Adult Cardiomyocytes Allograft To Protect The Cardiac Function After Myocardial Infarction

Background and objectivesCell transplantation is the hot point in the field of curing of myocardial infarction. Transplantation of stem cells has gained a great of achievements this decade, but some hidden troubles of safety and blind fields of technology limit the development of stem cell transplantation. Cardiomyocytes transplantation as the supplement of stem cell transplantation is more safe and provides function units directly. In this study, we tested the feasibility of allograft of cultured adult cardiomyocytes and the effect on cardiac function. In same time, we hope to build a model of culturing high purified cardiomyocytes.MethodsPART I :Culture of high grade pured of cardiomyocytes. Isolated heart perfusion technique isolate cardiomyocytes, and gradient centrifugation, Ficoll separating medium purify the cardiomyocytes. And then were purified again using the cell adhesion technique. In culture, calcium ionophore A23187 were used repeatedly to purify the cardiomyocytes. Then identified by α-sarcomeric actin.PART II : Adult cardiomyocytes allograft. The purified cardiomyocytes were transfected by calcium phosphate coprecipitation technique with pRSV-lacZ containing β -galactosidase gene. 33 adult New-Zealand rabbits were divided 3 groups, normal groups(n=9);transplantation groups(n=13);control groups(n=11).Infarction models were made up by ligation, and transplantation by acupuncture. 5 weeks after transplantation , cardiac function was evaluated by ultrasonograph. Histological studies include HE staining and histochemical techniques.Result1. Build up a technique of culturing high purified adult cardiomyocytes in vitro. The key points of this technique include: (1)low concentrate of calcium disconnecting the cell-cell junction; (2)gradient re-calcium; (3)high potassium low calcium solution containing 2% albumin to protect and repair the cellular membrane; (4) appropriate density ofinoculation; (5) gradient centrifugation; (6) cell adhesion technique to purify cardiomyocytes; (7)repeat use of calcium ionophore A₂3187 to purify cardiomyocytes further.2. α-sarcomeric actin identifying the purity of cardiomyocytes is 95±3.4%(n=16).And rhythmic beating could be seen under microscope. And "dedifferentiation" could be seen with losing of columnar organization.3. Purified cardiomyocytes were transfected by calcium phosphate coprecipitation technique with pRSV-lacZ containing β -galactosidase gene, and the labeling efficiency was 19.2 ± 4.7%(n=6) similar with reports.4. Cardiac ultrasonograph show: Cell transplantation improved cardial systole function and limited thinning of ventricular wall. But not significant effect on diastole function had been seen.5. The tissue of all animals, transplanted with cardiomyocytes transfected with the β -galactosidase gene, contained ft -galactosidase activity.Conclusion1. We can get high purified cultured cardiomyocytes in vitro by several techniques controlling the number of other cells.2. Genetic mark could be used to label adult cardiomyocytes and could be identified through a long time in vivo.3. After cultured in vitro, adult cardiomyocytes transplantation to...