Transcript and tenascin-C
觀察、度量與紀錄
1
• 選擇觀察的事物與保證個別觀察的客觀性， 是每一個生物學家進行研究之前必須面臨的 考驗。 經驗主義 者要求知識應該建立在自然 事物的觀察上； 理性主義 則認為人的推理就 可以作為知識來源的基礎。生物學知識的累 積過程，實際上只是擺盪在這兩端。 2
物質科學裡所注重的觀察項目有兩 個特徵： 可經驗性及可描述性 。
• 科學討論對於觀察項目的可經驗性與可描述性的 要求越一致，則越有共識。大部分的科學爭議， 若能先回頭檢視立論中的觀察項目是否符合這兩 個特徵，多半可以省下許多爭吵的力氣。 3
有效率的觀察者
•
一般科學研究過程的心智活動 及順序，約略由「提出問題、 假說、觀察、度量和推理」這 五個步驟所組成。
4
四種思維歷程：一、理所當然型；二、 天真型；三、實際型；四、倒裝型。
文章發表 5
三種方式來描述觀察的事物
• 分類（ Classification ） : 黑白紅黃、金 銀銅鐵、草木蟲魚是分類的語言。 • 量化（ Quantification ） : 有單位、附 帶數字的是量化的語言。 • 比較（ Comparison ） : 大小、長短、 高低、冷暖、新舊、強弱等可以配成 對來使用的，是比較的語言。 6
3-dimentional migration of leukocytes
H&E NPC (CD3 stain) Why T cells infiltrate and accumulate here? Is there any intrinsic difference between tissue/cells?
7
第一種想法
被拉住了
!
8
Brownian motion or active movement?
Bacteria Ciliates Macrophages http://en.wikipedia.org/wiki/Chemotaxis 9
Diapedesis of leukocytes.
leukotriene B4 (can be produced by macrophages) as chemoattractant 10
Leukocytes migration through endothelia cells
11
Methods for cell motility assay (chemokinetic vs. chemotactic: as a matter of time and direction)
• • • • Agar-plate techniques (bacteria, ciliates) Two-chamber techniques – – Boyden chamber Dunn chamber (Zigmoid chamber) – Capillary method Wound healing assay Motility assay – Time lapse – Particle tracer
http://en.wikipedia.org/wiki/Image:Chtx twochamber-techn.PNG
uniform carpets of gold particles 12
Transmigration of T cells through the tumor monolayers.
13
Extracellular matrix recaptures the effect of tumor monolayers.
It is integrin-dependant.
ECM was extracted by 0.5% Triton X-100.
14
Jurkat cells spread poorly on glioma ECM.
(F-actin accumulated at front of lamellipodia)
15
Extracellular matrices
• • • •
Consist of different combinations of fibrous collagen proteins, hyaluronic acid, fibronectin, etc. Type 1 collagen is the major ECM in the dermis, up to 30 mg/ml.
Are required for proliferation, differentiation, morphogenesis, etc.
Cell-surface receptor: integrin, laminin receptors, etc.
16
The transcripts of collagen IV (COL IV), fibronectin (FN), laminin g1 (LAM) and tenascin-C (TN-C) were detected by RT-PCR.
Glioma cells express high amount of tenascin-C.
Immunohistochemical stain for tenascin-C on a glioma tumor sample.
17
Tenascin-C can interact with perlecan and fibronectin, and cell surface receptors including integrins
a
2
b
1,
a
v
b
3 and
a
9
b
1, and annexin I.
Nature Reviews Cancer 5, 436-446 (2005) 18
Tenascin-C gene of glioma cells is knocked down by shRNA method.
Tenascin-C low tranfectant cells allow better transmigration of Jurkat cells.
19
A better spreading of Jurkat cells on monolayer and ECM of U-118MG(TNCshRNA) cells.
Question: how does tenascin-C work?
20
Tumor contact activates ERK activities in Jurkat cells, which are required for the transmigration of Jurkat cells.
21
Tumor contact activates ERK activity in Jurkat cells, which is required for the transmigration of Jurkat cells.
ERK and actin colocalize at the front of lamellipodia of Jurkat cells.
22
A surrogated evidence: breast tumors with invasive phenotype express tenascin-C.
CD3+T cells are trapped in tenascin-C rich area.
Tenascin-C CD3+ cells 23
Conclusion:
Tenascin-C inhibits polarization and transmigration of T cells, that consequently prevents a direct contact of tumor and T cells and maintains immune suppressive status. • Hwang JY, Cheng YJ, Lin YP, Lin HC, Su CC, Juliano R, and Yang BC (2010) Extracellular matrix of glioblastoma inhibits polarization and transmigration of T cells: a role of tenascin-C in immune suppression. J Immunol. 185:1450-1459.
24
3-dimentional migration of leukocytes
H&E NPC (CD3 stain) Why T cells infiltrate and accumulate here? Is there any intrinsic difference between tissue/cells?
25
第二種想法
喜歡待在那裡
!
26
Our in vitro 3D migration setting
We purified the collagen from pig skin.
Tumor spheroids are generated.
Time-lapse recording.
MCF-7 spheroid U-118MG/H&E SEM of U118MG spheroid Culture on 0.5% Nobel Agar Yuhas JM, et al. (1977) Cancer Res 37:3639-3643.
27
repulsion attraction The T cells final position relative to a common origen.
The surface of spheroid is set as 0 of X axis/Y axis 28
29
• Lin YP, Cheng YJ, Huang JY, Lin HC, and Yang BC (2010) Zap70 controls the interaction of talin with integrin to regulate the chemotactic directionality of T cell migration. Mol Immunol. 47(11-12):2022-9.
30
Zap70 first at the protrusion site!
定位 (focal adhesion) 前撲 (focal complex extension) 回縮 (retraction) Images processed by ImageJ: http://a-chien.blogspot.com/search/label/ImageJ 31
Vinculin controls focal adhesion formation by direct interactions with talin and actin.
JD Humphries et al. JCB, 179, 1043–1057 (2007) NIH3T3 cells model 32
以上的觀察程序如何
?
33
三種方式來描述觀察的事物
• 分類（ Classification ） : 黑白紅黃、金 銀銅鐵、草木蟲魚是分類的語言。 • 量化（ Quantification ） : 有單位、附 帶數字的是量化的語言。 • 比較（ Comparison ） : 大小、長短、 高低、冷暖、新舊、強弱等可以配成 對來使用的，是比較的語言。 34
以上實驗的度量與紀錄 有什麼特徵
?
35