骨髓间充质干细胞表达肿瘤坏死因子刺激蛋白-6对大鼠肝脏移植排斥反应的影响

目的 探讨骨髓间充质干细胞(BMSC)表达肿瘤坏死因子刺激蛋白-6(TSG-6)在大鼠原位肝移植排斥反应的作用.方法 Kupffer细胞(KCs)和BMSC分离培养,慢病毒载体(LV3-shTSG-6)感染下调TSG-6的表达,RT-PCR以及Western blot检测BMSC的TSG-6mRNA以及蛋白表达情况,KCs与BMSC共培养,检测上清液TNF-α、TSG-6含量;建立大鼠原位肝移植急性排斥反应模型,术后1d、3d、7d处死3只受体,检测血清AST、ALT、TBIL、γ-GGT、TNF-α、IL-6、IL-4含量,RT-PCR以及Western blot检测肝组织TSG-6mRNA以及蛋白表达情况,苏木精-伊红染色观察肝组织结构,各组剩余大鼠观察术后生存率.结果 BMSC慢病毒转染率＞70％;KCs和BMSC共培养,TSG-6-shRNA-BMSC+ KCs组各时间点分泌TSG-6明显低于BMSC+ KCs组和TSG-6-NC-BMSC+ KCs组(P＜0.05),BMSC+ KCs组和TSG-6-NC-BMSC+ KCs组分泌TNF-α于6h达高峰,12 h与6h比较明显降低(P＜0.05),但TSG-6-shRNA-BMSC+ KCs组12 h分泌TNF-α明显高于BMSC+ KCs组和TSG-6-NC-BMSC+ KCs组(P＜0.05);移植术后,TSG-6-shRNA-BMSC组ALT、AST、TBIL、γ-GGT、TNF-α、IL-6明显高于TSG-6-NC-BMSC组和BMSC组(P＜0.05),与PBS组比较差异无统计学意义;TSG-6-shRNA-BMSC组、TSG-6-NC-BMSC组和BMSC组IL-4明显高于PBS组(P＜0.05),而TSG-6-shRNA-BMSC组IL-4低于TSG-6-NC-BMSC组和BMSC组(P＜0.05);在肝脏病理中,TSG-6-shRNA-BMSC组与PBS组肝组织排斥反应程度明显较高,Banff评分Ⅱ～Ⅲ级;TSG-6-shRNA-BMSC组与PBS组大鼠肝移植术后存活时间分别为(16.6±4.6)d、(15.4±6.7)d,明显低于TSG-6-NC-BMSC组(69.6±28.1)d和BMSC组(69.2±28.2) d(P ＜0.05).结论 BMSC分泌表达TSG-6在一定程度上具有减轻肝移植急性排斥反应的作用.

Effects of TSG-6 gene expression mesenchymal stem cells on liver transplantation rejection in rats

Objective To investigate the effects of silencing TSG-6 gene modified bone marrow mesenchymal stem cells (BMSC) transplantation on liver allotransplantation rejection in rats.Methods BMSC and KCs were isolated from rats and cultured in complete medium.We down-regulated TSG-6 expression of BMSC with lentiviral vectors carrying short hairpin RNA (LV3-shTSG-6).TSG-6mRNA and protein level of BMSC were tested respectively by quantitative real-time PCR and western blot analysis.After co-cuhured between BMSC and KCs,the expression of TNF-α and TSG-6 in cell supernatants were tested.Then,we established the orthotopic liver transplantation models in rats,the rats of each group were killed at 1 days,3 days and 7 days after operation.The serum levels of AST,ALT,TBIL,γ-GGT,TNF-α,IL-6 and IL-4 were tested with ELISA in each group,TSG-6mRNA and protein level of liver tissues obtained from each group were tested by quantitative real-time PCR and western blot analysis.The 1iver tissues of each group were stained with HE,then microstructure of liver tissues were observed under light microscope.The postoperative survival rates in other rats of each group were observed.Results The lentivirus transfection efficiency of mesenchymal stem cells was beyond 70 percent;After co-cultured between BMSC and KCs,the expression of TSG-6 in TSG-6-shRNA-BMSC + KCs group supernatant was significantly lower in different time point than that in BMSC + KCs group and TSG-6-NC-BMSC + KCs group (P ＜0.05),and the expression of TNF-α peak in BMSC + KCs group and TSG-6-NC-BMSC + KCs group supernatants were at 6 hours,which were significandy lower than those at 12 hours (P ＜0.05),but the expression of TNF-α in TSG-6-shRNABMSC + KCs group at 12 hours was significantly higher than those in BMSC + KCs group and TSG-6-NC-BMSC +KCs group (P ＜0.05);After transplantation,the serum levels of AST,ALT,TBIL,γ-GGT,TNF-α and IL-6 in TSG-6-shRNA-BMSC group was significantly higher than those in TSG-6-NC-BMSC group and BMSC group in different time point (P ＜ 0.05),but had no significant difference compared with PBS group;The serum levels of IL-4 in TSG-6-shRNA-BMSC group,TSG-6-NC-BMSC group and BMSC group was significantly higher than that in PBS group(P ＜ 0.05),but TSG-6-shRNA-BMSC group compared with TSG-6-NC-BMSC group and BMSC group,the serum levels of IL-4 was significantly lower (P ＜ 0.05);In pathological changes,we found that the degree of liver rejection in TSG-6-shRNA-BMSC group and PBS group were seriously obvious,and were graded Ⅱ-Ⅲ with Banff schedule;Comparation of postoperative survival time in each group,TSG-6-shRNA-BMSC group and PBS group were (16.6 ±4.6) d and (15.4 ± 6.7) d respectively,which were signifcantly lower than those in TSG-6-NCBMSC group (69.6 ± 28.1) d and BMSC group (69.2 ± 28.2) d (P ＜ 0.05).Conclusion Transplantation of BMSC secreted TSG-6 could,to some extent,mitigate acute liver transplantation rejection.