An Experimental Study On Implantation Of Bone Marrow Cells Which Transfected Connexin43 Gene Into Infarct Myocardium In Rats

Bone marrow cells implanting into the damaged myocardium to replace theinjured myocardium and fibrous tissue had achieved encouraging results bothin animal experiments and in the pilot clinical studies. These researcheshave already provided a new strategy for the treatment of terminal cardiacdiseases. There is sufficient evidence that in ischemia myocardium or otherheart diseases, the Cx43, one of the gap junction forming proteins, expresssignificantly decreasing, and this alteration will become the foundation ofdysrhythmia and cardiac insufficiency. In the present experimental study,bone marrow cells form SD rats were isolated, purified and labeled in vitro.We have already constructed the transgenic cells which were transfected Cx43gene in vitro.The acute myocardial infarction model of rats was establishedand the transgenic cells were implanted into the acute infarct site via focalinjection; the effect of allogeneil graft transgenic cells on the cellsurvival, myocardial regeneration and cardiac function was assessed. Theresults could provide theoretical support at the cellular level for futureclinical application in the treatment of myocardial diseases with allogeneilgraft transgenic bone marrow cells. The study consists of the following threeparts.Part 1: Bone Marrow Cells Isolation, Culture, Purification and IdentificationObjective To prepare the cell line for implanting into the acute infarctsite. Methods The bone marrow cells were isolated, cultured, purified andidentified with flow cytometry, light microscope and electron microscope.Results The rat BMSCs showed active proliferate capacity in vitro in primaryand passage cultures. They still remained the status of homogenousundifferentiating after passage six. Cell markers CD44 and CD90 are positive,CD11b/c were weak, but CD34 negative. The purity of cells was over 97% in thepassage three. Conclusion The cells cultured in the study are bonemesenchymal cells; the cells of passage six are the optimal selection as thetransplanting cell line. Part2: Transfection to BMSCs with Eukaryotic Expression Plasmid ofpBudCE4.1_Cx43 and Evaluation of Their Expression Product and FunctionObjective To study the expression and function of rat BMSCs after the cellswere transfected with eukaryotic expression plasmid of pBudCE4.1_Cx43 invitro. Methods The passage six rat BMSCs were transfected with eukaryoticexpression plasmid of pBudCE4.1_Cx43 together with lipofectamineTM2000, weadopted hemiquantitative RT-PCR to analysis the expression of Cx43mRNA,immunocytochemical method to analysis the expression of Cx43 protein, SLDTto analysis the cell gap junction communication. Results There was nosignificant diversity in morphology, proliferate capacity betweentransgenic cells and BMSCs, while the RT-PCR showed that the expression ofCx43mRNA in BMSCs after transfection were higher than that of the controlgroup, Map analysis indicated the Cx43 protein expression in the transfectedBMSCs were higher than that of the control group. The difference of Cx43 mRNAand the protein between the two group was significant (P<0.05).SLDTdemonstrated that the fluorescence intensity of transgenic BMSCs was higherthan that of the control group (P<0.05). Conclusion Transfected BMSCs couldeffectively express the active Cx43 and produce biological efficiency,offering the ideal genetically engineered cell for cellularitycardiomyoplasty.Part3: Effect on Left Ventricular Function of Allogeneil Graft of Bone Marrow Cells Transfected Cx43Objective To study the effect on left ventricular function and the survivalof allogeneil graft of BMSCs transfectecd with Cx43. Methods Eleven shamoperated SD rats served as sham group. Forty nine rats with a model ofmyocardial infarction created by ligating LAD for 1to 3 hours were randomlydivided into four groups. The rats were transplanted with DMEM medium (n=11),BMSCs only (n=12), BMSCs after transfected pbudCE4.1_Cx43 (n=13) and BMSCsafter transfected pbudCE4.1 (n=13), respectively. The animals weresacrificed 6 weeks after coronary artery ligation and the heart function andother indices were evaluated. Results Allogenic BMSCs could survive in theinfracted myocardium, orientating themselves with accord to host cardiacmuscular cells .and BrDU was detected by immunohistochemistry. Compared withcontrols, the systolic function of group2,3,4,5 was significantly improved,respectively(P<0.05,each), the difference between group3 and 2 or group3and 4 was significant (P<0.05), the difference between group2 and 4 was notsignificant (P>0.05). Conclusion Allogeneil graft BMSCs to repairinfracted myocardium was feasible and efficacious. Allogeneil graft BMSCsafter transfected Cx43 was more effective than BMSCs only in improving thefunction of the infracted myocardium.To sum up, we drew conclusions as follows:1. We isolated and cultured rat bone mesenchymal cells successfully.2. We have already transfected BMSCs with pBudCE4.1_Cx43 mediated by lipofectamine 2000. There was no significant diversity in morphology,proliferate capacity between transgenic cells and BMSCs. The transfected BMSCs could effectively express the active Cx43 and produce biological efficiency, offering an ideal genetically engineered cell line for cellularity cardiomyoplasty.3. We have already established the model of rat myocardial infarction by ligating LAD successfully.4. Allogeneil graft BMSCs to mend infracted myocardium was feasible and effective. Allogeneil graft BMSCs after transfected Cx43 was more effective than BMSCs only in improving the function of the infracted myocardium. Transplantation of genetically engineered cells may offer a new technique for the treatment of myocardial infarction, and provid a potential application in the future.