骨髓间充质干细胞与内皮祖细胞对大鼠大部分肝切除合并缺血再灌注后损伤影响

Effect of bone marrow mesenchymal stem cells and endothelial progenitor cells on hepatic ischemia reperfusion and partial hepatectomy injury in rats

目的通过制造大鼠肝脏缺血再灌注损伤合并大范围肝切除模型,探讨将骨髓间充质干细胞(BM-MSCs)与骨髓内皮前体细胞(BM-EPCs)分别经门静脉移植入模型肝内,观察其对于鼠肝脏缺血再灌注肝损伤修复和肝脏再生中的作用,以及将两种细胞联合移植观察两种细胞是否存在协同作用促进肝脏缺血再灌注损伤修复和肝脏再生。方法通过细胞贴壁法从骨髓液中获得BM-MSCs和BM-EPCs。利用慢病毒携带绿色荧光蛋白(GFP)及红色荧光蛋白(RFP)分别对BM-MSCs及BM-EPCs进行细胞标记,为下一步将细胞移植入动物体内观察细胞的分布情况做准备。制作SD大鼠70%肝缺血再灌注损伤合并大范围肝切除手术模型,将SD大鼠随机分为四组:单独注射BM-MSCs(n=12)、单独BM-EPCs(n=12)、联合注射BM-MSCs+BM-EPCs组(n=12)、单独注射PBS空白对照组(n=12),按不同分组进行细胞移植,分别经大鼠门静脉注射GFP标记的BM-MSCs/200μL、RFP标记的BM-EPCs/200μL、BM-MSCs+BM-EPCs(BM-MSCs∶BM-EPCs=1∶1)/200μL、对照组注射PBS/200μL。以上四组分别在不同的时间段:术后第1天(n=4),第2天(n=4),第3天(n=4)各取腔静脉血检测血清AST、ALT及TNF-ɑ水平指标。肝脏组织做冰冻切片后利用免疫荧光方法动态观察BM-MSCs和BM-EPCs在肝脏内的分布。各组肝脏组织行常规病理切片观察肝脏病理损害及恢复的程度。最后各组取第3天的肝组织使用Tunnel法检测肝内细胞凋亡情况。结果与对照组比较,BM-MSCs组、BM-EPCs组、BM-MSCs+BM-EPCs组在肝缺血再灌注损伤合并大部分肝切除术后第1天、第2天、第3天大鼠血清AST及ALT、TNF-ɑ水平均明显下降(P<0.05),其中BM-MSCs组大鼠血清AST及ALT、TNF-ɑ水平均最低(P<0.05)。通过免疫荧光方法观察BM-MSCs及BM-EPCs可以特异性分布至肝脏损伤部位促进肝脏组织修复再生。肝组织HE染色病理及评分显示与对照组比较,BM-MSCs组、BM-EPCs组及BM-MSCs+BM-EPCs组肝内细胞损伤程度明显降低,然而与BM-MSCs+BM-EPCs组相比较,BM-MSCs组和BM-EPCs组肝内细胞损伤程度明显降低(P<0.05),BM-MSCs组与BM-EPCs组相比较肝内细胞损伤程度无统计学意义。与对照组比较BM-MSCs组、BM-EPCs组及BM-MSCs+BM-EPCs混合组均能降低肝内细胞的凋亡数量,但BM-MSCs组肝内凋亡细胞数量最少(P<0.05)。结论单独细胞移植组与联合移植组均对肝脏大部分切除合并缺血再灌注损伤起保护作用。但在本实验中联合移植组对肝功能以及肝内细胞的保护并未体现出协同作用。BM-MSCs和BM-EPCs可能通过降低血清TNF-ɑ水平抑制炎症反应从而在大部分肝切除合并肝缺血再灌注损伤后保护肝内细胞。

Objective To investigate the transplantation of bone marrow mesenchymal stem cells(BM-MSCs)and bone marrow endothelial progenitor cells(BM-EPCs)into the model liver through portal vein by establishing a rat model of hepatic ischemia-reperfusion injury combined with extensive hepatectomy.To observe its effect on the repair of liver injury and liver regeneration after hepatic ischemia-reperfusion in rats,and to observe the synergistic effect of the two kinds of cells on the repair of liver injury and liver regeneration after hepatic ischemia-reperfusion in rats,and to observe the synergistic effect of the two kinds of cells on the repair of hepatic ischemia-reperfusion liver injury and liver regeneration.Methods BM-MSCs and BM-EPCs were obtained from bone marrow fluid by cell adhesion method.Lentivirus carrying green fluorescent protein(GFP)and red fluorescent protein(RFP)were used to label BM-MSCs and BM-EPCs,respectively,in order to prepare for the next step of transplantation of cells into animals to observe the distribution of cells.The model of 70%hepatic ischemia-reperfusion injury combined with extensive hepatectomy was established in SD rats.SD rats were randomly divided into four groups:single injection of BM-MSCs(n=12),single BM-EPCs(n=12),combined injection of BM-MSCs+BM-EPCs group(n=12)and single injection of PBS blank control group(n=12).GFP labeled BM-MSCs/200μL,RFP labeled BM-EPCs/200μL,BM-MSCs+BM-EPCs(BM-MSCs∶BM-EPCs=1∶1)/200μL and PBS/200μL were injected into the portal vein of rats,respectively.Serum AST,ALT and TNF-ɑlevels were measured at 1 day(n=4),2 days(n=4)and 3 days(n=4)after operation in the above four groups.After frozen section of liver tissue,the distribution of BM-MSCs and BM-EPCs in liver was observed dynamically by immunofluorescence method.the apoptosis of liver tissue was detected by Tunnel method on the 3rd day in each group.The degree of liver pathological damage and recovery was observed by routine pathological section of liver tissue.Results Compared with the control group,the levels of serum AST,ALT and TNF-ɑin BM-MSCs group,BM-EPCs group and BM-MSCs+BM-EPCs group were significantly decreased on the 1st,2nd and 3rd day after hepatic ischemia-reperfusion injury combined with most hepatectomy(P<0.01).Serum AST in BM-MSCs group.The levels of ALT and TNF-ɑwere the lowest(P<0 05).Immunofluorescence method was used to observe that BM-MSCs and BM-EPCs could specifically distribute to the site of liver injury and promote liver tissue repair and regeneration.The pathology and score of HE staining in liver tissue showed that the degree of intrahepatic cell injury in BM-MSCs group,BM-EPCs group and BM-MSCs+BM-EPCs group was significantly lower than that in control group.However,the degree of intrahepatic cell injury in BM-MSCs group and BM-EPCs group was significantly lower than that in BM-MSCs+BM-EPCs group(P<0.05),but there was no significant difference in the degree of intrahepatic cell injury between BM-MSCs group and BM-EPCs group.Compared with the control group,BM-MSCs group,BM-EPCs group and BM-MSCs+BM-EPCs mixed group could reduce the number of apoptotic cells in liver,but the number of apoptotic cells in BM-MSCs group was the least(P<0 05).Conclusion Both the single cell transplantation group and the combined transplantation group had a protective effect on partial hepatectomy with ischemia-reperfusion injury.However,in this experiment,the combined transplantation group did not show a synergistic effect on the protection of liver function and intrahepatic cells.BM-MSCs and BM-EPCs may protect intrahepatic cells after most hepatectomy combined with hepatic ischemia-reperfusion injury by reducing the level of serum TNF-ɑto inhibit inflammation.