Over-expression Of CXCR4 And IL-35 In Vitro Migration Of Bone Marrow Mesenchymal Stem Cells And Their Immunoregulation On CD4~+T Cells And Synergistic Effect Of Compound Sophora Decoction Containing Serum

Part ? Over-expression of CXCR4 and IL-35 Bone Marrow Mesenchymal Stem cells CultureObjective: CXCR4 and IL-35 lentiviral vectors were constructed and then infected with rat bone marrow mesenchymal stem cells(BMSCs)to obtain bone marrow mesenchymal stem cells(BMSCs)overexpressing CXCR4 and IL-35.Methods: Rat bone marrow mesenchymal stem cells were cultured and isolated by whole bone marrow adherent method and expanded in vitro and the surface of MSCs were identified by flow cytometry(FCM).The linearized vector was obtained by restriction endonuclease digestion.The CXCR4 and IL-35 genes were inserted into two different lentivirus tool vectors,and the plasmids were extracted by PCR identification and sequencing.Two lentiviral particles,CXCR4 and IL-35,were obtained according to lentivirus packing manual and tested for titers.The two lentivirus particles were used to infect rat bone marrow mesenchymal stem cells successively,and the expression of CXCR4,IL-35 in the transfected bone marrow mesenchymal stem cells was detected by PCR,Western blot,ELISA.Results: Rat bone marrow mesenchymal stem cells(BMSCs)were isolated and identified successfully.CXCR4 and IL-35 lentivirus were successfully used to infect the same rat bone marrow mesenchymal stem cells,and the expression of CXCR4 and IL-35 in CXCR4-IL-35-BMSCs were higher than those in null-BMSCs(P < 0.05),which were detected by PCR,Western blot,ELISA.Conclusion: Rat bone marrow mesenchymal stem cells(BMSCs)expressing CXCR4 and IL-35 were successfully obtained.Part ? Study on the Migration of Bone Marrow Mesenchymal Stem Cells in vitro by CXCR4 and IL-35 lentivirus and Compound Sophora Decoction containing Medicinal Serum and its immune Regulation on CD4~+T cellsObjective: To investigate the effect of lentivirus overexpressing CXCR4 and IL-35 on the migration of bone marrow mesenchymal stem cells in vitro and the synergistic effect of compound Sophora decoction containing serum and its immune Regulation on CD4~+T cells.Methods: Eight male SD rats(200 g or so)were taken to prepare for the serum containing compound Sophora decoction,and 32 4-week-old male SD rats were used to extract bone marrow mesenchymal stem cells(BMSCs).They were randomly divided into4 groups: CXCR4-IL-35-BMSCs group,Compound Sophora decoction containing serum group,blank control group,CXCR4-IL-35-BMSCs and compound Sophora decoction group containing blood group.Using Transwell technique,100 ul bone marrow mesenchymal stem cells suspension was added to the upper chamber and 20% FBS DMEM cell medium containing SDF-1 was added to the lower chamber.The cells were cultured in CO2 saturated humidity incubator with 5% volume fraction of CO2 for 24 hours at 37 ?.The number of bone marrow mesenchymal stem cells migrated downwards was observed statistically.The expression levels of N-cadherin,E-cadherin,Vimentin and SMO m RNA in BMSCs cells of each group were detected by PCR technique.Co-culture technique was used to co-culture BMSCs:T at 1:10 for 24 hours.Cell supernatant was used to detect the expression of TNF-and IL-10,and flow cytometry was used to detect the ratio of Th17.Results: Compared with the blank control group,the number of downward chamber migration of bone marrow mesenchymal stem cells in CXCR4-IL-35-BMSCs group,compound Sophora decoction containing serum group and CXCR4-IL-35-BMSCs compound Sophora decoction group was significantly higher than that in control group(P <0 05).CXCR4-IL-35-BMSCs compound Sophora decoction containing serum group was the most significant(P < 0.0001).Compared with the blank control group,the content of N-cadherin and Vimentin MRNA in the other groups increased and the content of E-cadherin m RNA decreased(P < 0 05).There was no significant difference in the content of SMO m RNA between the other groups(P > 0 05).Compared with control group A,the expression levels of TNF-? and IL-10 in other groups were significantly lower than those in group A(P < 0.05),and the expression level of TNF-? in group B and group D was significantly lower than that in group A(P < 0.01).The expression of IL-10 increased significantly(P < 0.001).Compared with the group A,the Th17 of group B and group D was significantly lower than that of group(P < 0.01).There was no significant difference between group A and group C(P > 0.05).Conclusion: Compound Sophora decoction containing serum and overexpression of CXCR4 and IL-35 can promote BMSCs migration,by up-regulating N-cadherin and Vimentin,and down-regulating E-cadherin,CXCR4-IL-35-BMSCs promotes the expression of IL-10 and decreases the expression of TNF-? by inhibiting the ratio of Th17.Furthermore,compound Sophora decoction containing serum plays a synergistic role in enhancing the migration and immunosuppressive action of BMSCs.