肝细胞生长因子在人肝细胞中的表达及对肝细胞损伤的保护作用

目的　构建肝细胞生长因子 (HGF)真核细胞表达载体 ,并观察其在人正常肝细胞中的表达以及对细胞增殖和抗损伤能力的影响。方法　利用基因重组技术将HGF与绿色荧光蛋白 (GFP)融合并构建真核表达质粒 ,通过脂质体介导法 ,导入正常人肝细胞系中。用荧光显微镜以及免疫组织化学方法观察HGF表达情况。采用 3 H] TdR掺入DNA法、PCNA免疫组织化学观察肝细胞DNA合成 ,了解肝细胞增殖能力的变化。用CCl4造成急性肝细胞损伤 ,通过测定细胞存活率、细胞内丙氨酸氨基转移酶 (ALT)、K+ 的漏出量了解肝细胞的抗损伤能力。结果　酶切鉴定证实HGF片断已克隆到pEGFP N3 的BamHⅠ和SalⅠ位点之间 ,在转染人正常肝细胞后 ,利用荧光显微镜观察可见到绿色荧光蛋白的表达 ,用免疫组化方法进一步证实了HGF蛋白在细胞中的表达。 3 H] T转导HGF的肝细胞的DNA合成明显增加 (转染 96h后 ,3 H] TdR掺入量为对照组的 7倍 )。PCNA免疫组化结果显示转导HGF的肝细胞阳性率明显增加 ,肝细胞增殖活性增加。转导HGF的肝细胞抗CCl4损伤的能力明显增强 ,肝细胞存活率显著提高 (83%与 6 1% ,P <0 0 5 ) ,细胞内丙氨酸氨基转移酶 (ALT)、K+ 的漏出显著降低 (分别为 35 16U·L-1与 6 5 31U·L-1,P <0 0 1,5 5 9mmol/L与 6 0 2mmol/L ,P

Autocrine expression of hepatocyte growth factor and its cytoprotective effect on hepatocyte poisoning

OBJECTIVES: To construct pEGFP-hepatic growth factor (HGF) expression vector, detect its transient expression in transfected human hepatocytes, and to investigate the influence of autocrine HGF expression on the proliferative potential and cytoprotective effects in human hepatocyte. METHODS: Human HGF cDNA was ligated to pEGFP vector. The recombinant plasmid was transfected into human hepatocyte line QZG with liposome. The expression of HGF protein was observed by fluorescence microscopy and immunohistochemistry. Hepatic cells were collected 24, 48, and 72 hours after transfection to detect the number of (3)H]-TdR uptake in DNA. DNA synthesis was observed by using PCNA stain immunohistochemistry. Acute liver cell damage was induced by carbon tetrachloride. The supernatant of culture 10 days after transfection of pEGFP-HGF was collected to put in the normal culture of hepatic cells. Another sample of supernatant was added with anti-HGF antibody to block the HGF activity as control. Cytoprotective effect was observed by examining the survival rate of hepatocytes and leakage of intracellular alanine transaminase (ALT) and potassium ions. RESULTS: HGF identification of pEGFP-HGF by enzyme digestion showed that HGF fragment had been cloned into BamH I and Sal I sites of pEGFP-N3. Expression of GFP in transfected hepatocytes was observed with fluorescence microscopy. The (3)H]-TdR uptake became 7 times as much as in the control group 96 hours after transfection. After HGF transfection, survival rate of hepatocytes poisoned by CCl4 significantly increased (83% vs 61%, P < 0.05), and leakage of intracellular alanine transaminase and potassium ions decreased (35.16 U x L-1 vs 65.31 U x L-1, P < 0.01; and 5.59 mmol/L vs 6.02 mmol/L, P < 0.01 respectively). Culture of transfected hepatic cells promoted the proliferation of other non-transfected cells. This effect was blocked by anti-HGF antibody. CONCLUSION: Transfected HGF is expressed in hepatic cells and has the activity of promoting cell division and protecting hepatic cells against poisoning.