【作者】 唐琳；

【导师】 刘杞；

【作者基本信息】 重庆医科大学 ， 内科学， 2005， 博士

【摘要】 目的:肝再生增强因子(augmenter of liver regeneration, ALR)是Hagiya 等于 1994 年对肝刺激因子(Hepatic stimulatory substance,HSS) 的进一步研究时克隆的一种非特异性、具有热稳定性、不同于肝细胞生长因子(Hepatocyte growth factor , HGF)的促肝细胞再生因子。前期研究发现 ALR 在体外能刺激肝癌细胞株增殖,并呈剂量依赖性,并且将 ALR 表达质粒转入肝癌细胞中,发现 ALR 能明显促进肝癌细胞增殖和增强其抗损伤的能力,这些研究结果提示 ALR 与肝癌的发生、发展可能存在一定关系。我们新近对正常肝组织和肝癌组织的研究中发现正常肝细胞几乎不表达 ALR,而肝癌细胞却高表达 ALR,但是高表达的意义尚不明确。并且还发现 ALR 对 ConA 激活的单个核细胞具有很强的抑制作用。因此,推测 ALR 作为一种生长因子和免疫抑制因子可能在肝癌的自主性生长以及肝癌逃避机体免疫监视中发挥了重要的作用。但是其在肝癌发生、发展中的具体作用及地位尚不清楚。本研究拟在体内、外通过在核酸及蛋白质水平阻断内源性人肝再生增强因子(human augmenter of liver regeneration, hALR)的表达和分泌后,观察肝癌的生长是否受影响;以及观察 rALR 对肿瘤细胞膜抗原激活的大鼠外周血单个核细胞增殖作用的影响,以明确 ALR 在肝癌发生、发展中的作用及地位。因此,本实验根据 hALR 的基因序列,设计、构建针对 hALR 基因编码区能够产生发夹结构的小干扰 RNA(small interfering RNA, siRNA)的载体质粒,将其转染 HepG2 细胞,观察对hALR 表达的影响,以及对体外培养的 HepG2 细胞和裸鼠移植瘤生长的影响;同时观察 anti-hALR McAb (anti-hALR monoclonal antibody)中和 hALR 后对 HepG2 细胞增殖和裸鼠移植瘤生长的抑制作用;用更多还原

【Abstract】 Objective: Augmenter of liver regeneration (ALR), a heat-stable hepatotrophic growth factor, was first discovered and cloned from neonatal rat livers. Although its action is similar to hepatocyte growth factor (HGF) in promoting liver regeneration, its molecular structure is different from HGF. Present research indicated ALR could stimulate the proliferation of hepatoma cells in a dose-dependent manner in vitro. Moreover, ALR could increase the proliferation of cells and protect cells against damage after ALR expression plasmid was transfected into hepatoma cells. It was also showed by immunohistochemistry that the staining of ALR was strongly positive in the liver tissue of patients with hepatocellular carcinoma. They all demonstrated that ALR was related to the carcinogenesis of hepatocellular carcinoma. Moreover, we also observed ALR could inhibit the proliferation of mononuclear cells induced by ConA or specific antigens in vitro. Why hALR was highly expressed in hepatocellular carcinoma? We suspected ALR might play an important role in the carcinogenesis of hepatocellular carcinoma because of being involved in autocrine growth of hepatocellular carcinoma and immune suppression in hepatocellular carcinoma. However, the association of ALR and carcinogenesis of hepatocellular carcinoma was not fully understood. Our objective in this study was to investigate that biological behavior of hepatocellular carcinoma was influenced by siRNA targeting hALR and anti-ALR McAb on nuclei acid and protein level in vitro and in vivo. In this study, the RNAi plasmid pSIALR-A and the unrelated control plasmid pSIALR-B were transfected into HepG2 cells respectively. After transfection, the expression of hALR was measured. The proliferation of HepG2 cells after treated with pSIALR-A and anti-hALR McAb was detected. The growth of the xenograft tumor was observed after being treated with pSIALR-A and anti-hALR McAb in nude mice. The effect of rALR on proliferation of rat peripheral blood mononuclear cells was detected after rat peripheral blood mononuclear cells were activated by membrane of HepG2 cells. This study not only furthered an understanding of the mechanism of carcinogenesis of hepatocellular carcinoma but also provided potential candidates for gene therapy of hepatocellular carcinoma. Methods: 1. Expression of hALR in hepG2 cells: The expression of hALR in HepG2 cells was observed with immunocytochemistry on protein level. The expression of hALR in HepG2 cells was detected with RT-PCR on nuclei acid level. 2. To observe effects of anti-hALR McAb on biological behavior of hepatocellular carcinoma: 1) The proliferation of HepG2 cells treated with anti-hALR McAb was detected by 3H-TdR incorporation approach. 2) HepG2 cells were resuspended in 1:500 anti-hALR McAb and 1:500 ascites induced by SP2/0 cells, respectively (anti-hALR McAb/HepG2 cells and ascites induced by SP2/0 cells /HepG2 cells). Subcutaneous tumors in nude mice were induced by inoculation anti-hALR McAb/HepG2 cells and ascites induced by SP2/0 cells /HepG2 cells. Every other day, each group of mice were treated with 1:500 anti-hALR McAb or 1:500 ascites induced by SP2/0 cells by i.p. The growth of the xenograft tumor was observed after inoculation. The expression of hALR in thexenograft tumor was observed with immunocytochemistry. 3. Construction and identification of expressing siRNA plasmid of hALR and unrelated control plasmid pSIALR-B: After being constructed successfully, the expressing siRNA plasmid pSIALR-A, which targeted the cDNA of hALR, and the unrelated control plasmid pSIALR-B were transfected into HepG2 cells respectively (After transfection, the cells were nominated pSIALR-A / HepG2 and pSIALR-B / HepG2). At 24 hours after transfection, the cells were examined for the presence of GFP by an inverted fluorescent microscope. At 48 hours after transfection, the protein level of hALR was measured with immunocytochemistry; Meanwhile, the reverse transcriptive PCR (RT-PCR) was performed to detect the expression of hALR mRNA. 4. To observe更多还原