骨髓间充质干细胞体外心肌样细胞分化过程中结构蛋白发育的实验研究

发布时间：2018-07-09 12:54

本文选题：骨髓间充质干细胞 + 心肌细胞　；参考：《重庆医科大学》2005年硕士论文

【摘要】：背景:随着干细胞移植治疗心脏疾病的研究不断深入,骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMMSCs)由于具有易获得、易扩增、几乎无移植物抗宿主反应、无伦理道德限制等优点,成为目前研究的热点。体外诱导和体内移植方面均取得了令人鼓舞的成就[1-3],本课题组前期研究发现:BMMSCs 体外经 5-氮杂胞苷(5-azacytidine,5-aza)诱导后能表达心脏发育早期转录因子 GATA4,NKx2.5,骨骼肌增强因子-2C和转化生长因子-β等[2]; BMMSCs移植入心肌病模型鼠体内,病鼠心功能明显改善。但在对 BMMSCs 移植后心功 [3]能改善的机理探讨中还存在很大的争议,争议的焦点在于移植后的BMMSCs 能否分化成具有正常心肌细胞结构并具备主动收缩功能的细胞。由于无法直接在宿主心肌内观察移植细胞的收缩,就现阶段的技术水平而言,只有从侧面去间接说明这一点。即建立体外诱导模型,阻断心肌特异结构蛋白肌联蛋白 titin 基因的表达,以此来观察诱导后的BMMSCs是否还能分化成具有相应结构及具有收缩功能的心肌样细胞。目的:构建能干扰正常心肌细胞结构蛋白 titin 的重组质粒,将其转染入经 5-aza 诱导后的 BMMSCs,观察 BMMSCs 能否进一步分化成心重庆医科大学硕士研究生学位论文 4 肌样细胞并表达 titin、肌球蛋白重链(myosin heavy chain, MHC)、肌动蛋白 actin、心肌肌钙蛋白 T(cardiac troponin T,cTnT)等心肌特异蛋白标志物,同时就 titin 在心肌结构蛋白发育和肌小节组装过程中所起的作用进行初步探讨。材料和方法: 1. 分别构建携带针对 Wistar 大鼠 titinN2B 区设计的 siRNA 重组质粒 pSITITIN 和携带针对人β-actin 设计的 siRNA 重组质粒 pSIACTIN,利用脂质体将其转染入体外分离培养 1w 的正常新生 Wistar 大鼠心肌细胞中,免疫荧光检测 titin 的表达情况。 2. 将上述重组质粒分别转染入经 5-aza 诱导后 2w 的 BMMSCs,免疫荧光检测 titin 的表达情况。 3. 免疫荧光检测 titin 被阻断后 MHC、actin、cTnT 的表达情况。结果: 1. 酶切和 DNA 序列测定证实重组质粒构建成功。 2. 重组质粒 pSITITIN 转染入正常新生鼠心肌细胞后,titin 的表达减弱(P0.001)。而对照 pSIACTIN 转染入正常新生鼠心肌细胞后, titin 的表达无明显变化(P0.05)。 3. 重组质粒 pSITITIN 转染入经 5-aza 诱导后 2w 的 BMMSCs 后, titin 的表达减弱。而对照 pSIACTIN 转染入经 5-aza 诱导后 2w 的 BMMSCs 后,titin 的表达无明显变化。titin 的表达被阻断后,MHC、 actin 的表达无明显变化,cTnT 的表达减弱。结论:
[Abstract]:Background: with the development of stem cell transplantation in the treatment of heart disease, bone marrow mesenchymal stem cells (BMMSCs) have the advantages of easy to obtain, easy to expand, almost no graft versus host reaction, no ethical restrictions and so on. It has become the hot spot of current research. Encouraging achievements have been made in both in vitro induction and in vivo transplantation [1-3]. Our previous studies have found that in vitro, 5 azacytidine 5-aza can express GATA4NKx2.5, skeletal muscle enhancer factor -2C, a transcription factor GATA4NKx2.5and skeletal muscle enhancer factor -2C, after induction by 5-azacytidineine 5-aza in vitro. And transforming growth factor- 尾 [2]; BMMSCs were transplanted into cardiomyopathy model mice, The heart function of the diseased rats was improved obviously. However, there is still a lot of controversy on the mechanism of cardiac function [3] improvement after BMMSCs transplantation, the focus of which is whether BMMSCs can differentiate into cells with normal cardiomyocyte structure and active contractile function. Since it is not possible to observe the contraction of transplanted cells directly in the host myocardium, this can only be explained indirectly from the side in view of the present technical level. That is to establish an in vitro induction model to block the expression of myocyte specific structural protein (titin) gene in order to observe whether the induced BMMSCs can also differentiate into cardiomyoid cells with corresponding structure and contractile function. Objective: to construct a recombinant plasmid that interferes with the structural protein titin of normal cardiomyocytes. The BMMSCs were transfected into BMMSCs induced by 5-aza, and the differentiation of BMMSCs into a master's degree thesis of Chongqing Medical University was observed. 4 myoid cells expressed titin, myosin heavy chain (myosin heavy chain, MHC, actin actinin, Cardiac troponin T (cardiac troponin TnT) and other myocardial specific protein markers, At the same time, the role of titin in the development of myocardial structural protein and the assembly process of muscle section was discussed. Materials and methods: 1. Construction of siRNA recombinant plasmid pSITIN designed for titinN2B region of Wistar rat and siRNA recombinant plasmid designed for human 尾 -actin PSIACTIN was transfected with liposome into normal neonatal Wistar rat cardiomyocytes cultured for 1 week in vitro. Immunofluorescence detection of titin expression. 2. The recombinant plasmids were transfected into BMMSCs at 2 weeks after induction by 5-aza, and the expression of titin was detected by immunofluorescence. Immunofluorescence was used to detect the expression of cTnT after titin was blocked. Results: 1. Restriction endonuclease digestion and DNA sequencing confirmed that the recombinant plasmid was successfully constructed. After transfection of recombinant plasmid pSITITIN into normal neonatal rat cardiomyocytes, the expression of titin was weakened (P0.001). The expression of titin did not change significantly after transfection of control pSIACTIN into normal neonatal rat cardiomyocytes (P0.05). The expression of titin decreased after transfection of recombinant plasmid pSITITIN into BMMSCs at 2 weeks after induction by 5-aza. However, there was no significant change in the expression of titin after transfection of pSIACTIN into BMMSCs at 2 weeks after transfection with pSIACTIN, and the expression of titin was blocked after the transfection of pSIACTIN into BMMSCs. There was no significant change in the expression of actin and the expression of cTnT was decreased. Conclusion:
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R329

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