Comparation About Division Of Cardiomyocytes,Cardiac Fibroblasts And Cardiac Stem Cells In Vitro

BackgroundTraditional theory claims that myocardial cells are highly differentiated cells which have lost the ability to proliferate. Recent studies have found that, under normal or abnormal conditions, myocardial cells can proliferate by division, which lay a theoretical foundation for the clinical treatment of myocardial injury. However, how the division characteristics of different cells in myocardial tissues are, how the division forms of myocardial cells are, and whether the division modes of cells and tissues in vitro culture are in the same way are still unclear. These problems significantly restricted the study and application of cardiac regeneration.ObjectivesObservation and comparison about division modes of cardiac cells, cardiac fibroblasts and stem cells in vitro culture provide the research of myocardial tissue engineering and treatment of myocardial injury with cytological and theoretical basis.Methodsl.We isolated the cardiomyocytes, cardiac fibroblasts and cardiac stem cells from myocardial tissue of neonatal rat1to3days in vitro, using a primary culture. Observe the growth, morphological differences, and beating of the cardiomyocytes in the case of an inverted microscope.2. The adherent time, growth, the way and time of proliferation, and when the split is completed of the isolated cardiomyocytes, cardiac fibroblasts and cardiac stem cells were real-time monitored in vitro with living cell station. 3. Cardiomyocytes, cardiac fibroblasts and cardiac stem cells isolated and cultured in vitro were double labled with specificity immunofluorescence.We compared the ability and the way of cell proliferation, dynamic changes of cell morphology and tubulin in mitotic of the three kind of cells.Results1. Primary cultured cardiomyocytes grow aggregately and form cell clusters or single cells with synchronous pulse on a frequency of40-170times/min. Primary cultured cardiac fibroblasts keep a great growing state, have varied shapes and are rich in binucleated cells. These cells become fecund after3-4days and it is easy to find a large number of cells which were swelled up and round into mitosis phase. Primary cultured cardiac stem cells are mostly small, round, bright cells. These cells keep a spherical shape and high refractive index and its nucleus is not visible. The distribution of cells is concentrated and these cells grow as clusters or scattered colony.2. Cardiac fibroblasts was the first to stick wall and extended pseudopodia within0.5-1h observed by living cells workstation, whereafter myocardial cells began to stick wall growth and formated a rhythmic beat gradually. Fibroblasts entered into the logarithmic phase and proliferation massive cultured by2-3days. Cells in the interphase period was in flat which stick to the culture dish and extended pseudopodia walk. Cells began to uplift and became round in Mitosis prophase while reture bact to flat after cytokinesis. Time lasts about20-30min from Mitosis prophase to cytokinesis.The fission of pulsating myocardial cells have not been obsetved. Cardiac stem cells always remain small and round throughout the process of mitosis not extending pseudopodia.3. The double-labelling immunofluorescence shows that Cardiomyocytes in miosis maintain squamous. Most of cardiac fibroblasts in every period of mosis will swelled up but a few still maintain squamous. Nanog positive cardiomyocytes stem cells are small and round at interkinesis and division stage, completing cytokinesis. The nuclear shape in mitosis prophase was regular, the chromatin became condensation and H3P begin to express. At prometaphase, karyotheca broken and spindle formed. Chromosomes were arranged in the equatorial plane and the shape of spindle was very typical at metaphase. The character at anaphase was that the chromatids separate and moved towards the poles and the spindle extended. At telophase, daughter nucleus came into being and parallel microtubule distributed between two daughter nucleus. At cytokinesis period, two daughter cells were generated and a small amount of silky microtubules located between two daughter cells could be seen after the two daughter cells separation. A few cardiac fibroblasts were seen amitosis. The nucleus concave from the middle and split into two daughter nucleis.There is no change of chromosome and spindle. Cytokinesis can be seen in cardiac fibroblasts with amitosis but not in cardiomyocytes.ConclusionsCardiomyocytes, cardiac fibroblasts and cardiomyocytes stem cells exist miosis but division form is different from each other.1. Both of them can finish cytokinesis and form daughter cells. Cardiomyocytes mainly exist miosis and multiplication capacity of cardiomyocytes is weaker than cardiac fibroblasts. After miosis, cells including two nuclears is little and also can finish cytokinesis and form daughter cells. Cardiomyocytes in miosis don’t swell up and get round.2. Multiplication capacity of cardiac fibroblasts increase and exist miosis (including swelling up and not swelling up) and amitosis.3. The shape of cardiomyocytes stem cells is small and round and exist completely miosis.