The Effect Of Nitric Oxide Combined With Rat Bone Marrow Mesenchymal Stem Cells On The Mice Liver Cirrhosis

Objective:Builds stable system of isolation,culture and identification of rat bone marrow mesenchymal stem cells(BMSCs) and studies the effects of nitric oxide(NO) combined with rat bone marrow mesenchymal stem cells on the mice liver cirrhosis.Methods:(1) Isolation,culture and identification of rat BMSCs: the rat BMSCs were isolated by the whole bone marrow adherent method,passaged and cultured continuously.The third passage BMSCs were taked to identify phenotype by flow cytometry,to measure the proliferation activity by MTT and to identify the differentiation of osteogenesis.And the third passage BMSCs were taked to test the role of NO on the proliferation activity of BMSCs by MTT.(2)The effect of nitric oxide combined with rat bone marrow mesenchymal stem cells on the mice liver cirrhosis in vivo:68 female KM mice were divided into blank group(n=10)、model group(n=10)、SNP group(n=12)、BMSCs group(n=12) 、 BMSCs+SNP(one time)group(n=12) 、 BMSCs+SNP(more than one time)group(n=12) 。By intraperitoneally injection CCL4 to make liver cirrhosis model. BMSCs group、BMSCs+SNP( one time)group、BMSCs+SNP(more than one time)group were transplanted BMSCs through tail vein after 10 weeks.SNP group 、 BMSCs+SNP(one time)group 、BMSCs+SNP(more than one time)group were injected SNP by intraperitoneal injection,PBS took the place of CCL4 and SNP for blank group and model group.All mice were sacrificed after 4weeks of translation BMSCs.And transplantation blood used for AST、ALT、ALB test,RT-PCR to analyze Sex-determining Region on the Y Chromosome(SRY) expression,HE-staining to assess liver fibrosis.Reults:(1) In vitro experiment: the rat BMSCs were isolated successfully,and growth curve was portrayed.The phenotypes identified by flow cytometry were in line with the mesenchymal stem cell phenotypes. Differentiation of osteogenesis was stained by alizarin red. There was no significant influence of NO with various concentration and different dosing frequency on proliferation activity of BMSCs(P>0.05).(2) In vivo experiment: liver cirrhosis model was made successfully.ALT 、 AST were lower in BMSCs group 、 BMSCs+SNP(one time) group 、BMSCs+SNP(more than one time)group than other groups and ALB was higher at the sametime(P<0.05) there was no statistically significant difference between model group with SNP group;ALT、AST were lower in BMSCs+SNP(more than one time)group than BMSCs+SNP(one time) group and BMSCs group and ALB were higher at the same time(P<0.05);there was no statistically significant difference between BMSCs+SNP(one time) group with BMSCs group.There was no expression of SRY mRNA in blank group、model group and SNP group. It could be detected of SRY mRNA in BMSCs group 、 BMSCs+SNP(one time) group 、BMSCs+SNP(more than one time)group.The expression of SRY mRNA was higher in BMSCs+SNP(more than one time)group than BMSCs+SNP(one time) group and BMSCs group(P<0.05);there was no statistically significant difference between BMSCs+SNP(one time) group with BMSCs group in expression of SRY mRNA(P>0.05).Significant liver fibrosis could be observed in blank group、model group and SNP group in HE-staining.AT the same time, liver cirrhosis obviously improved in BMSCs group 、 BMSCs+SNP(one time) group 、BMSCs+SNP(more than one time)group.Conclusion: The rat BMSCs can be isolated by the whole bone marrow adherent method,and passaged、cultured and induced differentiation steadily; There was no significant influence of NO on proliferation activity of BMSCs;The transplanted BMSCs could improve live cirrhosis,and nitric oxide augments BMSCs ability to repair liver cirrhosis.