肝细胞生长因子通过促进内源性干细胞增殖治疗大鼠肝硬化的研究

HGF promotes liver cirrhosis in rats by boosting the proliferation of endogenous stem cells

目的 探讨不同剂量肝细胞生长因子（hepatocyte growth factor,HGF）促进内源性干细胞增殖治疗肝硬化的疗效.方法 通过腹腔注射50％四氯化碳橄榄油溶液构建肝硬化模型,100只模型大鼠随机分成四个实验组：分别为腹腔注射HGF低剂量组、中剂量组、高剂量组以及空白对照组,采用血清学检测比较四个实验组大鼠丙氨酸氨基转移酶（alanine transaminase,ALT）、天门冬氨酸氨基转移酶（aspartate transaminase,AST）、白蛋白（albumin,ALB）以及总胆红素（total bilirubin,TBIL）水平来评估肝脏功能,应用RT-PCR、Western blot、免疫组织化学检测ALB、角蛋白18（cytokeratin 18,CK18）、肝细胞核因子-4α（hepatocyte nuclear factor-4α,HNF-4α）蛋白变化,评估肝细胞增殖情况.通过EdU荧光标记内源性干细胞,评估其向肝样细胞分化情况.结果 血清学检测表明治疗第4周各治疗组较空白对照组AST、ALT、TBIL均有显著降低,中剂量组与低剂量组之间差异具有统计学意义（均P〈0.05）.ALB各治疗组于第4周均较对照组显著升高,且各治疗组间差异具有统计学差异（均P〈0.05）.免疫组织化学、RT-PCR、Western blot半定量分析表明HNF-4α、ALB以及CK18蛋白表达在中、高剂量组与对照组及低剂量组之间差异具有统计学差异（均P〈0.05）.免疫荧光半定量分析,中、高剂量组ALB、CK18蛋白阳性细胞数与对照组及低剂量组相比,差异均具有统计学意义（均P〈0.05）.结论 多次反复腹腔注射HGF可促进内源性干细胞增殖并缓解大鼠肝硬化,且最佳腹腔注射剂量为20 mg/kg/d.

 Objective To explore the effect of different doses of hepatocyte growth factor （HGF）on promoting the proliferation of endogenous stem cells in the treatment of liver cirrhosis. Methods All of 100 rats were injected intraperitoneally with 50% carbon tetrachloride to construct the model of liver cirrhosis,and were randomly divided into 4 experimental groups：blank control group,low dose group,middle dose group,high dose group of intraperitoneal injection of HGF. Liver functions were evaluated by serological test of Aspartate Trans-aminase （AST）,Alanine Transaminase （ALT）,Total Bilirubin （TBIL）and Albumin （ALB）. Changes of ALB,Cytokeratin 18 （CK18）and Hepatocyte Nuclear Factor-4α （HNF-4α）expression were detected by RT-PCR,western blot and immunohistochemistry to evaluate the proliferation of hepatic cell. Endogenous stem cells were labeled by EdU to evaluate the proliferation and differentiation to hepatic cell. Results Compared with the control group,AST,ALT and TBIL in experimental groups were significantly lower in the fourth wee （P 〈0. 05）. The difference between middle dose group and low dose group was statistically significant. While,ALB in experimental groups were significantly higher than control group in the fourth week（P 〈 0. 05）. There were sig-nificant differences among all experiment groups（all P 〈 0. 05）. Semi-quantitative analysis of expression on HNF-4α,ALB and CK18 loci in immunohistochemistry,RT-PCR,and Western blot showed that both control group and low dose group between middle and high dose group were statistically significant（all P 〈 0. 05）. Con-clusion Repeated intraperitoneal injection of HGF may promote the proliferation of endogenous stem cells and alleviate cirrhosis in rats. The best dosage of intraperitoneal injection is 20 mg/ kg/ d.