觀察、度量與紀錄
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• 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。
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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。
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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。
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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
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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。
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3-dimentional migration of leukocytes
NPC (CD3 stain)
H&E
Why T cells infiltrate and accumulate here? Is there
any intrinsic difference between tissue/cells?
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第一種想法
被拉住了!
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Brownian motion
or active
movement?
Bacteria
Ciliates
Macrophages
http://en.wikipedia.org/wiki/Chemotaxis
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Diapedesis of leukocytes.
leukotriene B4 (can be produced by macrophages) as chemoattractant
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Leukocytes migration through endothelia cells
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Methods for cell motility assay
(chemokinetic vs. chemotactic: as a matter of time and direction)
• Agar-plate techniques (bacteria,
http://en.wikipedia.org/wiki/Image:Chtxtwochamber-techn.PNG
ciliates)
• Two-chamber techniques
– Boyden chamber
– Dunn chamber (Zigmoid chamber)
– Capillary method
• Wound healing assay
• Motility assay
– Time lapse
– Particle tracer
uniform carpets of gold particles
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Transmigration of T cells
through the tumor
monolayers.
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
Extracellular matrix recaptures the effect of tumor
monolayers.

It is integrin-dependant.
ECM was extracted by
0.5% Triton X-100.
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Jurkat cells spread poorly on glioma ECM.
(F-actin accumulated at front of lamellipodia)
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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.
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
The transcripts of collagen IV (COL IV), fibronectin (FN), lamining1 (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.
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Tenascin-C can interact with perlecan and
fibronectin, and cell surface receptors including
integrins a2b1, avb3 and a9b1, and annexin I.
Nature Reviews Cancer 5, 436-446 (2005)
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
Tenascin-C gene of glioma cells is knocked down by shRNA method.

Tenascin-C low tranfectant cells allow better transmigration of Jurkat
cells.
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A better spreading of Jurkat cells on monolayer and ECM
of U-118MG(TNCshRNA) cells.
Question: how does tenascin-C work?
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Tumor contact activates ERK activities in Jurkat cells,
which are required for the transmigration of Jurkat
cells.
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
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.
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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
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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.
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3-dimentional migration of leukocytes
NPC (CD3 stain)
H&E
Why T cells infiltrate and accumulate here? Is there
any intrinsic difference between tissue/cells?
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第二種想法
喜歡待在那裡!
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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.
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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
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• 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.
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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
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Vinculin controls focal adhesion formation by direct interactions with talin and
actin.
JD Humphries et al. JCB, 179, 1043–1057 (2007)
NIH3T3 cells model
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以上的觀察程序如何?
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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。
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以上實驗的度量與紀錄
有什麼特徵?
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