Experimental Study Of Myocardial Reconstruction By Converting Cardiac Fibroblasts Into Cardiac Myocyte-like Cells

PART ONE 5-Azacytidine Induce Cultured Rat Cardiac Fibroblasts to Convert into Cardiac Myocyte-like Cells In Vitro First section Collection and culture of rat ventricular fibroblasts Objective: To establish the methods for culture of ventricular fibroblasts. Method: ventriculars from 10 Sprague-Dawley neonatal rats were minced and subjected to serial trypsin digestion to release single cells, the cells were preplated for 1 hour in DMEM with 10% fetal calf serum, attached cells were then incubated in the same medium and passaged by the methods of trypsin enzymic digestion when they were grown to confluence. Result: Ventricular fibroblasts were circular, triangular or polygonal-shaped and grown slowly in the beginning of incubation; they were triangular or polygonal-shaped in 24hrs and spindle-shaped 48 to 72hrs later with enlarged physical volume; their passages began to attach in 30mins and adhered on the bottom of the flask completely after 12hrs, and then, they were aligned in spindle, parallel-form gradually and grown quickly to confluence in about 3 to 4 days. Conclusion: Trypsin enzymic digestion and differential attachment was suitable for culture of ventricular fibroblast.Second section 5-Azacytidine induce cultured rat cardiacfibroblasts to convert into cardiac myocyte-like cellsObjective: To determine whether 5-Azacytidine can induce cultured ratcardiac fibroblasts to convert into cardiac myocyte-like cells. Method:Ventricular fibroblasts were obtained from 10 Sprague-Dawley neonatal rats as previously described; the third passage were then cultured to confluence and divided into 7 groups: control, 5-Azacytidine in 2.5, 5, 7.5, 10, 15, 20umol/L respectirely; the cells were serum-starved for 24hrs and treated with different concentration of 5-Azacytidine for 24hrs and then cultured in standard condition for 2 weeks. Cells were harvested and determined for their morphology and phenotypic markers using immunohistochemistry, reverse transcription polymerase chain reaction(RT-PCR), differ and transmission electron microscopy. Result: Compared to control group, the cells treated with 5-azacytidine, especially in 10umol/L concentration, contracted and polymerized to form clone unit with abundant mitochondria and actin filaments but no intercalated discs. This cells were positive with cellular immunochemical stain and RT-PCR on β muscle heavy chain, cardiac troponin I and connexin43. Conclution: 5-Azacytidine can induce cultured rat cardiac fibroblasts to differentiate into cardiac myocyte-like cells. PART TWO Experiment Research Of Rat Ventricular Fibroblasts Treated With 5-Azacytidine in Transplantation for Treatment Of Myocardial InfarctionFirst section Establish ment of rat myocardial infarction modelObjective: To study the method of establishing rat myocardial infarction model. Method: 20 Sprague-Dawley adult rats were devided into operation(n=10) and sham-operation(n=10) groups. Operated animals were anesthetized intraperitoneal injection, incubated and mechanically ventilated. The heart was exposed by sterile anterolateral thoracotomy inthe fourth left intercostal space and pericardiotomy, and the left anterior descending(LAD) coronary artery distal to the first diagonal branch (one third distal to its origin) was ligated, the lungs were inflated and the chest was closed. Sham-Operated animals underwent a similar procedure except that the LAD was not ligated. The electrocardiogram (ECG) was constantly monitored throughout the entire operation. The rats were killed and their hearts were harvested to undergo histopathological examination 2 weeks later. Result: In the operated animals, macroscopic analysis demonstrated that left ventricular free wall and apex bulged, turned from vermeil to purple just after LAD ligation, and were thin, offwhite in postmortem; S-T elevation to single-phase curve appeared on the ECG during operation; light microscopy examination showed that cardiomyocytes were curdy necrosis with nuclei weak dyed or disappeared, accompanied by inflammatory infiltration and fibrous connect...