组成； 粘液性腺泡
——
由粘液性细胞组成； 混合性腺泡
——
由两种细胞组成。 消化系统和呼吸管道的外分泌腺可分为： 浆液性腺： 分泌部全由浆液性腺泡组成：腮腺 粘液性腺： 分泌部全由粘液性腺泡组成：十二指肠腺 混合性腺： 由三种腺泡组成：颌下腺、舌下腺
大部分混合性腺泡主要由粘液性腺泡组成， 少量浆液性腺泡位于腺泡底部，切片中呈半 月形
—
浆半月（
serous demilune
） 。
粘液性细胞间隙局部扩大，形成分泌小管， 浆半月的分泌物经分泌小管释放入腺泡腔。 在汗腺、乳腺和唾液腺分泌部与基膜间可有 肌上皮细胞（
myoepithelial cell
） ：扁平， 有突起，胞质含肌动蛋白丝
—
收缩，帮助 分泌物排出。
2、导管（duct）： 单层或复层上皮构成。有的可分泌或吸收水、 电解质。 单腺：1个或几个分泌部连1条不分支的导管 复腺：导管分支，最小的导管末端连分泌部 腺体分类：分泌部形状+导管
(
二
)
上皮组织的特殊结构 Specializations of the cell surface in epithelium
游离面： 侧 面： 基底面： 微绒毛 纤毛 紧密连接 中间连接 桥粒 缝隙连接 基膜 质膜内褶 半桥粒
（
1
）微绒毛（
microvillus
） 细胞游离面的 微细指状突起 。 小肠上皮细胞
:
密集、整齐排 列成 纹状缘（
striated border
异。 ） 。直径
0.1μm
，长 度因细胞种类、生理状态而
1
、上皮细胞的游离面 结构： 细胞膜，胞质，许多纵 行微丝
—
上端附于微绒毛顶 部，下端插入胞质，附于 终 末网（
terminal web
）
—
微 绒毛基部胞质中与细胞表面 平行的微丝网，边缘部附于 细胞侧面的中间连接处。 微丝 ：为肌动蛋白丝，终末网 中还有肌球蛋白，微丝收缩 使微绒毛伸长或缩短。 功能：增加表面积。
纹状缘
2、纤毛（cilium）：
上皮细胞游离面 粗而长 的突起，有节律定向摆动。 长
5-10μm
直径
0.2μm
。
外包 细胞膜 ， 细胞质，
9+2
结构： 中央
2
条 单独的微管，周围
9
组二联微管，二联 微管一侧伸出
2
条动 力蛋白臂
动力蛋白（
dynein
）： 有
ATP
酶活性
—
分解
ATP
后动力蛋白臂附于 相邻的二联微管
—
微管间产生位移或滑 动
—
纤毛整体运动。 纤毛基部有一致密的 基体（
basal body
）
—
似 中心粒，基体微管与纤毛微管连续。 功能：许多纤毛协调摆动把上皮表面的粘液 及粘附颗粒定向推送
—
利于排出。
（二） 上皮细胞的侧面Lateral surface of epithelial cell
细胞相邻面，间隙狭窄，无明显的细胞外 基质，相邻细胞以 钙粘蛋白（
cadherin
） 互相结合。 钙粘蛋白 ：为细胞粘附分子，镶嵌于细胞 膜，胞外部分在钙离子存在下才能结合。 细胞侧面的特化结构为细胞连接
——
只有 电镜下才能观察到，并也都依赖钙离子。
1
、紧密连接（
tight junction
） 又称闭锁小带（
zonula oocludens
） 位细胞侧面顶端。相邻细胞膜形 成
2-4
个点状融合
—
此处间隙消失， 非融合处有极窄的间隙
—
观察： 冷冻蚀刻复型法
—
劈开细胞膜双 层脂质，暴露膜内的蛋白质，透 射电镜观察：紧密连接处的膜内， 蛋白颗粒排列成
2-4
条线形结构， 交错成网格，带状环绕细胞。相 邻细胞连接面上，这种网格互相 吻合，蛋白颗粒与蛋白颗粒对接， 封闭细胞间隙
—
屏障作用：阻挡 物质通过。
2
、中间连接（
intermediate junction
） 又称粘着小带（
zonula adherens
）： 位紧密连接下方， 环绕细胞顶部 。 相邻 细胞 间 隙
15-20nm
