Objective To explore the differences in genes expression associated with rat cardiac fibroblasts(CFs) differentiation into cardiomyocyte-like cells induced by 5-azacytidine(5-aza) at 5, 7, 14, 21 and 28 days. Methods CFs were isolated from twelve 1-3-day neonatal SD rats by using combined type Ⅰ collagenase and trypsin digestion method . Cell purity was determined by CFs molecular marker discoidin domain receptor 2(DDR2) immunofluorescence assay. Myocardial induction solution containing 15 μmol/L 5-aza was incubated with passage 3 of CFs. The expression differences of related genes including vimentin, DDR2, Nanog, sox2, c-kit, sca-1, Tbx5, CD73, CD34, cMyc, klf4, Gata4, Oct4, Mef2c and cardiac troponin T(cTnT) were detected by Real-time PCR at different induction time. Cell immunofluorescence for cTnT was observed at day 5, 7, 14, 21 and 28 after myocardial induction. Morphological ultrastructure of induced CFs at day 28 was observed by transmission electron microscopy. Results The cell growth rate was slowed down at 3 days after 5-aza induction, and the morphology of some cells were transformed from triangular, spindle shaped to round and rod shaped. Compared with the CFs before induction, induced after 7, 14, 21 and 28 days, DDR2 had stable expression, while vimentin expression decreased at day 14. But Nanog, c-kit, sox2 and Tbx5 declined accompanied by myocardial process, while CD73, Mef2c, CD34, Gata4, Oct4 and cTnT gradually increased, sca-1 rose at 7 days and then falled, cMyc and klf4 increased at 14 days and declined. At day 5, there were a few cTnT positive cells and increased significantly at day 14-21. cTnT positive cells were stable at day 28 and positive cells had no obviously change cultured from 35 days to 40 days. The cytoplasm appeared in a large amount of collagen fiber bundle, arranged regularly and rich mitochondria under transmission electron microscope. It showed end to end connection between induced fibroblasts. There were a large number of myofibril s in the cytoplasm. However, sarcomere was not obvious, and the H band, Z line and M line were not clear. Conclusion CFs can differentiate into cardiomyocyte-like cells but do not form mature cardiomyocytes induced by 5-aza.
