Experimental Study Of Sodium Butyrate To Enhance The Differentiation Of BMMSCs And Promote BMMSCs To Improve Liver Injury

Objective:1.To establish one of a method for isolation,culture,proliferation and cryopreservation of rabbit bone marrow mesenchymal stem cells(BMMSCs)in vitro,and to identify its immunophenotype.2.To explore the effects of 24 h pretreatment and continuous treatment with different concentrations of histone deacetylase inhibitor sodium butyrate on differentiation of BMMSCs into hepatocytes in the environment of hepatogenic induction,so as to select the appropriate concentration and induction method.3.An acute liver injury model was established to study the effect of sodium butyrate on the liver injury of BMMSCs induced by HGF and EGF in vitro,and to investigate whether intravenous sodium butyrate affects paracrine secretion of BMMSCs to ameliorate liver injury through an IL-6 dependent pattern involving the NF-κB pathway.4.To investigate the effect of sodium butyrate(Na B)on HGF and EGF induced BMMSCs in vivo in acute liver injury model and its possible mechanism.Methods:1.Rabbit BMMSCs were isolated and cultured by whole bone marrow cell adherence screening method.The morphology of the cells was observed by light microscopy.The immunophenotype of the cells was identified by flow cytometry.2.The third generation of BMMSCs cells were selected for hepatogenesis induction.There were 4 groups:(1)control group(control): BMMSCs;(2)Sodium butyrate free hepatogenic induction group(0m M Na B): BMMSCs+HGF+EGF;(3)24h Sodium butyrate pretreatment group(Na B 24h):BMMSCs+HGF+EGF+Na B 24 h.(4)Sodium butyrate continuous treatment group: BMMSCs+HGF+EGF+Na B continuous treatment.According to the study in the previous chapter,the concentration of sodium butyrate in group 3 and group 4 was successively divided into two subgroups(0.5mm and 1m M).3.Except for control group,hepatocyte growth factor(HGF)and epidermal growth factor(EGF)were added in each well with the same concentrations as before(HGF concentration was 60.0μg/L,EGF concentration was 45μg/L).Sodium butyrate was pretreated for 24 h in each subgroup.Sodium butyrate was removed after intervention for 24 h,washed twice with PBS,and HGF+EGF were added respectively for further culture.4.At the 7th,14 th and 21 st day of induction,RT-PCR was used to detect the expression levels of AFP and ALB specific proteins in each group,Western-blot was used to detect the protein expression levels of AFP and ALB genes,and PAS and urea were used to detect the function of liver cells,and the activity of cells was detected by CCK-8 method.5.Rabbit model of acute liver injury was prepared.There were 4groups:1)control;2)control+:Acute liver injury model +Basic medium;3)BMMSCs:Acute liver injury model +BMMSCs+EGF+HGF;4)Acute liver injury model +BMMSCs+EGF+HGF+0.5m M Na B for 24h;6.The corresponding drugs were injected intravenously through the ear edge of rabbits at the peak of liver function damage 24 hours after the successful modeling。After one week of administration,the indexes of the samples were as follows: The levels of alanine aminotransferase(ALT),total protein(TP)and total bilirubin(TBIL)were detected by histopathological examination.The m RNA expression of IL-6 was detected by RT-PCR and the content of NF-кB p65 was detected by Western-blot analysis.7.Peak in 24 hours after the success of the modeling function of liver damage,through the guidance of ultrasound,percutaneous liver puncture injection groups corresponding drugs,after dosing week each index of test specimens,and the overall level of observation,and histo-pathological examination and detection of serum liver function markers alanine aminotransferase(ALT),total protein(TP)and total bilirubin(TBIL)level,TUNEL kit was used to detect the apoptotic cells.Western blot was used to detect the m RNA expression levels of apoptosis related factors Bax,Bcl-2 and Caspase-3,and q RT-PCR was used to detect the m RNA expression levels of cytokines IL-6 and plultipotent transcription factor(SOX2).Results:1.BMMSCs were adherent,with an uniform spindle-shaped fibrocyte-like structure,and could be expanded in vitro.Flow cytometry immunophenotypic analysis showed that these cells significantly expressed CD29(97.21%±3.2%,n=3))and CD90(99.89%±4.1%,n=3),but lacked hematopoietic cell marker CD45(1.78%±1.02%,n=3)and CD34(1.06%±0.27%,n=3),which was consistent with the characteristics of BMMSCs.2.CCK-8 method was used to detect each subgroup of Na B continuous treatment group and Na B 24 h pretreatment group,and compared with the blank group,the growth activity of BMMSC was measured.Using the blank control group as the base of 1,it was found that the OD value of 1.5m M and 2m M subgroups of Na B continuous treatment decreased significantly at day 7,14 and 21.It is suggested that inhibition of cell proliferation has cytotoxicity.The OD values of the0.5m M and 1m M subgroups and the 24 h 2m M subgroups treated by Na B decreased significantly only on the 21 st day,suggesting that cell proliferation was inhibited.There was no statistical difference between Na B 24 h pretreatment(0.5mm and 1m M)group and between the Na B-group3.RT-PCR expression: ALB m RNA levels in the Na B Continuous treatment group were significantly lower than those in the 0m M Na B group,while AFP and ALB m RNA levels in the 24 h Na B group were