Experimental Study Of Adipose Tissue-Derived Mesenchymal Stem Cell On Treatment Of Acute Myocardial Ischemia Injury

This study was divided into two parts. In the first part, we investigated the processes and feasibility to obtain, culture, proliferate and label with CM-Dil the human and mini-swine adipose-derived mesenchymal stem cells (AD-MSCs). We investigated the differentiation ability of human AD-MSCs in vitro. We investigated the effectiveness of human and mini-swine AD-MSCs on post-ischemic heart injure. On the basis of first part, the second part was carried out to investigate the mechanisms of human and mini-swine AD-MSCs underlying the effectiveness on post-ischemic heart injure in mini-swine acute myocardial infarction models. We investigated the ability of AD-MSCs to survive, differentiate and secret cytokines after being transplanted into infracted region.The part1:The effectiveness of AD-MSCs on ventricular remodeling after acute myocardial infarction and heart function evaluating.Objective:Myocardial infarction, which is characterized by reducing blood supply to the heart and loss of functioning cardiomyocytes, has become the mainly cause of morbidity and mortality in modern society. The limited capacity of the adult heart to self-regenerate and persistent deteriorate of the heart function yield poor prognosis for myocardial infarction. AD-MSCs are a promising source of stem cells which are used for myocardium regeneration and repair of infarction heart, which also have multi-lineage differentiation potential. In this study, we investigated the feasibility to obtain, culture, proliferate and label with CM-Dil the human and mini-swine AD-MSCs and in vitro differentiation ability of them. We investigated the effectiveness of AD-MSCs on ventricular remodeling after myocardial infarction after being transplanted.Method:Human and mini-swine adipose tissues were collected for culturing and proliferating AD-MSCs. The prepared AD-MSCs were labeled with CM-Dil. The prepared AD-MSCs were tested by flow cytometer and induced to differentiate in virto.Anterior descending branch of left coronary artery was ligated to set up AMI model in mini-swines. The animals were randomly divided into sham operation group, the animals received no treatment (n=6); control group, PBS were injected into the infracted area of left ventricular (n=6); study group1, xenogenic AD-MSCs were injected into the infracted area of left ventricular (n=6); study group2, allogenic AD-MSCs were injected into the infracted area of left ventricular (n=6); study group3, autologus AD-MSCs were injected into the infracted area of left ventricular (n=6).1) Ultrasonic cardiogram was carried out before the operation,2hours after the operation and6weeks after the operation.2) Myocardial perfusion SPECT scan was carried out2hours after the operation and6weeks after the operation.3) Masson trichrome stain of the infarcted myocardium was carried out6weeks after the operation to observe the distribution of viable myocardium and collagen under the microscope.All the dates collected were analyzed.Result:The prepared AD-MSCs could be labeled with CM-Dil easily and effectively. The prepared AD-MSCs were proved to have the features of MSCs according to the ISCT criterion for MSCs by flow cytometer test and could be induced to differentiate into mature adipose cells and chondrocytes.Six weeks after the AD-MSCs being transplanted, the MWTD, MWTS and WTR in study group1, study group2and study group3were increased significantly compared with the sham operation group and control group (p＜0.001).Six weeks after the AD-MSCs being transplanted, the myocardial perfusion were increased in study group1, study group2and study group3, while in sham operation group and control group the myocardial perfusion were decreased.The mass of viable myocardium in study group1, study group2and study group3were more than those in sham operation group and control group significantly (p＜0.001).Conclusion:The processes to obtain, culture, proliferate and label with CM-Dil the human and mini-swine adipose-derived mesenchymal stem cells (AD-MSCs) were simple, effective and economical, which could be manipulated easily. Transplanting AD-MSCs into infarcted myocardium could not only prevent the left ventricular enlarging, thinning of the infarcted left ventricular wall and reversing left ventricular remodeling, but also could improve the myocardial perfusion and protect the myocardium. The Part2:The mechanism underlying the effects of AD-MSCs on repair of post-ischemic heart injureObjective:To investigate the mechanism underlying the effectiveness of AD-MSCs on repair of post-ischemic heart injure after being transplanted on the basis of part1. Including to investigate the AD-MSCs’ability to differentiate into mature myocardial cells and vascular endothelial cells; the ability to secrete cytokine via paracrine effect; the ability to inhibit cell apoptosis.Method:Anterior descending branch of left coronary artery was ligated to set up AMI model in mini-swines. The animals were randomly divided into sham operation group, the animals received no treatment (n=6); control group, PBS were injected into the infracted area of left ventricular (n=6); study group1, xenogenic AD-MSCs were injected into the infracted area of left ventricular (n=6); study group2, allogenic AD-MSCs were injected into the infracted area of left ventricular (n=6); study group3, autologus AD-MSCs were injected into the infracted area of left ventricular (n=6).1) The mRNA gene expression levels of VEGF、 vWF、 TGF-β3、 HGF、 CXCR4and SDF-1in each group were tested by RT-PCR,6weeks after the AD-MSCs being transplanted. All datas collected were analyzed.2) The microvessel density was studied by immunohistochemichal staining of vWF in each group and the level of cell apoptosis around infracted myocardium was studied by TUNEL staining in each group6weeks after the AD-MSCs being transplanted. All datas collected were analyzed.3) The immunofluorescent staining was used to observe the AD-MSCs differentiation into mature myocardial cells and vascular endothelial cells; the intercalated disc formation between new differentiating myocardial cells and local surviving myocardial cells; the level of cell proliferation around the infracted myocardium in each group6weeks after being transplanted. Result:1) Six weeks after the AD-MSCs being transplanted, the mRNA gene expression level of VEGF、 vWF、 TGF-β3、 HGF、 CXCR4and SDF-1in study group1, study group2and study group3were increased significantly compared with the sham operation group and control group (p<0.001).2) Six weeks after the AD-MSCs being transplanted, the microvessel density of study group1, study group2and study group3were increased significantly compared with the sham operation group and control group (p＜0.001); the cell apoptosis in study group1, study group2and study group3were decreased significantly compared with the sham operation group and control group (p<0.001).3) The number of surviving cells in study group2and study group3was larger than that in study group1significantly (p＜0.001). The AD-MSCs differentiation into mature myocardial cells and vascular endothelial cells were observed in study group2and study group3, the intercalated discs were also observed between new differentiating myocardial cells and local surviving myocardial cells. There is no evidence for the AD-MSCs differentiation in study group1. Obvious cell proliferations were observed in study group1, study group2and study group3, and many proliferating cells were CM-Dil negative.Conclusion:The AD-MSCs could increase the microvessel density and inhibit cell apoptosis in infarcted myocardium after being transplanted. The AD-MSCs could promote angiogenesis, mobilize local stem cell and induce them differentiating to repair the injured myocardium via paracrine effect. The allogeneic and autologous AD-MSCs could differentiate into mature myocardial cells and vascular endothelial cells after being transplanted, there are intercalated discs formation between the new differentiating myocardial cells and local surviving myocardial cells.