Résumé
Objective To investigate the survival and differentiated situation of transplanted mesenchymal stem cells ( MSCs) transfected with Sonic hedgehog (SHH) gene after chronic myocardial infarction (MI).Methods MSCs were obtained from bone marrow of limb bones by density gradient centrifugation combined with attachment culture method .SHH gene was cloned and eukaryotic expression plasmid pcDNA 3.1-SHH was constructed .Then nucleofector TM was used to transfer SHH gene into MSCs and the expres-sions of mRNA and protein of SHH gene were detected in vitro .Myocardial infarction model was established by ligating the left anterior descending branch .A total of 150μl (3 ×106 ) of MSCs transferred with SHH gene was injected into ischemic area at the fourth week after MI model was set up .Brdu immunohistochemical staining and electron microscope examination were used to detect the survival and differentiation of MSCs with SHH in vivo at the 1st, 2nd, 4th, and 8th week after MSCs SHH transplantation.Results It was con-firmed that SHH gene clone and the recombinant eukaryotic expression vector pcDNA 3.1-SHH construction were successful by gene se-quence analysis and double digestions with EcoR I and Bamh I .Reverse transcriptase polymerase chain reaction ( RT-PCR) and West-ern-blot demonstrated that the expression of SHH mRNA was significantly increased in transfected MSCs compared with untransfected MSCs.Brdu immunohistochemical staining confirmed MSCs with SHH were survived from one to eight weeks in MI area after transplan -tation.Electron microscope examination showed that MSCs had the trend to be differentiated into myocardiac -like cells in MI area at the 4th and 8th week after transplantation .Conclusions The expression of SHH mRNA was significantly increased while MSCs were transfected with SHH gene , and MSCs SHH could survive and differentiate in good condition in the transplanted recipients .Those data provide the experimental basis for further transgenic cell therapy research of ischemic heart disease .
Résumé
Objective To evaluate the feasibility of transfection of Sonic hedgehog gene (SHH)into bone marrow mesenchymal stem cells(BMMSC).Methods After the SHH gene was transfected into BMMSC by electroporation apparatus,the transfection rate was evaluated by fluorescence inverted microscope.The growth curves of untransfected and transfected BMMSC were drawn,respectively,to observe the influence of transfection on cells.The expression of SHH gene in the BMMSC was detected by PCR,RT-PCR,Western-blot analyses.Results Through fluorescence inverted microscope,the observed transfection rate was appropriately 30％,PCR showed a obvious increase of SHH expression in transfected cells than that in untransfected cells,and it is quantified by qPCR for appropriately 7 times.Western-blot further demonstrated that the SHH protein expression in transfected cells had a distinct increase.However,it was observed that the exponential phase of BMMSCSHH growth curve delayed.The growth curves of both overlap 12 days after transfection.Conclusions This electroporation method can transfect exogenous SHH gene into BMMSC sufficiently with the effective protein expression in BMMSCSHH.It is the foundation of further research of genetic therapy for ischemic heart disease.