significantly higher than those in the 0m M Na B group.There were no significant differences in AFP and ALB m RNA levels between 0.5 m M24 h Na B group and 1.0 m M 24 h Na B group.4.Western-blot analysis : AFP and ALB proteins were expressed in a time-dependent manner in 0 m M Na B group and 24 h Na B group(0.5m M,1 m M).AFP and ALB increased on the 7th day,peaked on the 14 th day,and decreased slightly on the 21 st day.The continuous Na B treatment group showed lower AFP and ALB protein expression levels than 24 h Na B group,and continuous Na B treatment group showed no regularity in the levels of ALB and AFP.There was no significant difference between 0.5 m M 24 h Na B group and 1.0 m M 24 h Na B group.5.PAS staining and urea detection: the glycogen synthesis function of 0.5m M 24 h Na B group was significantly higher than that of the other groups.The urea level of 0 m M Na B group and 24 h Na B groups(0.5 m M,1 m M)increased gradually on the 14 th and 21 st days,and were significantly higher than that in control group.The urea secretion level of0.5m M Na B 24 h group on the 14 th and 21 st day were significantly higher than those of other induction groups.6.Route of intravenous injection:Compared with acute liver injury model group,the degree of liver cell necrosis began to decrease in BMMSCs group,and the degree of liver cell necrosis was further reduced in group of BMMSCs+ Na B,and the division,proliferation and regeneration of liver cells were more active than that in BMMSCs group.Serum liver biochemical markers,ALT and total bilirubin(TBIL)in BMMSCs group were significantly decreased,ALT and TBIL in group of BMMCs+Na B were further decreased compared with BMMSCs group。Serum total protein in BMMSCs group began to significantly increase.The increase of serum total protein in BMMCs+ Na B group was more significant than that in BMMSCs group.The level of IL-6 m RNA and protein content of NF-кB p65 in BMMSCs group were significantly decreased,and the level of IL-6 m RNA level and protein content of NF-кB p65 in BMMCs+Na B group were significantly lower than that in BMMSCs group,and the differences were significant.7.Route of percutaneous hepatic puncture therapy:Compared with control+ model of acute liver failure,the general condition of BMMSCs group and Na B +BMMSCs group were better than that of medium group.After the injection of BMMSCs,the degree of degeneration and necrosis of liver cells was reduced,and a large number of binuclear cells and lymphocytes were infiltrated in portal area.Cell division and proliferation were more active after BMMSCs+Na B injection than BMMSCs group.In serum liver biochemical markers,ALT and TBIL of BMMSCs group were significantly decreased,ALT and TBIL of BMMCs+Na B group were further decreased compared with BMMSCs group,and serum total protein of BMMSCs group was increased.The increase of serum total protein in BMMCs+Na B group was more significant than that in BMMSCs group.Apoptosis index(AI)was significantly increased in acute liver failure model group(Control +),and the expression levels of Bax and Caspase-3 were significantly increased(#p<0.05 vs Control)on day 7,while the expression levels of Bcl-2 were significantly decreased(#p < 0.05 vs Control).BMMSCs attenuated these effects and Na B+BMMSCs further reversed these effects(#p < 0.05 vs Control+).IL-6 level was significantly increased in the control+ group,and significantly decreased in the Na B induced group and BMMSCs group(*p<0.05 vs control+).IL-6 was lower in the Na B +BMMSCs group than in the BMMSCs group(#p<0.05 vs BMMSCs group).BMMSCs group increased SOX2 gene expression(*p<0.05 vs Control+).However,Na B +BMMSCs group further increased SOX2 gene expression(#p <0.05 vs BMMSCs group).Conclusions:1.It is feasible to isolate,purify and amplify BMMSCs in vitro by the improved whole bone marrow differential adherent method.2.In the hepatogenic induction environment,the persistence of high concentration of Na B(1.5m M and 2m M)was detrimental to the hepatocyte differentiation of BMMSCs.3.0.5m M sodium butyrate pretreatment for 24 h could promote the differentiation of BMMSCs induced by HGF and EGF.4.0.5m M Na B pretreatment for 24 hours enhanced the differentiation of BMMSCs induced by HGF and EGF,significantly reduced IL-6 and inhibited the activation of NF-κB signaling pathway during liver injury.These factors may be involved in the mechanism of BMMSCs promoting liver injury repair and liver regeneration.5.Intrahepatic transplantation of 0.5m M sodium butyrate for 24 hours pretreated hepatogenesis induced BMMSCs,improved liver function,decreased the gene levels of pro-apoptotic protein Bax and the core protease caspase,which mediate cell death,increased the gene expression of anti-apoptotic protein Bcl-2,and reversed a large number of apoptosis of liver cells.At the same time,it caused the activation and large expression of SOD2,promoted the differentiation of liver cells and liver regeneration,and also promoted the reduction of the concentration of inflammatory factor IL-6,suggesting that the suspension of hepatoblast differentiated BMMSCs induced by percutaneous liver puncture injection of sodium butyrate not only promoted the parocrine of BMMSCs and improved liver failure.Moreover,it can enhance liver regeneration by promoting hepatocyte differentiation.Figures 19,Tables 2,References 233...