研究肝癌細胞對於內皮前驅細胞及衍生內皮細胞分化、移動、入侵及管狀結構形成之影響

Effect of Hepatocellular Carcinoma Cell on Differentiation, Migration, Invasion, and Tubular Structure Formation of Endothelial Progenitor Cell and Outgrowth Endothelial Cell

陳庭芳(Chen, Ting Fang)

指導教授 ： 陳令儀 ； 裘正健

國立清華大學/生命科學院/分子醫學研究所/碩士(2009年)

摘要

血管新生在胚胎發育或組織修補上扮演重要的角色，也是腫瘤惡化過程中重要的步驟。近年研究顯示，來自血液的內皮前驅細胞及衍生內皮細胞會因為受到腫瘤細胞釋放的訊息因子刺激之後，移動與侵入至腫瘤組織附近，並逐漸分化成內皮細胞而幫助血管新生發展。在多篇的文獻中，也都證明利用骨髓移植或皮下注射內皮前驅細胞及衍生內皮細胞到帶有腫瘤的動物體內，內皮前驅細胞及衍生內皮細胞會移動到腫瘤組織的週邊並促進血管新生。然而，內皮先驅細胞和衍生內皮細胞與腫瘤之間的交互作用，存在著許多待解的謎題。因此，本實驗將利用體外共同培養系統，觀察內皮前驅細胞與其衍生內皮細胞受到腫瘤細胞的影響後，其分化、移動、侵入、細胞週期與管狀結構的能力變化。
在臨床相關的結果裡顯示，肝腫瘤是個會釋放大量促進血管新生訊息及好發血管新生的惡性腫瘤。首先，我們利用流式細胞儀偵測細胞表面特殊蛋白的變化，發現肝腫瘤細胞株Huh7與Hep3B能夠促進內皮前驅細胞分化走向更成熟的內皮細胞；進一步，我們利用活細胞攝影系統與計算移動細胞數，發現肝癌細胞株Huh7與Hep3B，具有促進並引導內皮先驅細胞的移動與侵入走向腫瘤細胞的能力，在管狀結構的形成方面，肝癌細胞株並不會促進內皮前驅細胞的管狀結構能力；但相反的，肝癌細胞株Huh7與Hep3B而對於衍生內皮細胞在移動與侵入甚至是管狀結構形成的影響卻是呈現抑制的現象。不僅如此，肝癌細胞會調節衍生內皮細胞的細胞週期並抑制進入S及M的階段，促使細胞停留在G0/G1的階段。
從以上研究結果，我們認為肝癌細胞株會影響內皮先驅細胞與其衍生內皮細胞的許多血管新生的相關功能，但也許內皮先驅細胞與其衍生內皮細胞在血管新生過程中擔任的角色不同，而造成肝癌細胞株對於兩者在分化、移動、入侵及管狀結構形成的影響能力不同。

關鍵字

內皮前驅細胞 ； 衍生內皮細胞 ； 共同培養 ； 肝癌腫瘤細胞 ； 血管新生過程 ； 內皮細胞

並列摘要

Angiogenesis not only play a critical role in embryonic development and tissue repair, but also is an important process for tumor growth and metastasis. Recent studies have shown that tumor cells could induce the mobilization of endothelial progenitor cell (EPC) and outgrowth endothelial cell (OEC) from bone marrow via many cytokines, such as GM-CSF、SDF-1 and VEGF. Furthermore, those EPC could migrate and invade to tumor micro-metastatic niche, and gradually differentiate into endothelial like-cell incorporate the growing vasculature. Using ex vitro culture-expanded EPC or OEC transplanted in to the tumor bearing mice, many researches suggest that EPC control the angiogenic switch and OEC facilitate the neoangiogenesis in the tumor progression. However, the underlying interaction mechanism of EPC/OEC and carcinoma cells in the progression of tumor cell remains unclear. Thus, the aim of the present study to elucidate the effect of tumor cell on differentiation, migration, invasion, and tubular structure formation of EPC and OEC, using in vitro co-cultured system.
Firstly, we need to identified EPC and OEC derived from peripheral blood mononuclear cells. We found both of EPC and OEC displayed several commonly accepted EPC phenotypes, including spindle/cobblestone morphology, ac-LDL incorporation, UEA-1 binding, and CD31/KDR/Flt-1 reactivity. The previous clinical reports have indicated that hepatocellular carcinoma (HCC) is one of malignant tumor with rich neovascularization, which can be clearly observed in hepatic angiography. On the basis of co-culture transwell model and time-lapse video microscopy system, we show that migration and invasion capability of EPC was augmented by HCC, and also can induce EPC express higher intensity of endothelial markers to promote the differentiation of EPC. On the other hand, the migration, invasion and tubular structure formation of OEC was inhibited by HCC. In addition, HCC did not alter cell cycle of EPC, whereas they prevented OEC from entering S and G2/M phases and induce OEC cell cycle G0/G1 arrest. The present study suggests that the different effect of HCC on EPC and OEC may mediate their different contribution to tumor angiogenesis. Our findings may provide new insights into the interaction mechanism of EPC/OEC and HCC involve in the progression of angiogenesis.

並列關鍵字

endothelial progenitor cell ； outgrowth endothelial cell ； coculture ； hepatocellulae carcinomas cell ； angiogenesis processing ； endothelial cell

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