【作者】 王勃；

【导师】 白小涓；

【作者基本信息】 中国医科大学 ， 内科学， 2005， 博士

【摘要】 前言 心肌梗死等缺血性心脏病占心血管病死因的1/2,也是充血性心衰的主要病因。目前对缺血性心脏病的治疗主要包括药物治疗、介入治疗、外科手术等,但均不能解决功能心肌细胞减少等根本问题。目前心衰的根治性治疗只有同种心脏移植,美国因心衰做同种心脏移植2500例,需做的有30000例,主要原因是供体来源缺乏。而其他方法价格昂贵,大多数患者不能承受,如:1例左室辅助装置须花费25万美元,1例人工心脏须10万美元。因此需要有一种全新的治疗方式从根本上解决上述问题。大量的动物实验发现:干细胞可在体内、体外横向分化为心肌细胞,特别是自体骨髓干细胞,与其他侯选细胞(胚胎干细胞、脐带血干细胞、卫星细胞、心肌细胞、平滑肌细胞、骨骼肌细胞)相比,更适合临床应用,原因是骨髓干细胞容易通过常规途径获取,没有伦理道德问题,使用病人自体骨髓干细胞,避免了异体移植所致的免疫排斥反应,且与宿主心肌细胞形成电-机械耦联。临床试验也初步表明:骨髓干细胞自体移植可以增加心肌血供,减少心肌梗死面积,提高缺血损伤后的心功能,改善预后。基于上述理论,我们选用Wistar大鼠作为研究对象,研究自体骨髓干细胞原位移植对损伤心肌的修复作用;同时探讨体外培养的骨髓间充质干细胞向心肌样细胞的诱导、分化与其电生理功能。本实验分三部分,下面分述如下: 论文一 启动大鼠自体骨髓干细胞修复梗死心肌 目的:应用细胞因子动员Wistar大鼠自体骨髓池中的干细胞大量增殖,并释放至外周血,根据迁移、归巢、损伤微环境理论,探讨骨髓干细胞原位移植对坏死心肌的修复作用。 方法:更多还原

