骨髓巨噬细胞M2亚型共培养后的骨髓间充质干细胞移植治疗肝硬化大鼠模型的效果分析

Therapeutic effect of transplantation of bone marrow mesenchymal stem cells co-cultured with bone marrow M2 macrophages on a rat model of liver cirrhosis

目的探讨骨髓巨噬细胞M2亚型(M2-BMDM)共培养后的骨髓间充质干细胞(BMSCM2)移植对四氯化碳/2-乙酰氨基芴(CCl4/2-AAF)诱导肝硬化大鼠模型进展的影响。方法分离大鼠BMDM并极化为M2表型;分离大鼠BMSC,培养至第3代时与M2-BMDM共培养后获取BMSCM2。CCl4皮下注射6周建立大鼠肝硬化模型。将模型大鼠随机分为模型组(M组)、BMSC组、BMSCM2组,同时设有正常组(N组),每组6只。第7周开始,模型大鼠于CCl4皮下注射的同时予以2-AAF灌胃,分组干预,10周末取材,观察肝功能、肝组织病理、肝组织羟脯氨酸(Hyp)含量,以及肝星状细胞、肝祖细胞、胆管细胞、肝细胞标志物的变化情况。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果与N组比较,M组大鼠血清ALT、AST活性均显著升高(P值均<0.01);与M组比较,BMSC组和BMSCM2组大鼠ALT、AST均显著降低(P值均<0.01),且BMSCM2组显著优于BMSC组(P值均<0.05)。与N组比较,M组大鼠肝脏Hyp含量、α-SMA mRNA及蛋白表达均显著升高(P值均<0.01);与M组比较,BMSC组和BMSCM2组Hyp含量、α-SMA表达均显著降低(P值均<0.05),且BMSCM2组α-SMA水平显著低于BMSC组(P<0.01)。与N组比较,M组大鼠肝祖细胞标志物EpCam、Sox9以及胆管细胞标志物CK7、CK19 mRNA表达均显著增加(P值均<0.01),肝细胞标志物HNF-4α和Alb表达均显著降低(P值均<0.01);与M组比较,BMSC组和BMSCM2组EpCam、Sox9、CK7和CK19 mRNA表达均显著降低(P值均<0.05),HNF-4α和Alb mRNA表达均显著增加(P值均<0.05);且与BMSC组比较,BMSCM2组EpCam和CK19 mRNA表达均显著降低(P值均<0.05),而HNF-4αmRNA表达显著增加(P<0.05)。结论M2-BMDM可提高BMSC对CCl4/2-AAF诱导大鼠肝硬化的治疗效应,为进一步提高BMSC治疗肝硬化的作用提供了新思路。

Objective To investigate the effect of transplantation of bone marrow mesenchymal stem cells(BMSCs)co-cultured with bone marrow-derived M2 macrophages(M2-BMDMs),named as BMSCM2,on a rat model of liver cirrhosis induced by carbon tetrachloride(CCl4)/2-acetaminofluorene(2-AAF).Methods Rat BMDMs were isolated and polarized into M2 phenotype,and rat BMSCs were isolated and co-cultured with M2-BMDMs at the third generation to obtain BMSCM2.The rats were given subcutaneous injection of CCl4 for 6 weeks to establish a model of liver cirrhosis,and then they were randomly divided into model group(M group),BMSC group,and BMSCM2 group,with 6 rats in each group.A normal group(N group)with 6 rats was also established.Since week 7,the model rats were given 2-AAF by gavage in addition to the subcutaneous injection of CCl4.Samples were collected at the end of week 10 to observe liver function,liver histopathology,and hydroxyproline(Hyp)content in liver tissue,as well as changes in the markers for hepatic stellate cells,hepatic progenitor cells,cholangiocytes,and hepatocytes.A one-way analysis of variance was used for comparison of continuous data between multiple groups,and the least significant difference t-test was used for further comparison between two groups.Results Compared with the N group,the M group had significant increases in the activities of serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)(P<0.01);compared with the M group,the BMSC and BMSCM2 groups had significant reductions in ALT and AST(P<0.01),and the BMSCM2 group had significantly better activities than the BMSC group(P<0.05).Compared with the N group,the M group had significant increases in Hyp content and the mRNA and protein expression levels of alpha-smooth muscle actin(α-SMA)in the liver(P<0.01);compared with the M group,the BMSC and BMSCM2 groups had significant reductions in Hyp content and the expression ofα-SMA(P<0.05),and the BMSCM2 group had a significantly lower level ofα-SMA than the BMSC group(P<0.01).Compared with the N group,the M group had significant increases in the mRNA expression levels of the hepatic progenitor cell markers EpCam and Sox9 and the cholangiocyte markers CK7 and CK19(P<0.01)and significant reductions in the expression levels of the hepatocyte markers HNF-4αand Alb(P<0.01);compared with the M group,the BMSC and BMSCM2 groups had significant reductions in the mRNA expression levels of EpCam,Sox9,CK7,and CK19(P<0.05)and significant increases in the mRNA expression levels of HNF-4αand Alb(P<0.05),and compared with the BMSC group,the BMSCM2 group had significant reductions in the mRNA expression levels of EpCam and CK19(P<0.05)and significant increase in the expression level of HNF-4α(P<0.05).Conclusion M2-BMDMs can enhance the therapeutic effect of BMSCs on CCl4/2-AAF-induced liver cirrhosis in rats,which provides new ideas for further improving the therapeutic effect of BMSCs on liver cirrhosis.