The Study Of Cardiac Stem Cell Differentiation On Hydrogel Substrate Mechanical Environment

With the progress of population aging in China, the morbidity and mortality of myocardialinfarction and ischemic cardiomyopathy have risen obviously, and it is a serious threat to humanhealth. Cardiac Stem Cells (CSCs) with potentials of self-replicating, and with specificdirectional differentiation trend for myocardial cell line at the same time. Autologous stem celltransplantation therapy does not exist heart ethical issues, without immune rejection as well, so itis the ideal seed cells for the treatment of myocardial infarction and ischemic cardiomyopathy.There are very few number of myocardial cells in situ of the heart, and with low renewal rate, soas to not enough to fully restore myocardial injury and loss. The number of the myocardial cellsreduce gradually with aging, in order to carry out stem cell transplantation therapy, the first issueneeded to be solved is the separation, purification, amplification and specific differentiation ofCSCs in vitro, CSCs with high purity, strong vitality, enough amount and homologous biologicalperformance can meet the needs of clinical stem cell transplantation and tissue engineering. Allcells of the body exist in the mechanical microenvironment, so CSCs is not an exception, theproliferation and differentiation, and growth status will be affected by the mechanicalenvironment.The purpose of this paper is to explore the effects of basal stiffness on the proliferation anddifferentiation of CSCs. With different concentrations of methylene diacrylamide as crosslinkingagent, we polymerized acrylamide monomer into Polyacrylamide hydrogel, PAHG. By adjustingthe concentration of methylene diacrylamide with0.1%,0.3%,0.5%,0.6%, we makePolyacrylamide hydrogel of several rigidity gradients, and combined with commonly used glasssubstrates for cells culture, as basal for CSCs adhesion. Rigidity of the PAHGwith concentration of0.1%and0.3%are lower than the normal tissue; Rigidity of the PAHGwith concentration of0.5%and0.6%are similar to the normal tissue; Rigidity of the glassbase is higer than the normal tissue. The influence of basal rigidity to proliferation of CSCs are measured by EDU proliferationkit, the influence of differentiation assessed by the cell morphology and immunofluorescenceanalysis of the expression of cell surface specific protein. The results showed that on the PAHGsubstrate of0.5%,0.6%with modulus about15kPa, closing to the physiological modulus ofheart (18±2kPa), CSCs with high proliferation rate and maintain high differentiation potentialat the same time; on the PAHG substrate of0.1%,0.3%with modulus about2~7kPa, which arelower than the physiological modulus of heart, the proliferation and differentiation of CSCs arealmost at a standstill state; glass substrate with modulus higher than the physiological modulusof heart, with higer proliferation and launch the differentiation mechanism of CSCs.The effect of basal hardness on the proliferation and differentiation of CSCs, help usrecogonize the culture conditions and the environment of keeping the stemness of CSCs, andthen in vitro to amplify the separated and purificated CSCs, and provide experimental basis formeeting the needs of clinical stem cell transplantation.This paper mainly discusses in seven chapters, the first chapter is the introduction, aimed atreviewing the situation of each subtype CSCs and the impact of basal hardness on stem cellresearch progress; The second and third chapter is the experimental material, separation andpurification of CSCs, CSCs of high purity, strong activity are key to the success of the follow-upstudy. The fourth chapter is the preparation of PAHG substrate, which is the substrate we used inthe study, after completion of gel preparation, surface processing is the key to determine the cellswhether can attach on with a good growth situation or not; The fifth chapter discusses the impactof basal hardness on CSCs proliferation, and the influence mainly be measured by using theEDU proliferation kit; Chapter six discusses the impact of basal hardness on CSCsdifferentiation, and then the influence to the differentiation of it mainly be measured from twoaspects: the influence on the cell morphology and immunofluorescence analysis of theexpression of cell surface specific protein; Chapter7is the summary of the full text and makeexpectations for subsequent work.