阻断肝再生增强因子表达对HepG2细胞转化生长因子-α及表皮生长因子受体表达的影响  

Effect of blocking expression of human augmenter of liver regeneration (hALR) on the expression of transforming growth factor-α (TGF-α) and epidermal growth factor receptor in HepG2

作　　者：梁珊[1] 孙航[1] 王新国[1] 谢松丽[1] 刘杞[1] 

机构地区：[1]重庆医科大学病毒性肝炎研究所教育部感染性疾病分子生物学重点实验室,重庆400010

出　　处：《中国病原生物学杂志》2009年第6期401-404,F0003,共5页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.30570826);重庆市自然科学基金项目(No.2004-54)

摘　　要：目的用RNA干扰技术阻断人肝再生增强因子(human augmenter of liver regeneration,hALR)表达,观察人肝癌细胞HepG2中转化生长因子-α(transforming growth factor-α,TGF-α)及表皮生长因子受体(epidermal growth factor receptor,EGFR)的表达是否受影响,以阐明hALR在促进肝癌细胞生长的相关细胞因子网络中的作用。方法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。免疫细胞化学法检测转染细胞中hALR表达,确定抑制效果。根据转染质粒的不同,将HepG2细胞分为3组:转染组(转染pSIALR-A)、阴性对照组(转染pSIALR-B)和空白组(未转染重组质粒)。放射免疫法检测阻断肝再生增强因子后各组细胞培养上清中TGF-α水平,Western blot法检测阻断肝再生增强因子后各组细胞EGFR蛋白的表达。结果转染组、空白组和阴性对照组TGF-α分别为(5.27±0.86)pg/ml、(7.92±2.65)pg/ml和(6.50±1.28)pg/ml,转染组与空白组、阴性对照组比较差异有统计学意义(P<0.05);EGFR相对表达量分别为0.946±0.136、1.115±0.606和1.131±0.509,转染组与空白组、阴性对照组比较差异有统计学意义(P<0.05)。结论在促进肝癌细胞生长的细胞因子网络中,肝再生增强因子除了可以直接刺激肝癌细胞增殖外还可通过上调TGF-α及EGFR的表达水平参与肝癌的发生、发展。Objective To investigate the effect of blocking the expression of hALR on the expression of TGF-α and EG-FR of HCC cell line HepG2 with small interfering RNA （siRNA） targeting ALR. Methods 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 expression of green fluorescent protein was observed under a fluorescent microscope when calculating transfection efficiency. The protein level of hALR in the transfected cells was measured by immunocyto-chemistry to determine the inhibitory effect. According to different plasmid transfection, HepG2 cells were divided into three groups： the transfection group （transfected pSIALR-A）, the control group （transfected pSIALR-B） and the blank control group （non-transfected with the recombinant plasmid）. The expression of TGF-α in the cell culture supernatant was detected by radioimmunoassay, the expression of EGFR was detected by Western blot. Results In the transfection group, the blank group and the unrelated control group, the expression of TGF-α was （5.27±0.86） pg/ml, （7.92±2.65）pg/ml and （6.50±1.28） pg/ml, respectively. In this case, the difference between the transfection group and the：blank group as well as the transfection group and the unrelated control group was statistically significant （P〈0.05）. When the relative expression of EGFR is 0.946±0.136,1.115±0.606 and 1. 131±0.509, respectively, the difference between the transfection group and the blank group as well as the transfection group and the unrelated control group was again statistically significant （P〈0.05）. Conclusion In the complex eytokine network which promotes the growth of hepatomas, the augmenter of liver regeneration played an important role in the occurrence and development of hepatic carcinoma not only by stimulating hepatomas proliferation directly but also by upwardly regulating the expression level of TGF-α and EGFR.

关 键 词：人肝再生增强因子 RNA干扰 转化生长因子-Α 表皮生长因子受体 肝癌细胞HEPG2 

分 类 号：R735.7[医药卫生—肿瘤]