The Research Of Transformation Of Fibroblasts In The Myocardial After Infarction By Rat Bone Marrow-derived Mesenchymal Stem Cells And Its Mechanism Of Regulation

Objective:To observe the rol of the rat bone marrow-derived mesenchymal stem cells on myocardial infarction in the regulation of fibroblast transformation, and to explore its possible mechanism.Methods:1. Isolation, culture, amplification, identification MSCs of SD rat, and to choose the best method of MSCs to isolate and culture:(1) use of the two main existing MSCs isolation and culture methods:density gradient centrifugation, whole bone marrow adherent method to isolate and culture MSCs, and whole bone marrow adherent method was improved, choose the best state of MSCs cultured growth and proliferation of training methods to cultivate experimental MSCs; (2) identify MSCs by flow cytometry to detect surface expression of the two antigen molecules:CD29, CD25; (3) by adding into the bone, fat-induced differentiation culture system to observe the ability of MSCs to differentiate into bone cells, fat cells,to identify their multi-differentiation potential; (4) using Brdu labeling to tag the transplantation cells and detected cell viability.2. Production of rat myocardial infarction model and analysis of modeling failure by cardiac arrhythmia, production of myocardial homogenate:(1) by open-chest and under direct vision ligating the left anterior descending artery, to product acute myocardial infarction model; (2) analysis of early arrhythmias making modeling failure mechanisms, and to prevent improvement; (3) myocardial infarction homogenate production:using tissue Grinding myocardial tissue blocks to make, take homogenate supernatant and filter sterilization.3. Vitro simulate micro-environment after myocardial infarction, single and nurturing culture MSCs with cardiac fibroblasts to observe the differentiation situation of the two kinds of cells:(1)by adding myocardial infarction homogenate into medium to simulate micro-environment after myocardial infarction; (2) single and train two kinds of cells in culture or to observe the differentiation situation of the two kinds of cells by the application of immuno-chemical staining; (3) application ELASA training matrix to detect TGF-β1, TNF-α, PDGF concentration changing.4. Vivo experiment:after 14 days modeling of myocardial infarction in rats, the myocardial infarction border zone of rat hearts were multi-point injected with 150μLDMEM culture medium with TGF-β1 (n =13), PBS150μL(n=13),5×106MSCs/150μL(n=13). Post-transplant in 2 weeks, rats were killed to obtain the heart specimens, to identify whether there were accumulation of myofibroblasts in grafted sites by tissue pathology, immunohistochemistry and adopt the westen-blot, PCR analysis of the production of collagen.Results:1. MSCs cultured by the whole group of bone marrow adherent method,which passage and growth rate significantly faster than the density gradient centrifugation method group MSCs, the two groups of cells, flow cytometry identification, differentiation and cell activity were consistent with identification of test requirements;2. Young rats arrhythmia, ventricular fibrillation and early mortality rates were significantly lower than the middle-aged rats, the incidence of arrhythmia and two groups of rats the rate of increase is proportional to the value of serum NE;3. Preoperative intraperitoneal injection of lidocaine, the early decline in the incidence of arrhythmias, early modeling success rate of increase;4. Differential adhesion method can be used to separate and culture the second generation of fibroblasts, MSCs and cardiac fibroblasts were cultured together, and was added homogenate around the area of myocardial infarction 14 days,after the intervention in 7-28 days, smooth muscle actin protein (α-SMA) immunocytochemistry positive, ELASA method detected MSCs single culture group, MSCs and cardiac fibroblasts co-cultured group with myocardial infarction neighboring homogenate interfere 14 days after acute myocardial infarction,the concentration of cytokines TGF-β1 was significantly higher than other groups, and with the positive rate of myofibroblasts positive correlation;5. There were a large number of BrdU-positive markers' transplanted cells in the infarcted myocardium in MSCS transplantation group, Brdu positive area serial sections were made vimentin and smooth muscle actin immunohistochemical staining which was positive, Brdu negative zone serial sectioning line-smooth muscle actin immunohistochemical staining negative staining; MSCs grafts after transplantation, TGF-β1 injection zone, PBS injection zone collagenⅠ,ⅢmRNA and protein expression, PBS injection area significantly smaller than MSCs grafts, TGF-β1 injection zone collagenⅠ,ⅢmRNA and protein expression.Conclusion:1. The whole bone marrow adherent method group's growth rate and proliferation of MSCs significantly faster than the density gradient centrifugation method group MSCs, and in accordance with experimental use requirements;2. Thoracotomy look directly at the left anterior descending artery ligation in rats produced an effective model of acute myocardial infarction, arrhythmia to its early failure rate modeling, increased rate and serum values of NE are related to preoperative intraperitoneal injection of lidocaine can improve the success rate of modeling; 3. Differential adhesion method detachable train cardiac fibroblasts, myocardial homogenate around infarction region 14 days after myocardial infarction to interfere MSCs with cardiac fibroblasts,which were cultured after 7-28 days,could form myofibroblasts which transformed by the fibroblasts, interfered with neighboring myocardial homogenate 14 days after myocardial infarction MSCs (whether single or with cultured fibroblast co-culture) could be detected a certain amount of secretion of cytokines TGF-β1, TGF-β1 involved in transition of fibroblast to myofibroblast;4. Rat heart transplanted MSCs had no significant immune rejection, 14 days after transplant MSCs, MSCs still alive in the heart, MSCs transplantation district myofibroblast accumulation, MSCs transplantation can promote the migration zone of collagenⅠ,Ⅲsecretion, TGF-β1 may be involved, in vivo is verified in vitro on MSCs through the secretion of TGF-β1 in regulation of cardiac fibroblast to myofibroblast transformation conclusions.