【Abstract】 IntroductionIschemic heart disease, including myocardial infarction, is the major cause of congestive heart failure, accounting for 1/2 cause of death in cardiovascular diseases. Up to now, treatment strategy for ischemic heart disease are medication , interventional therapy and surgery, but they don’ t solve foundamental question of cardiac myocytes regeneration. Present, homogeneity heart transplantation is the only radical cure for heart failure. In American, 2500 cases of heart failure received homogeneity heart transplantation, but patients who really need are 30000. The cause is lack of donators. Other methods, such as left ventricular assistor and artificial heart, are very expensive, most of patients don t accept it. In the situation, it is very necessary to find a novel method to change basic questions. Mass animals trials showed that stem cells differentiated to cardiac myocytes in vitro and in vivo. Especial for bone marrow stem cells, they prior to other candidate cells ( embryonic stem cell, cord blood stem cells, satellite cells,cardiac myocytes,et al. ) and apply for clinics. Bone marrow stem cells can be harvested by routine way and no moral problems. The cells from bone marrow stem cells can form electric - mechanical couple with near host myocytes. In addition, acceptors avoid immunological rejection reaction. Dates from clinical trials showed that autological bone marrow stem cells transplantation improved heart function , reperfuse, and decreased infarct size. In view of above mentioned theories, we selected Wistar rats as objects and investigated recovery of situ transplantation for damaged myocardium. At the same time, we studied if bone marrow mesenchymal stem cells could be induced into cardiac -like myocytes and their electrophysiology properties. We divided experiment intothree parts, state as follow:(—) Mobilize autologous bone marrow stem cells to repair infarcted myocardiumObjective: Autologous bone marrow stem cells were mobilized and released by cytokines. On the basis of homing and injured microenvironment theories , we investigated the effective reparation of situ transplantation for acute my-ocardial infarction in rats.Methods.-(1) We divided 60 Wistar rats into situ transplantation group and control group; To duplicate rat’ acute myocardial infarction model by injection of drug; CK^LDH level were checked by automatic biochemistry analyzer;(2)30 Wistar rats were injected rhG - CSF 50 jig/kg, day;( 3 ) We did a control analysis on pathological section between two groups by histological staining technique and computer graphic analysis;(4) Heart function were checked by polygraph system after AMI model 4 weeks.Results:(1) Serum cardiac enzymes were higher after AMI model 48 hours than it before AMI model. We saw local necrosis region with grid and trabs shape in endocardium of left ventricular apex and papillary muscle;(2)1116 infarction size in situ transplantation group was smaller than one in control group;(3)The heart function parameters improved significantly in situ transplantation group compared with control group.Discussion:Stem cells are daughter cells that differentiation potency gradually grow down. They can amplify and remain undifferentiation in vitro. According to their differentiation potency, they are divided into totipotential stem cell, multipoten-tial stem cell, multipotent stem cell (adult stem cell). Bone marrow stem cells are multipotent stem cell ( adult stem cell). Bone marrow stem cells have abilityto transdifferentiation, which differentiate into another type of cells in cytokine and microenvironment. It is important for cardiomyocytes regeneration. Therefore , we mobilize rats bone marrow stem cells by granulocyte colony - stimulating factors, migrating and homing to zone of necrosis. It was found that area of myocardial infarction was smaller in situ transplantation group than in control group and heart function improved obviously in situ transplantation group. This method was simple and no invasive compared with interventional means, but further studies and evaluations will be necessary.Conclusions:(l)Rat’ s acute myocardial infarction model could be completed by injecting 10 mg/kg isoprenaline interaperitoneally;(2)infarcted myocardium were repaired and heart function improved by u-sing cytokines.Key words: acute myocardial infarction; cytokine; situ transplantation(H)Bone marrow mesenchymal stem cells can be induced to differentiate to cardiac - like myocytes in vitroObjective:To investigate the bone marrow mesenchymal stem cells induced and differentiated to cardiac - like myocytes in Wistar rats.Methods;(1) We selected Wistar rats to isolate and culture bone marrow mesenchymal stem cells;(2)The first generation of bone marrow mesenchymal stem cells were induced with lOujnol/L concentrations 5 - azacytidine agent;(3 ) We identified and evaluated expression of surface markers, including CD34,CD44,CDw90;(4) After bone marrow mesenchymal stem cells were treated with lOjimol/L concentrations 5 -azacytidine agent, they were checked by immunohistochemis-try staining of antibodies against a - sarcomeric actin and troponin T in 2^4 week.Results;(1) The first generation of bone marrow mesenchymal stem cells expressed CD^O arid CD34 was negtive;(2)The cardiac - like myocytes expressed a - sarcomeric actin and tropo-nin T.Discussion:Now, specific surface markers in bone marrow mesenchymal stem cells have not been discovered, which is difficult to identify and purify bone marrow mesenchymal stem cells. It is well known that bone marrow mesenchymal stem cells expresse SH2, SH3> CD29, CD44, CD71, CD90, CD120a, CD124, CD14, CD34, and CD45 is negtive. We cultured and purified bone marrow mesenchymal stem cells by adherence method. These stem cells were in good condition and easy to be induced in vitro.Conclusions: We verified that the first generation of bone marrow mesenchymal stem cells could be induced to differentiate to cardiac - like myocytes. This provided theory foundation and technique method for cardiomyocytes induced in vitro and applied in clinic.Key words: bone marrow mesenchymal stem cells; induce; cardiac - like myocytes(H) Changes of intraceUular calcium concentration in cardiac - like myocytesObjective; To study the effects of verapamil, endothelin on [ Ca2+ ]{ in cardiac - like myocytes derived of bone marrow mesenchymal stem cells. Methods:(1) Bone marrow mesenchymal stem cells and cardiac myocytes were cultured by primary method;(2) Bone marrow mesenchymal stem cells differentiated to cardiac - like myocytes by 5 - azacytidine induction agent;(3) We divided our test into three groups,including the first generation of bone marrow mesenchymal stem cells, cardiac - like myocytes and cardiac myocytes;(4) Hie alteration of [ Ca2+ ], affected by verapamil, endothelin was ob-更多还原