臺灣博碩士論文加值系統

細胞外基質是一種位在細胞外面的組織的成分，主要是提供細胞結構上的支撐，並且可以調控細胞的一些行為，如生存、發育、移行、增生及細細胞型態。細胞外基質包括間質性基質及基底質，基底質部分包括了第四型的膠原蛋白及非膠原蛋白的基質(如基膜粘連蛋白及纖維結合蛋白)。腫瘤會有異常的細胞外基質的表現，而細胞外基質也會在腫瘤生長中扮演阻止免疫細胞浸潤至腫瘤的角色。為了探討究竟腫瘤的細胞外基質對於腫瘤新生及免疫浸潤的影響如何，我們利用慢病毒攜帶干擾核醣核酸，抑制了B16F10 (老鼠黑色素瘤細胞株)中第四型膠原蛋白、纖維結合蛋白及基膜粘連蛋白的基因表現。這些細胞株分別命名為shCol, shFn, and shLm γ1。降低這些細胞外基質的基因表現並不會影響細胞的生長速率，但是會抑制傷口癒合、單一細胞的移行與基底膜基質的侵入能力。利用皮下注射腫瘤細胞到C57BL/6 老鼠後，腫瘤生長速度的順序為控制組 = shFn ＞ shLm γ1 ＞ shCol。我們也利用流式細胞儀的技術分析在這些腫瘤中免疫細胞浸潤的情形，發現在這些降低細胞外基質表現的腫瘤中，嗜酸性球的浸潤比例較高。實驗性腫瘤轉移模式的動物實驗，結果顯示在肺部腫瘤結合形成的數量順序為控制組 = shLm γ1 ＞ shFn = shCol。在體外三維的細胞培養系統中，降低第四型膠原蛋白表現的腫瘤細胞比控制組形成結構較鬆散的腫瘤球，並且細胞容易從腫瘤球上剝落。在蘇木素?伊紅染色及免疫螢光染色下，觀察腫瘤切片，可見降低纖維結合蛋白的腫瘤切片及降低基層粘連蛋白的腫瘤切片中，有較多的纖維母細胞聚在這些腫瘤的周圍，然而在降低第四型膠原蛋白的腫瘤切片中則這種細胞明顯減少。在transwell 實驗中，降低第四型膠原蛋白腫瘤細胞的條件培養液，和控制組相比吸引較少的老鼠纖維母細胞。綜合以上的結果顯示，腫瘤的細胞外基質可能透過影響細胞的移行能力及和纖維母細胞間的互動而影響了腫瘤新生的能力。

The extracellular matrix (ECM) is the extracellular part of tissue that provides structural support to cells and regulates the behaviors of cells, such as survival, development, migration, proliferation, and morphology. ECM includes the interstitial matrix and basement membrane. The basement membrane consists of type IV collagen and non?collagenous ECM (such as laminin and fibronectin). Many studies have demonstrated abnormal expressions of ECM components in tumors. Tumor ECMs play a role in preventing infiltration of immune cells. To understand the impact of tumor ECM on tumorigenesis and immune cell infiltration, we knocked down three ECM genes including type IV collagen, fibronectin, and laminin gamma 1 by Lentiviral?mediated RNA
interference. These ECM knock down clones derived from B16F10 (a mouse melanoma cell line) were named as shCol, shFn, and shLm γ1, respectively. Knocking down those
ECM genes did not significantly affect cell growth rate. ECM?suppression significantly impaired wound healing, single cell migration and matrigel invasive ability. By subcutaneous inoculation into C57BL/6 mice, tumor formation abilities of those ECM?suppressed B16F10 cells were in the order of control = shFn ＞ shLm γ1 ＞ shCol. We also analyzed infiltrating immune cells in ECM?suppressed tumors by FACS and found elevated neutrophils infiltration in ECM?suppressed tumors. Experiment tumor metastasis model showed that the number of tumor nodules in the lung was in the order of control = shLm γ1 ＞ shFn = shCol. By using 3D cell culture, we found that shCol cells formed loose structure and easily detached from 3D spheroid as compared with control group. By H＆E and immunofluorescence staining, fibroblasts were detected in the peripheral area of the control tumor, shFn tumor, and shLm γ1 tumor, but less in shCol tumor. Accordingly,conditioned medium of shCol cells attracted less NIH3T3 cells in transwell system. In
conclusion, tumor ECM may affect tumorigenesis by affecting tumor motility and interaction with the fibroblast.

摘要 I
Abstract II
誌謝 IV
Contents VI
Figure Index VII
Introduction 1
Materials ＆ Methods 6
Materials 6
Media ＆ Buffers 9
Methods 14
Results 25
Discussion 33
References 36
Figures 41
Appendix 57

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