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系統識別號 U0026-2801201121152500
論文名稱(中文) 肝癌細胞之層黏蛋白的功能性探討
論文名稱(英文) Functional Study of Laminin in Hepatoma Cells
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 99
學期 1
出版年 100
研究生(中文) 姚文欣
研究生(英文) Wen-Hsin Yao
學號 S4697409
學位類別 碩士
語文別 英文
論文頁數 42頁
口試委員 指導教授-楊倍昌
口試委員-凌斌
口試委員-洪義人
中文關鍵字 肝癌細胞  細胞外間質  層黏蛋白 
英文關鍵字 hepatoma cells  extracellular matrix  laminin 
學科別分類
中文摘要 在肝組織中,過量之細胞外間質沉積會造成肝纖維化,而此疾病與後續可能演發的肝癌有很高的相關性。層黏蛋白(laminin)是一種基底膜富含的細胞外間質,同時,它也被發現是纖維化之肝組織及肝癌組織中主要的細胞外間質之一。Laminins是由α,β及γ鏈所組成的異三元體醣蛋白,目前已發現16種異構體,而經由與細胞上的接受體結合,其所傳遞的訊息可以調控細胞生長、凋亡、黏附、移動,以及分化等現象。在正常的生理情況中,laminins主要由基質細胞所分泌,不過在肝癌組織中,肝癌細胞本身也會分泌大量的laminins。目前對於癌細胞本身分泌的laminin會對腫瘤形成造成什麼影響尚未完全明瞭。由於laminin的γ1鏈廣泛的存在於身體各處,因此我們以慢病毒感染sh-RNA的方式將HepG2與PLC/PRF/5這兩個肝癌細胞株中的γ1鏈的表現量減低。缺乏laminin γ1鏈的細胞,其顆粒性增加,約有5到10%的細胞死亡。待這些細胞適應缺乏laminin γ1的環境後,細胞增生的速度較控制組慢。另一方面,缺乏laminin γ1的表現並不影響細胞移動的能力與速度。但在皮下注射腫瘤細胞的動物模式中,我們卻發現缺乏lamininγ1表現的細胞反而較快形成腫瘤團塊,且團塊體積亦較控制組大,而它與促進血管新生的能力無關。對於缺乏laminin γ1的細胞較易在小鼠體內生成腫瘤的原因,仍需再做探討。
英文摘要 Liver fibrosis, an outcome of excessive extracellular matrix (ECM) deposition, is highly correlated with hepatocellular carcinoma (HCC). Among ECMs, laminins, being basement membrane components, are the major ECM components that have been found in fibrosis liver and HCC. Laminins are heterotrimeric glycoproteins composed ofα, βand γchains. To date, 16 laminin trimers are found, and they regulate various cellular functions such as growth, apoptosis, motility, adhesion, and differentiation. Laminns are normally secreted by stromal cells. However, hepatoma cells have been shown to be major laminins producers in tumors. At moment, the contribution of tumor-secreted laminins to tumor formation is not fully understood. Therefore, we established laminin-knockdown cells and analyzed the in vitro and in vivo effects on HCC cell lines as well as tumor formation. As being prevalent among laminins, the γ1 chain of laminin in HepG2 and PLC/PRF/5, two human hepatoma cell lines, was knockdown by sh-RNA strategy using lentiviral system. Lacking laminin γ1 (Lm-γ1) resulted in 5 to 10% of cell death, and the granularity of cells was increased. After Lm-γ1-knockdown cells adapted to such condition, the growth rate of the cells was still lower than that of control cells. Besides, Lm-γ1 did not affect random and polarized migration of PLC/PRF/5. In the subcutaneous tumor model on nude mice, we surprisingly discovered that tumors composed of Lm-γ1-knockdown cells had shorter latent duration and bigger size than control tumors did, and this was not due to increased angiogenesis. The reason of Lm-γ1-knockdown cells in promoting tumor formation still needs further analysis.
論文目次 摘要i
Abstract ii
致謝iii
Contents iv
Introduction 1
Extracellular matrix 1
Laminin 1
Laminin and tumor 3
Increased expression of laminin in hepatoma 3
Rationale 4
Materials and Methods 5
Materials 5
Media and buffers 8
Methods 13
Results 20
The expression of laminin-γ1 in mouse liver tissue and several hepatocellular carcinoma cell lines. 20
Knocking down of Lm-γ1 in PLC and HepG2 by transducing sh-RNA-carrying lentivirus. 20
Knocking down of Lm-γ1 affected the morphology and granulation of PLC/PRF/5 and HepG2 cells. 20
Knocking down of Lm-γ1 affected the survival rate of PLC/PRF/5 and HepG2 cells within about 20 days post transduction. 21
The growth rate of PLC/PRF/5 was decreased after knocking down of Lm-γ1. 22
Lacking of Lm-γ1 did not affect the migration of PLC/PRF/5 cells. 22
sh-L PLC/PRF/5 cells formed larger subcutaneous tumor than control cells in nude mice. 22
Number of CD31+ blood vessels was comparable between GFP- and sh-L-formed tumors. 23
Discussion 24
References 27
Figures and Figure Legends 31
Appendix 41
自述 42
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