【作者】 梁珊；

【导师】 刘杞；

【作者基本信息】 重庆医科大学 ， 内科学， 2009， 硕士

【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)的研究中发现的一种非特异性、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。前期研究中发现ALR可通过自分泌方式直接刺激肝癌细胞增殖,适当剂量的ALR可作为一种免疫抑制因子逃避机体免疫系统对肝癌的免疫效应。在核酸及蛋白水平抑制hALR的表达后,体内、外肝癌生长均受到显著抑制。肿瘤的发生、发展是由细胞网络调控的、多基因参与的、极其复杂的过程。转化生长因子-α(transforming growth factor-α,TGF-α)是参与细胞生长与转化的一类细胞因子,通过与细胞膜上的表皮生长因子受体(epidermal growth factor receptor,EGFR)结合,最终促使细胞增殖和分化。许多恶性肿瘤中均有TGF-α的高表达及EGFR表达上调,肿瘤细胞自分泌TGF-α作用于自身膜受体,形成其自身增殖的环路,对肿瘤的发生发展起着重要作用。正常肝细胞中ALR受体表达的数量及亲和力均低于肝癌细胞。ALR可以上调EGFR mRNA的表达,通过作用于EGFR来调节肝细胞的再生。EGFR可以介导尿激酶型纤溶酶原激活物受体(Urokinase plasminogen activator recepter ,uPAR)/整合素/纤连蛋白诱导的生长途径,从而促进肿瘤细胞的生长与转移,EGFR激酶(能水解EGFR)能阻断uPAR诱导的生长途径。尿激酶型纤溶酶原激活物受体(uPAR)是细胞表面一种锚连蛋白受体,活化后能影响肿瘤细胞的侵袭转移。这提示细胞因子间的相互作用可能在肿瘤的发生发展过程中起着重要作用。本研究拟在体外通过RNA干扰技术在核酸水平阻断内源性产生的人肝再生增强因子(human augmenter of liver regeneration, hALR)后,观察人肝癌细胞HepG2中TGF-α及EGFR的表达是否受影响,以阐明hALR在促肝癌细胞生长相关细胞因子网络中起何作用。方法:⒈将构建好的人肝再生增强因子siRNA表达质粒pSIALR—A及其阴性对照质粒pSIALR—B采用脂质体转染的方法分别转染至HepG2细胞,荧光显微镜观察绿色荧光蛋白的表达,计算转染效率。⒉免疫细胞化学法检测转染细胞中hALR表达,以确定抑制效果。⒊根据转染不同的质粒,将HepG2细胞分为三组:转染组(转染pSIALR-A),对照组(转染pSIALR-B),空白组(未转染重组质粒),每组设三个复孔。转染成功以后再进行以下两部分的研究:用放射免疫法检测各组细胞培养上清中TGF-α水平。用Western blot法检测各组细胞中EGFR表达水平。每个实验均重复三次。结果:1、转染后24 h,HepG2细胞有大量明亮的绿色荧光蛋白(GFP)表达,经两种质粒转染的细胞均有80%以上的细胞有绿色荧光的表达,转染效率高。HepG2细胞经两种质粒转染后,两组细胞的转染效率基本相同,无明显差异,可做后续实验。2、转染48h后HepG2细胞hALR在蛋白水平表达的变化转染pSIALR-A的HepG2细胞hALR表达明显减少。而转染阴性对照质粒pSIALR-B的HepG2细胞仍然有hALR表达。在蛋白水平证明hALR的siRNA具有显著的抑制hALR表达的作用。3、转染HepG2细胞上清中TGF-α表达的变化用放射免疫法检测转染组、阴性对照组、空白组细胞上清中TGF-α含量,发现转染组与空白组比较细胞上清中TGF-α水平明显降低,P<0.05,对照组与转染组比较P<0.05,对照组与空白组比较P﹥0.05。4、转染后HepG2细胞中EGFR表达的变化Western blot法检测转染组、阴性对照组、空白组细胞中EGFR含量,发现转染组与空白组比较EGFR蛋白表达水平明显降低,P<0.05,转染组与对照组比较P<0.05,对照组与空白组比较P﹥0.05。结论:1、以脂质体为载体介导重组质粒pSIALR-A及pSIALR-B转染人肝癌HepG2细胞,转染效率较高。2、hALR的siRNA具有显著和特异性的抑制hALR表达的作用。3、阻断人肝再生增强因子的表达后, HepG2细胞转化生长因子-α及表皮生长因子受体的表达均受到明显抑制。更多还原

