大鼠骨髓间充质干细胞来源HGF对肝星状细胞DR5及Caspase-8表达的影响*

 目的:观察大鼠骨髓间充质干细胞(BMSCs)来源的肝细胞生长因子（HGF）对原代肝星状细胞(HSCs)死亡受体-5(DR5)及Caspase-8的影响，初步探讨BMSCs来源HGF在促进HSCs凋亡中的部分作用机制。方法:贴壁法培养及纯化SD大鼠BMSCs，使用第3-4代细胞进行实验；复苏大鼠原代HSCs及传代，应用6孔培养板，每孔使用(Transwell insert)半透膜建立双层细胞共培养体系，常规培养24h、48h、72h。实验分为4组:A组:空白对照组; B组:BMSCs+HSCs共培养组; C组: TNF-a预处理组及D组:HGF-ShRNA干扰组。酶联免疫吸附法（Elisa）检测各组上清液中HGF及TRAIL的浓度；用流式细胞仪Annexin V-FITC/PI双染法检测细胞凋亡；分别使用荧光定量PCR(FQ-PCR)、Western blot检测各组HSCs内DR5、Caspase-8mRNA和蛋白的表达。结果：1. Elisa法检测结果示:TNF-a预处理组的HGF浓度明显高于BMSCs组及空白对照组（P<0.01）；BMSCs组中TRAIL的浓度明显高于空白对照组及TNF-a预处理组（P<0.01）;2. Annexin-V-FITC/PI双染法检测凋亡结果示：TNF-a预处理组HSCs的凋亡率明显高于其它3组（P<0.01）且呈时间依赖性；HGF-ShRNA干扰组中HSCs的凋亡率低于共培养组,较空白对照组高二者比较有统计学意义（P<0.05）。3. FQ-PCR、Western blotting结果示共培养组及TNF-a预处理组HSCs的DR5、Caspase-8mRNA及蛋白表达量明显高于空白对照组及HGF-ShRNA干扰组（P<0.01）。 结论： BMSCs与HSCs共培养后促进HSCs的凋亡，其可能与BMSCs来源HGF能上调HSCs的DR5及Caspase-8的表达有关。

HGF from bone marrow mesenchymal stem cells paracrine up-regulate the expression of DR5 and caspase-8 in rat hepatic stellate cells

Objective To observe the effect of HGF from rat bone marrow mesenchymal stem cells paracrine on hepatic stellate cells death Receptor-5 and caspase-8.Methods Six well plates were used to establish co-culture system.Cells were divided into group A,control group,group B,co-cultured group;group C:TNF-α pretreatment group;group D:HGF-ShRNA interference group.The concentrations of HGF and TRAIL were measured by ELISA.Apoptosis of HSCs was determined by Annexin-V-FITC/PI;the expression of DR5 mRNA,caspase-8 mRNA and protein were determined by FQ-PCR and Western blot respectively.Results 1) Concentration of HGF in TNF-α pretreatment group was obviously higher than that in BMSCs group and control group(P<0.01);Concentration of TRAIL in the BMSCs group was higher than that in control group and TNF-α pretreament group;2) Apoptosis rate of HSCs in TNF-α pretreatment group was obviously higher than that of the other three groups and had time-dependent(P<0.01);Apoptosis rate of HGF-ShRNA interference group was lower than that of in cocluture group and higher than that of control group(P<0.05).3) The expressions of DR5,caspase-8 protein in co-culture group and TNF-α pretreatment group were significantly higher than that of in control group and HGFShRNA interference group(P<0.01).Conclusions BMSCs paracrined HGF can up-regulate the expression of DR5 and caspase-8 which may be associated with the apoptosis of HSCs.