| objective ADMSCs as a novel seed cell for cytological engineering has been gradually accepted. But before clinical application, there are still many questions to be solved. For example, whether ADMSCs can differentiate into real cardiomyocytes with the normal functions of excitability, conductibility and contractibility? How to improve differentiation rate of ADMSCs into cardiomyocytes? In this study, firstly, we try to establish a mature method to isolate and culture sufficient ADMSCs form Wistar rat. Then, try to find an ideal method for inducing ADMSCs to differnentiate into cardiomyocytes. At last, try to detect the electrophysiological characters of induced AMDSCs.Methods According to the aims mentioned above, this study was divided into three parts.First part Cells were obtained by digesting adipose tissue of Wistar rat with trypsin and collagenase, and then cultured in vitro. Growth curve of passages from second to eighth was drawn and cell doubling times(Td) were calculated. The third passage cells were determined with immunocytochemistry methods for surface positive molecules of CD13, CD44, CD105 and negative molecules of CD34, CD45, factor VIII, HLA-DR and induced with lineage-specific induction factors to bone, fat and muscle which were determined with different staining methods of alkaline phosphatase, Oil red O and immunocytochemistry method. The cells were also stored in liquid nitrogen for 2 to 4 weeks and resuscitated and cell viability was measured by Trypan Blue Dye Exclusion Method. At last, cells were infected with human V adenovirus vector carrying fluorescent protein (GFP) and HGF.Second part The third passage of Wistar rat's ADMSCs were induced by three different methods of 5-aza, extracts of neonatal rat cardiomyocytes or both for 4 to 6 weeks, and differentiation rates were evaluated by detecting immunofluorescence labeled-α-actin positive cells through flow cytometry. After induced for 6 weeks, ADMSCs were detected on Gene Level (GATA-4, Nkx2.5, MyoD), protein level(α-actin, connexin 43, connexin 45) and ultrastructure with RT-PCR technology, immunofluorescence method and transmission Electron Microscope (TEM) respectively. DAPI labeled ADMSCs were were co-cultured with second passage of neonatal rat's cardiomyocytes and their growth and beat were observed.Third part ADMSCs induced 4 to 6 weeks or non-induced by an ideal method were subjected to multi-channel array system(MEA) and their excitability and conductibility were detected and compared. Induced ADMSCs co-cultured with neonatal rat's cardiomyocytes were also detected by MEA.Results3.6×10~5 of adherent cells were obtained from 0.3g rat's adipose tissue. These cells could be cultured for generations in vitro with stable population doubling time ranging from 55 to 63 hours. Immunocytochemistry showed that most of them were positive for CD13, CD44, CD105 and negative for CD45, VIII factor, HLA-DR, VWF. They could differentiate into osteogenic, adipogenic and myogenic cells in the presence of lineage-specific induction factors. After frozen deposition, over 90% cells survived and kept stable surface markers and well state for culture. The cells could be infected by human V adenovirus vector carrying GFP and HGF, but infected cells died soon.Three inducement methods(5-aza, neonatal rat cardiomyocytes or both) all could induce ADMCs differentiate into cardiomyocytes with differentiation rates of 18.58%, 58.67%,62.27% respectively. Cardiomyocyte related genes GATA-4, Nkx2.5 and MyoD were activated. Proteins of α-actin, connexin 43, connexin 45were expressed. Myoneme-liked ultrastructure was found. ADMSCs co-cultured with neonatal rat's cardiomyocytes beated passively with the cardiomyocytes' clone.Spontaneous population action potential(AP) could be recorded from cardiomyocytes. No signals of spontaneous AP from induced or non-induced ADMSCs were recorded by MEA. When exposed to outside electrical stimuli, non-induced ADMSCs reacted feebly, while induced ADMSCs reacted consumingly with their membrane potential being fluctuated markedly just like cardiomyocytes' AP. But this kind of fluctuated membrane potential could not conduct normally for serious delay and attenuation. Co-cultured ADMSCs with cardiomyocytes also didn't produce electrical action by themselves and stimuli of adjcent beating cardiomyocytes clone.Conclusion1. Large quantity of ADMSCs with multilineage differentiation potential can be easily obtained from Wistar rat's adipose tissue by enzyme digestion. Obtained cells are easy to be cultured, expanded, deposited long and transfected with targeted gene in vitro.2. In vitro, both 5-aza and extracts of neonatal rat cardiomyocytes can induce ADMSCs from Wistar rat differentiate into cardiac-like myocytes, but co-inducement with the both is the most ideal method.3. Excitability of induced ADMSCs increases, but conductivity is poor for its serious delay and attenuation.
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