PAd-RFP/BMP2-Mbs Complex Transfected Into BMSCs And Promote Their Differentiation Into Cardiomyocytes In Vitro

Objective: To investigate the feasibility of transfection of rat bone marrow mesenchymal stem cells(BMSCs)by biotinylated targeted microbubble localization release technology(UTMD)in vitro using recombinant adenovirus-microbubble complex labeled with biotinylated bone morphogenetic protein2 and red fluorescence(PAd-RFP/BMP2-Mbs).Method: BMSCs were isolated,purified and purified from rat bone marrow.The P3 BMSCs were randomly divided into 5 groups:Ⅰ group:simple BMSCs group(control group);Group Ⅱ: ultrasound+microbubble+BMSCs group(BMSCs+UTMD);Group Ⅲ : ultrasound+BMSCs group(BMSCs+U);Group Ⅳ :microbubbles+BMSCs group(BMSCs+M);Group V: ultrasound+microbubbles+BMSCs+PAd-RFP/BMP2 group(BMSCs+UTMD+BMP2);The transfection of BMSCs into cardiomyocytes was induced by ultrasound irradiation in vitro.The expression of BMP2 was observed 24 h,48h,72 h after transfection.The transfection rate of PAd-RFP/BMP2 in BMSCs was detected 72 h after transfection.The CCK8 assay BMSCs were detected.The mRNA and protein expression of PAd-RFP/BMP2 in BMSCs were detected by RT-PCR and Western blot at the 72 h after transfection.On the 14 th day after transfection,the cardiac-specific troponin cTnT,Cx43,α-MHC and α-actin were detected by Western blot.The cardiac-specific homologous transcription factor(Nkx2.5)and zinc finger transcription factor(GATA-4)mRNA expression detected by RT-PCR.Result: After 24 h,48h,72 h after transfection,the group Ⅴ BMSCs were observed,the red fluorescence gradually increased,the group Ⅴ fluorescence protein expression was80%,group Ⅴ PAd-RFP / BMP2 transfection rate was 85%,CCK8 value : There was no significant difference in absorbance(A)between transfection group and untransfected group(P> 0.05).The results of RT-PCR and Western blot 72 h after transfection showed that the expression of BMP2 mRNA and protein were only high in group Ⅴ,P <0.05 compared with untransfected group;The results of Western blotting on day 14 aftertransfection showed that cTnT The expression of Cx43,α-MHC and α-actin in group V was higher than that in group V(P <0.05).The result of RT-PCR showed that Nkx2.5 and GATA-4 mRNA was higher in group Ⅴ compared with that in non-transfected group(P<0.05).Conclusion: BMSCs can be successfully transfected with PAd-RFP/BMP2-Mbs adenovirus-microbubble complex combined with UTMD technology.After transfection,BMP2 can induce BMSCs to differentiate into cardiomyocytes successfully.