肝星状细胞对大鼠肝部分切除术后肝再生修复的影响

Experimental study on the effect of hepatic stellate cells on rat liver regeneration and repair after partial hepatectomy

目的 探讨肝星状细胞（hepatic stellate cells,HSCs）在大鼠肝部分切除术后对肝损伤修复的影响.方法 体外实验部分：将大鼠肝星状细胞（HSCs）与大鼠肝细胞系（BRL-3A）共培养,CCK-8比色法检测细胞增殖情况;ELISA法检测培养肝星状细胞上清液中细胞因子,即肝细胞生长因子（hepatic growth factor,HGF）、胰岛素样生长因子（insulin growth factor,IGF）、表皮细胞生长因子（epidermal growth factor,EGF）、转化生长因子-α（transfactor growth factor-α,TGF-α）含量.体内实验部分;将45只260 ～330 g健康雄性SD大鼠随机分为三组：正常组（生理盐水组）、对照组（肝星状细胞培养液组）、实验组（肝星状细胞培养上清液组）.各组分别于肝大部切除术后第3、7、12天取血清检测肝功能情况并计算肝再生指数;HE染色切片显微镜下观察肝脏病理结构改变情况;免疫组织化学染色法检测增殖细胞核抗原（proliferating cell nucleus antigen,PCNA）、平滑肌肌动蛋白（α-smooth muscle actin,α-SMA）、甲胎蛋白（a1pha feta1 protein,AFP）及白蛋白（albumin,ALB）的表达情况.结果 ①CCK-8法检测肝星状细胞能促进肝细胞增殖（P＜0.05）;②上清液较培养液中细胞因子HGF、TGF-α含量多,差别有统计学意义（P＜ 0.05）;③实验组肝再生指数比对照组大,差别具有统计学意义（P＜0.05）;④实验组大鼠AST随着时间的推移,较对照组下降明显,差别有统计学意义（P＜0.05）;⑤实验组PCNA表达量较正常组、对照组增多,差别有统计学意义（P＜ 0.05）;⑥平滑肌肌动蛋白、白蛋白、甲胎蛋白三组间差别不显著（P＞ 0.05）.结论 ①体外肝星状细胞能促进肝细胞增殖;②肝星状细胞培养上清液能促进大鼠肝部分切除术后肝再生,其分泌的多种细胞因子在肝损伤再生修复中起了重要作用.

Objective To investigate the effect and mechanism of hepatic stellate cells （HSCs） on rat liver regeneration and repair after partial hepatectomy.Methods HSCs were co-cultured with hepatocytes （BRL-3a） in vitro.The proliferation of BRL cells was detected by CCK-8 test kit.The cytokines secreted by hepatic stellate cells were tested by ELISA test kit.In vivo experiment：Forty-five male Sprague Dawley rats were randomly assigned to three groups （n=15 per group）：normal group （saline group）,control group （hepatic stellate cells culture medium group） and the experimental group （hepatic stellate cells culture supernatant group）.The content of ALT,AST,albumin,total protein in serum were detected on 3 d,7 d and 12 d after partial hepatectomy and the liver regeneration index was calculated.The specimens from each group were investigated with HE and immunohistochemical staining for proliferating cell nucleus antigen （PCNA）,alpha SMA,alpha fetal protein （AFP） and albumin.Results ① HSCs could significantly promote hepatocytes proliferation in vitro （P＜0.05）.② The levels of the cytokines in supernatant medium secreted by hepatic stellate cells were higher than those in nutrient solution,the difference was statistically significant （P＜0.05）.③ Compared with control group,the liver regeneration index in experimental group was higher,the difference was significant （P＜0.05）.④ It was distinct that the AST synthesised by hepatocytes in experimental group decreased gradually,compared with other groups （P＜0.05）.⑤The difference of PCNA was significant among three groups,but there were not significant differences among α-SMA,ALB and AFP.Conclusion ① HSCs can significantly promote hepatocytes proliferation in vitro; ② The cytokines HGF,TGF-α secreted by hepatic stellate cells play important roles in liver regeneration after partial hepatectomy.