关键词: 骨髓间充质干细胞, 原代肝星状细胞, 肿瘤坏死因子相关凋亡诱导配体, 肝细胞生长因子, 死亡受体-5

Abstract: Objective: To observe rat bone marrow mesenchymal stem cells (BMSCs) derived hepatocyte growth factor (HGF) effects on primary hepatic stellate cells (HSCs) death receptor-5 (DR5), Caspase-8. To study part of the mechanism of BMSCs derived HGF to promote apoptosis of HSCs. Methods: BMSCs were isolated from SD rats, cultured and purified in vitro. Primary HSCs were cultured in plastic plates. BMSCs were cultured in the Transwell insert and the HSCs were cultured in the plastic plates(6-wells) establish the upper and lower double-cell co-culture system. Cultured 24h，48h，72h；divided into four groups: Group A: HSCs blank group: HSCs cultured alone; ② Co-cultured group: BMSCs co-cultured with HSCs; ③ TNF-a Pretreatment group: BMSCs stimulated by TNF-a six hours later discard the supernatants and then co-cultured with HSCs with fresh medium ; ④ HGF-ShRNA interference group. Cell supernatants were harvested to determine the concentration of HGF and TRAIL by ELISA. Apoptosis of HSCs was determined by Annexin-V-FITC/PI; the expression of DR5 mRNA、Caspase-8 mRNA and two kinds of protein in HSCs was determined by FQ-QPCR and Western blot respectively. Results: 1. Concentration of HGF and TRAIL in each group Supernatants were determined by Elisa: Concentration of HGF in TNF-a pretreatment group obviously higher than that in BMSCs group and HSCs blank control group (P<0.01); Concentration of TRAIL in the BMSCs group was higher than that in HSCs blank control group and TNF-a pretreament group; 2. Flow cytometric Annexin-V-FITC/PI test shew: apoptosis rate of HSCs in TNF-a 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 BMSCs+HSCs cocluture group and higher than HSCs blank control group (P<0.05). 3. The expressions of DR5、Caspase-8 protein and mRNA in HSCs was lower than BMSCs+HSCs cocluture group (P<0.01); Those of TNF-a pretreatment group was significantly higher than HSCs blank control group and HGF-ShRNA interference group (P<0.01). Conclusions: BMSCs co-cultured with HSCs to promote apoptosis of HSCs, which may be associated with BMSCs derived HGF increasing the expression of HSCs DR5 and Caspase-8.

Key words: bone marrow mesenchymal stem cells, primary hepatic stellate cells, tumor necrosis factor-related apoptosis-inducing ligand, hepatocyte growth factor, Death receptor-5