The Biological Research Of Cardiac Fibroblast Under The Condition Of Hydrogel Substrate Mechanical Environment

In recent years, the incidence of cardiovascular disease increased abruptly, becoming oneof the three major threats to human health. Research on the field of cardiovascular disease hasreceived more attention. Myocardial infarction, heart failure and other cardiovascular diseases,at the end of the course, have emerged such common pathology of myocardial fibrosis, andled to heart stiffness increase. Thereby affecting cardiac fibroblast proliferation and promotemyocardial fibrosis occurs, ultimately leading to heart failure. In this study, we simulatedifferent subtratum mechanical environment, and evaluate the effects on cardiac fibroblastproliferation and differentiation. The purpose of this research is to provide basic clues for thefuture deep explore for the cause of disease such as myocardial fibrosis development and mayprovide the experimental basis for new therapeutic strategies.Discussed the biological responses of cardiac fibroblasts under different substratestiffness for adhesion morphology, proliferation and differentiation. Produced differentsubstrate stiffness polyacrylamide hydrogel(PAHG) by changing the crosslinkerbisacrylamide concentration with0.1%,0.3%,0.5%,0.6%. To study the biological responseof myocardial fibroblasts in different stiffness of the PAHG substrate for cell morphology,proliferation, differentiation. The results showed, with neonatal rat cardiac fibroblasts culturein0.1%,0.3%substrate (less than the Young’s modulus of heart, modulus of about3-10kPa),cell adhesion and proliferation is sequestrated, and there is no significent myofibroblastdifferentiation. In0.5%,0.6%(simulating the Young’s modulus of heart, modulus of12-20kPa)and glass culture dish substrate((more than the Young’s modulus of heart, modulus is GPa),cell adhesion and proliferation rapidly. With the increase of substrate modulus, cardiacfibroblast transition to myofibroblast is obvious. In glass substrate almost all cardiacfibroblasts differentiate into myofibroblasts. The experimental data displayed: theproliferation rate between0.1%and0.3%substrate is significant, P<0.05. The proliferationrate between0.5%and0.6%substrate is no statistical significance, P>0.05. The proliferationrate of0.1%,0.3%substrate between0.5%,0.6%substrate is extremely significance, P<0.001.The proliferation rate of0.1%,0.3%,0.5%,0.6%between glass substrate is extremelysignificance, P<0.001. The se results suggest that cardiac fibroblasts can make a differentresponse to the change of substrate stiffness, on a soft substrate, the proliferation anddifferentiation of cell is relatively low. Closed to the heart modulus substrate the proliferation rate increase. With modulus is much higher than the heart, The proliferation anddifferentiation rate is extremely high.Cardiac fibroblast can make different responses to substrate stiffness. On this basis, toinvestigate the signaling pathways associated with cell proliferation and differentiation will beimplemented. Three-dimensional mechanical environment will be carried out the study ofcardiac fibroblasts proliferation and differentiation in further research. Three-dimensionalenvironment is close to the real environment in the body, can reflect the real proliferation anddifferentiation due to mechanical factors, and the related signal pathway involved in thisprocess, all these cluse direct to better understand the basis, such as myocardial infarctionmyocardial fibrosis progression, thus may development new strategies for treatment ofmyocardial fibrosis and other fibrosis related diseases.The main content of this paper is divided into six chapters. the first chapter is theintroduction, mainly reviewed in recent years, the effects of the mechanical environment(such as tensile strength, hardness of the substrate) on cell morphology, proliferation,migration and other biological functions. The second chapter, the separation and purificationof cardiac fibroblast cells, mainly introduces the neonatal rat cardiac fibroblasts separationand purification. The third chapter, cardiac fibroblasts identification, mainly on theexperimental materials used in this paper, cardiac fibroblasts were identified, with vimentin、DDR2immunohistochemical staining. The fourth chapter, the different PAHG substratepreparation and cell culture, using acrylamide as raw material, through changing thecrosslinker bisacrylamide concentration, to produce different stiffness of the PAHG substrate,and the cells were cultured on the substrate. The fifth chapter, the proliferation anddifferentiation of cultured fibroblasts on different substrate, proliferation detection with EdUkit, the differentiation analysis with alpha-SMA immunohistochemical staining. The sixthchapter, the summary and prospection, summarizes the main conclusions obtained from theexperiment, the follow-up work is prospected.