【作者】 林楚伟；

【导师】 周胜华；

【作者基本信息】 中南大学 ， 介入心脏病学， 2010， 博士

【摘要】 目的：观察大鼠骨髓间充质干细胞对梗死后心肌中成纤维细胞转型的调控,并探讨其可能机制。研究方法：1.体外分离、培养、扩增、鉴定SD大鼠MSCs,并选择最佳分离培养MSCs的方法：(1)采用现行两种主要分离培养MSCs的方法：密度梯度离心法、全骨髓贴壁法分离培养MSCs,并对全骨髓贴壁法加以改进,选择MSCs培养生长增殖状态最佳的培养方法来培养实验用MSCs;(2)使用流式细胞仪技术检测MSCs表面两种抗原分子的表达情况：CD29、CD45;(3)加入成骨、成脂肪诱导分化培养体系,观察MSCs向成骨细胞、成脂肪细胞分化的能力,以鉴定其多向分化潜能；(4)采用Brdu标记法标记移植用细胞,并检测其细胞活力。2.大鼠心梗模型的制作及心律失常至造模失败分析、心肌匀浆的制作：(1)采用开胸直视下结扎左前降支的方法制作大鼠急性心肌梗死模型；(2)分析早期心律失常至造模失败机制,并加以预防改进；(3)心梗后心肌匀浆制作：使用组织匀浆机研磨心肌组织块,取匀浆上清并过滤除菌。3.体外模拟心梗后微环境,单培养及和培养MSCs与心肌成纤维细胞,观察两种细胞的转化情况：(1)使用心梗后心肌匀浆加入培养基,模拟心梗后微环境；(2)单培养或和培养两种细胞,应用细胞免疫化学染色的方法观察两种细胞的转化情况；(3)应用ELASA法检测培养基质中TGF-β1、TNF-α、PDGF的浓度变化。4.体内实验：大鼠心梗模型建模14天后,对大鼠心脏心梗周边区多点分别注射含TGF-β1 150μLDMEM培养液(n=13), PBS150μL (n=13),5×106MSCs/150μL(n=13)。移植后一月,处死大鼠,获取心脏标本,采用组织病理、免疫组化的方法鉴定移植部位有无肌纤维母细胞的聚集,并采用westen-blot、PCR分析胶原蛋白的产生情况。结果：1.全骨髓贴壁法组MSCs传代、生长速度明显快于密度梯度离心法法组MSCs,两组细胞流式细胞仪鉴定、分化能力鉴定及细胞活力均符合实验要求；2.青年组大鼠心律失常、室颤及早期死亡率均明显低于中年组大鼠,心律失常发生率与两组大鼠血清NE值增加速度成正比；3.利多卡因术前腹腔注射后,早期心律失常发生率下降,早期造模成功率升高；4.差速贴壁法可以分离培养的第二代成纤维细胞,MSCs与心脏成纤维细胞共培养,加用心梗14天后心梗周边区域心肌匀浆干预7-28天后行平滑肌肌动蛋白(α-SMA)免疫细胞化学染色呈阳性,ELASA法检测到急性心梗后14天心梗周边去心肌匀浆干预MSCs单培养组、MSCs与心脏成纤维细胞共培养组细胞因子TGF-β1浓度明显高于其他组,且与肌纤维母细胞阳性率正相关；5、MSCs移植组的梗死心肌内存在大量BrdU标记阳性的移植细胞,Brdu阳性区连续切片行波形蛋白及平滑肌肌动蛋白免疫组化染色阳性,Brdu阴性区连续切片行平滑肌肌动蛋白免疫组化染色阴性；移植后MSCs移植区、TGF-β1注射区、PBS注射区胶原蛋白Ⅰ、ⅢmRNA及蛋白的表达,PBS注射区明显小于MSCs移植区、TGF-β1注射区胶原蛋白Ⅰ、ⅢmRNA及蛋白的表达。结论：1.全骨髓贴壁法组MSCs生长增殖速度明显快于密度梯度离心法法组MSCs,并符合实验使用要求；2.开胸直视结扎左前降支能有效制作大鼠急性心肌梗死模型,其早期心律失常至造模失败率与血清NE值升高速度正相关,术前腹腔注射利多卡因能提高造模成功率；3.差速贴壁法可分离培养出心脏成纤维母细胞,急性心梗后14天心梗周边去心肌匀浆干预MSCs与心脏成纤维细胞共培养组细胞7-28天后可形成肌纤维母细胞,并由成纤维细胞转化而来,急性心梗后14天心梗周边去心肌匀浆干预MSCs(无论单培养还是与成纤维细胞共培养)可分泌一定量的细胞因子TGF-β1,TGF-β1参与成纤维细胞向肌纤维母细胞的转型；4.大鼠心脏对移植MSCs无明显免疫排斥,移植MSCs14天后,MSCs仍存活于心脏,MSCs移植区有肌纤维母细胞的聚集,MSCs移植能促进移植区胶原蛋白Ⅰ、Ⅲ的分泌,TGF-β1可能参与其中,体内实验验证了体外实验关于MSCs通过分泌TGF-β1调控心肌内成纤维细胞向肌纤维母细胞转型的结论。更多还原

【Abstract】 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.更多还原