| The cardiac myocytes couldn't differentiate and regenerate, and there were also no cardiac stem cells in the heart.So when the heart was damaged, it was repaired by scar which might cause heart failure and death.Recently many researchers have begun to treat heart failure,ischemia heart deseases and dilated cardiomyopathy by cell transplantation.It could prevent ventricularreconstruction,reduce mortality rate and improve life quality.There were many kinds of seed cells,which could be implanted into hearts,including myoblasts(skeletal satellite cell), mesenchymal stem cells,cardiac cells(embryonic, neonatal, adult), smoothmuscle(embryonic, neonatal),and etc.The studies and application of myoblasts were much more than other seeds.It was argued that how they changed and whether they could form electro-mechanical coupling when they were transplanted into hearts.It was complicated and influenced by many factors in vzvo.We established a practical coculture model of myoblasts and cardiac myocytes of rats in vitro to study how both act to each other in vivo.Besides this,we also introduced means of isolating,purifying and identifying myoblasts,and described myoblasts' biological characters in detail.Methods We cultured primary myoblasts.Myoblasts were digested with collagenase I of 1 g L-1 and Trypsin of 2.5 g L-1.Also myoblasts were isolated and purified with discontinuous density centigugation (Percoll gradient liquid).We observed their biologicalcharacters by inverted microscope and their ultrastructure by transmission electron microscope and described their growth curve.They were tested by a -actin and desmin monoclonal antibodies by SABC immunochemistry stains. Isoproterenol was added into the culture bottles,in which myoblasts could auto-contract, with the final density as 0.1 umol L-1 so as to compared the contracting rates between before addition and after addition.We established coculture model of myoblasts and cardiac myocytes in vitro. Myoblasts and cardiac myocytes were isolated and purified individually and were mixed with proportion of one to one. The mixture was seeded into culture plates and their characters were observed. On the fifth day of coculture,we collected,dehydrated and fixed the cells,which were observed by transmission electron microscopy and found if there were desmosomes and gap junctions.Also we studied them by microinjection under inverse microscopy.When micro pole was injected into myoblasts,biocytin diffused with freedom and electrophoresis of square wave .After the cells were fixed,Avidin labeled with Texas Red was applied to detect where showed biocytin under fluorescence microscopy.Results: When myoblasts were digested with mixture of collagenase I and Trypsin isolated and purified with discontinuous density centigugation,myoblasts purity could be as high as 95 % .Myoblasts showed as spindle and fused into myotubes at the forth or fifth day of culture.By a -actin immunochemistry stains tests, myoblasts were stained as red,and it was similar by desmin. The majority of them had one round nucleus and few had two nuclei.Under transmission electron microscopy myoblasts had one to three nucleoli in nucleus.There were irregular myofilament and none in some zones in cell plasm. Mature chondriosome,hepatin,free ribosome and lysosome also could be found.Myoblastes could auto-contract on the 2nd or 3rd day after they were fused and the contracting rates ranged from 50 to 160 beats per minute for the most part. When isoproterenol was added, the mean contracting rateincreased by 22.5 percents compared with that before the addition of isoproterenol.And we get the result of p<0.05 by statistics analysis,which illustrated that IP had a positive chronotropic effect on myoblasts.We could find both myoblasts and cardiac myocytes contracted synchronously when cocultured together.Desmosomes were easily found while gap junctions were not found between the two kinds of cells by transmission electron microscopy. We also could find... |
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