，内 有 中等电子密度的丝状物 连接相 邻细胞膜，膜的胞质内面有 薄 层致密物质和微丝附着 ，微丝 组成 终末网 。 功能：粘着，保持细胞形状，传递 细胞收缩力。 此连接也见于心肌细胞间的闰盘。
3
、桥粒
desmosome
或黏着斑
macula adherens
斑状 ，大小不等。细胞 间隙
20 30nm
，有 隙中央有一条与细胞膜平行的 中间线
—
低密度的丝状物 ，间 由丝状物质交织而成。 细胞膜的胞质面有较厚的致密 物质构成的 附着板 ，胞质中有 许多 直径 回胞质
— 10nm
的角蛋白丝（张 力丝）附于板上，折成袢状返 起固定，支持作用。 功能：连接牢固。 分布：易受磨擦部位
—
皮肤、 食管等复层扁平上皮中发达。 半桥粒：在后面介绍
病例
2-2
寻常型天疱疮 皮肤棘层松解，表皮内裂隙和水泡。 免疫病理显示角质形成细胞间
IgG,IgA,IgM
或
C3
网状沉积，血清中存在针对桥粒成分的天 疱疮抗体 本型天疱疮的抗原主要是桥粒芯糖蛋白
3(Dsg3)
天疱疮抗体
+
桥粒芯糖蛋白
3(Dsg3)—
通过细胞 信号传导途径激活一系列蛋白水解酶
—
细胞 间连接结构（桥粒）水解，棘层细胞分离、 棘层松解及表皮内水泡
4
、缝隙连接
gap junction
连接处相邻细胞膜高度平行， 细胞间隙
3nm
， 内有许多间 隔大致相等的 连接点 。 冷冻 蚀刻复型：缝隙连接处胞膜 中有规律分布 柱状颗粒
—
连 接小体（
connexon
） ，聚集 为大小不等的斑状。 小 体 直 径
7-9nm
，有
6
个 杆 状连接素（
connexin
）分子 围成，中央有直径
2nm
管腔 。 连接小体贯穿细胞膜双层脂 质，突出细胞表面
1.5nm
， 相邻细胞膜中连接小体对接， 管腔相通 。
在钙离子和其它因素作用下，管道可开、 闭。分子 量
<1500D
的物质：离子、
CAMP
等信息分子、氨基酸、葡萄糖、维生素等 可通过使细胞在营养代谢、增殖分化、功 能 等 方 面 成 为 统 一 体
—
又 称 通 讯 连 接 （
communication junction
）。 连接复合体（
junctional complex
） ：上述连 接，只要有
2
个或以上同时存在，即成为。 细胞连接的存在、数量随器官的发育阶段、 功能状态、病理变化而改变。
（三）上皮细胞的基底面
1、基膜（basement membrane）：薄，HE 片不易分辨。假复层纤毛柱状上皮、复 层扁平上皮厚。镀银：黑色。
电镜： 基板（basal lamina） ：靠上皮，上皮细胞产生， 50-100nm，包括： 透明层（lamina lucida）
—
紧 贴细胞基底面，电子密度低； 致密层（lamina densa） ：深面，电子密度高。 基板主要成分： 层粘连蛋白laminin,LN、纤维黏 连蛋白fibronectin,FN、 IV型胶原蛋白和硫酸肝 素蛋白多糖 层粘连蛋白： 大分子粘连性糖蛋白，具有与上皮 细胞、与IV 型胶原蛋白、硫酸肝素蛋白多糖等细 胞外基质成分结合的部位
—
在细胞与细胞外基质连接中起媒介作用，促进细 胞粘着在基膜上并铺展开。
网板reticular lamina ： 由结缔组织成纤维细胞分 泌产生
—
由 网状纤维、基 质构成 ，可有少许胶原纤 维。 某些部位
—
毛细血管内皮 下、肌细胞和某些神经胶 质细胞周围，仅有基板。 功能： 支持，连接，固着， 半透膜作用
—
利于细胞与 结缔组织物质交换， 引导 上皮细胞移动，影响细胞 增殖、分化 。
2、质膜内褶plasma membrane infolding
上皮细胞基底面胞膜向胞 质形成许多内褶，与细胞 基底面垂直。 褶间有纵行长线粒体
—
光镜下形成基底纵纹。 主要见于肾小管。作用： 扩大基部表面积，利于 水、电解质转运。
半桥粒（hemidesmosome）：
位上皮细胞基底面。为桥粒的一半，质膜内有附着板， 张力丝附着其上，折成袢状返回胞质。作用：将上皮 细胞固着于基膜上
.