【Abstract】 Objectives Augmenter of liver regeneration (ALR), a heat-stable hepatotrophic growth factor, was first discovered and cloned from rat livers. Its action is different from hepatocyte growth factor (HGF). In Preliminary study we found that ALR could stimulate hepatoma proliferation directly by autocrine, the appropriate dose of ALR can be used as an immune inhibitor of the immune system to evade the immune response of hepatocellular carcinoma. When the level of expression hALR of nucleic acid and protein were inhibited, the liver tumor were both inhibited significantly in vivo and vitro.There are complicated cellular network , multi-gene involvement in the occurrence and development of tumor . Transforming growth factor-α(TGF-α) takes part in cell growth and transformation, combined with the epidermal growth factor receptor (EGFR) cause cell proliferation and differentiation. Many malignant tumors were both of the high TGF-αexpression and EGFR expression, tumor cell autocrine TGF-αreceptor on their membranes to form a loop of its own proliferation of tumors plays an important role in the occurrence and development. The number and affinity of ALR receptor in hepatocytes were lower than in hepatomaes. ALR can up-regulate the expression of EGFR mRNA, through the role of the EGFR to regulate regeneration of liver cells.EGFR can mediate urokinase-type plasminogen activator receptor (uPAR) / integrin / fibronectin-induced growth, thereby promoting tumor cell growth and metastasis, EGFR kinase (to hydrolysis EGFR) can block uPAR-induced growth of channels. UPAR is a cell surface anchor protein receptor, can affect the invasion and metastasis of tumor cells after stimulating. This suggests that the interaction between cytokines may play a role in the tumor development process.This study was to investigate the effect of blocking the expression of hALR on the expression of TGF-αand EGFR of HCC cell line HepG2 with small interfering RNA (siRNA) targeting ALR. This study furthered an understanding of the mechanism of carcinogenesis of hepatocellular carcinoma.Methods⒈The expressing siRNA plasmid pSIALR—A targeting hALR and the unrelated control plasmid pSIALR-B were constructed successfully and transfected into HepG2 cells with lipofectamine 2000 methods ,respectively.the expression of green fluorescent protein was observed under a fluorescent microscope to calculate transfection efficiency. ⒉The protein level of hALR in the transfected cells was measured by immunocytochemistry to determine the inhibitory effect.⒊According to different plasmid transfection, HepG2 cells were divided into three groups: transfection group (transfected pSIALR-A), the control group (transfected pSIALR-B), blank control group (non-transfected with the recombinant plasmid). there are three duplicated wells in each group. The expression of TGF-αin the cell culture supernatant was detected by radioimmunoassay. The expression of EGFR was detected by Western blot. Each experiment was repeated for three times.Resluts1. At 24 hours after transfection, there are a large number of bright green fluorescent protein (GFP) expression in HepG2 cells .There are are more than 80% of the cells which has the expression of green fluorescent protein after transfected by the two plasmids.The transfection efficiency is high. The two groups of cells have basically the same transfection efficiency.2. At 48 hours after transfection, the protein level of hALR was measured with immunocytochemistry. In HepG2 cells which transfected pSIALR-A , the level of hALR protein expression decreased significantly. HepG2 cells which transfected the negative control plasmid pSIALR-B still has the expression of hALR. In the level of protein ,it suggests that siRNA targeting hALR can significantly inhibit the expression of hALR.3. The expression level of TGF-αin the transfection group is significantly lower than the blank group and the control group (P <0.05).4. The relative quantity of EGFR expression of the transfection group,the blank group, the control group detected by Western blot was 1.115±0.606,1.131±0.509,0.946±0.136,respectively.The relative quantity of EGFR in the transfection group is significantly lower than the blank group and the control group (P <0.05).Conclusion1. The expressing siRNA plasmid pSIALR—A targeting hALR and the unrelated control plasmid pSIALR-B were transfected into HepG2 cells with lipofectamine 2000 methods,respectively.The transfection efficiency is high.2. The pSIALR-A inhibited the expression of hALR in HepG2 cells significantly as compared with pSIALR-B.3. The Expression of Transforming Growth Factor-α(TGF-α) and Epidermal Growth Factor Receptor in HepG2 was inhibited after transfection in vitro.更多还原