【作者】 李松南；

【导师】 冯义柏； 王祥； 曾秋棠；

【作者基本信息】 华中科技大学 ， 内科学， 2010， 博士

【摘要】 目的：构建含有增强型绿色荧光蛋白(EGFP)基因和大鼠缝隙连接蛋白43基因(Cx43)的重组慢病毒,并进行包装、纯化。方法：用RT-PCR技术获得大鼠的Cx43基因,并将其连接到含有EGFP基因的慢病毒表达载体pGC-FU中,重组获得慢病毒载体质粒pGC-FU-Cx43,采用限制性内切酶酶切法和DNA测序鉴定构建的载体是否正确。将pGC-FU-Cx43与辅助包装质粒载体(pHelper1.0、pHelper2.0)一起共转染293T细胞,包装生产病毒并测定其滴度。结果：酶切证实目的基因Cx43已经定向连入目的载体,测序结果显示,所构建的pGC-FU-Cx43重组慢病毒质粒序列与目标序列完全一致。最终获得的病毒滴度为2x109 TU/ml。结论：成功构建并包装获得较高滴度的含有EGFP基因和目的基因Cx43的重组慢病毒pGC-FU-Cx43,为后续的体外及在体研究奠定基础。目的：分离、培养、扩增大鼠BMSC,并转染重组慢病毒pGC-FU-Cx43,观察BMSC中Cx43的表达及细胞间通讯功能的变化。方法：用密度梯度离心法结合贴壁培养法分离、纯化培养大鼠BMSCs,选取P3代细胞,以流式细胞仪鉴定其表面标记。重组慢病毒pGC-FU-Cx43转染P3代细胞,荧光显微镜观察EGFP的表达,以Western blot法检测转染后BMSC中Cx43的蛋白含量,以荧光漂白恢复技术(FRAP)检测转染后细胞间通讯功能的变化。结果：经分离、纯化得到细胞纯度较高的BMSC; pGC-FU-Cx43转染BMSC后,可见大量EGFP表达,目的蛋白Cx43的表达量明显增加,且细胞间通讯功能增强。结论：密度梯度离心法结合贴壁培养法可以有效地分离纯化BMSC。重组慢病毒pGC-FU-Cx43成功转染BMSC,目的蛋白的表达增加且细胞间通讯功能增强。目的：建立大鼠心肌梗死模型,观察移植过表达Cx43的BMSC对心梗后心衰的治疗作用。方法：120只SD大鼠随机分为四组,Sham组：假手术组,n=30; DMEM/F12组,注射DMEM/F12, n=30; EGFP组：移植转染EGFP基因的BMSC,n=30; Cx43组：移植转染Cx43基因的BMSC, n=30。采用结扎前降支的方法,建立心肌梗死模型,于梗死后30min,行细胞移植。移植后四周,超声心动图检测心脏功能并取心脏组织检测各项指标。在Langendorff离体心脏灌注系统下行心脏电生理检查,诱发心动过速。结果：与DMEM/F12组相比,EGFP组和Cx43组心功能明显改善,心肌梗死面积缩小,胶原纤维含量降低。尤以Cx43组上述心脏重构指标改善更明显。RT-PCR和Western blot显示DMEM/F12和EGFP组大鼠心肌组织Cx43表达明显降低；移植Cx43转染BMSC后,Cx43表达量增加。激光共聚焦显示EGFP组和Cx43组有BMSC存活,并可见BMSC与宿主心肌组织间有缝隙连接形成。电生理检查显示,DMEM/F12组室性心律失常的诱发率明显增高,EGFP组的心律失常的诱发率与DMEM/F12组无明显差异,Cx43组的心律失常诱发率较DMEM/F12组和EGFP组明显降低。结论：同种异体BMSC移植延缓心室重塑,改善心脏功能,且对可诱导的心律失常无明显影响；过表达Cx43的BMSC移植改善心肌梗死后心衰的效果更为明显,且可以明显降低可诱导的心律失常的发生。这可能是因为Cx43基因过表达可以改善缝隙连接重构,增加BMSC与宿主心肌细胞的电机械耦联。更多还原

【Abstract】 Objective:To construct, package and purify the recombinant lentivirus carrying green fluorescent protein (GFP) gene and the rat connexin 43 gene (Cx43).Methods:The rat Cx43 gene was amplified by RT-PCR and connected to the lentiviral expression vector pGC-FU, which is carrying the EGFP gene, to construct the lentiviral vector plasmid pGC-FU-Cx43. Restriction digestion analysis and DNA sequencing was used to verified the correction. The viral particles were generated by cotransfection of 293T cells with the pGC-FU-Cx43 and two packaging vector (pHelper1.0, pHelper2.0) and the virus titer was determined by counting the percentage of GFP positive cells.Result:The insert orientation of the Cx43 gene in the lentiviral vector plasmid pGC-FU-Cx43 was verified by restriction digestion analysis.Target Cx43 sequences was confirmed by the bulk sequencing. The final titer obtained was 2x109TU/ml.Conclusion:The pGC-FU-Cx43 recombinant lentivirus carrying GFP gene and Cx43 gene with high viral titer was constructed and packaged successfully and would pave the way for the further study in vitro and in vivo. Objective:Transfecting pGC-FU-Cx43 recombinant lentiviral into amplified rat BMSC to observe the Cx43 expression level and the function change of intercellular communication in BMSC.Methods:Rat BMSCs were isolated and purified by density gradient centrifugation and adherent cell culture. Flow cytometry was used to identify the surface markers in P3 cells. After transfection of pGC-FU-Cx43 recombinant lentiviral into P3 cells, the expression of EGFP was observed by fluorescence microscope, the level of Cx43 protein in BMSC was determined by western blot assay and the changes of cell communication was measured by FRAP assay.Results:High purity BMSC was obtained after isolation and purification. Increased EGFP expression was observed, expression level of Cx43 protein was significantly increased, and communication between cells was remarkably enhanced in pGC-FU-Cx43 transfected BMSC.Conclusion:The combination of density gradient centrifugation and adherent cell culture method can isolate BMSC efficiently. pGC-FU-Cx43 recombinant lentiviral was transfected into BMSC successfully to increase the expression of target protein and enhance the communication between cells. Objective:The goal of the present study was to explore the effect of transplantation of bone marrow mesenchymal stem cells over-expressing Cx43 on heart failure in post-infarction rats.Methods:120 rats were randomly divided into four groups:sham group consisting of 30 rats, DMEM/F12 group consisting of 30 rats injected with DMEM/F12, EGFP group consisting of 30 rats transplanted EGFP transfected BMSC, and Cx43 group consisting of 30 rats transplanted Cx43 transfected BMSC. Myocardial infarction models were built by ligating the anterior descending branch and then the cells were transplanted after 30 minutes. The rats were sacrificed and heart function was measured by echocardiography after 4 weeks. Cardiac electrophysiology examination was operated in the Langendorff isolated heart perfusion system and then tachycardia arrhythmia was induced.Results:Compared with DMEM/F12 group, heart function was improved significantly, myocardial infarct size reduced and collagen fiber content was decreased significantly in EGFP group and in Cx43 group especially. After myocardial infarction, expression of Cx43 in myocardial tissue reduced significantly, but increased after transplantation of BMSC over-expressing Cx43. Survival BMSC and the formation of gap junction between BMSC and the host myocardium could be found using confocal laser both in EGFP group and Cx43 group. Electrophysiological examination showed that arrhythmia prone to be induced in DMEM/F12 group. There was no significant difference in EGFP group and DMEM/F12 group. The rate of arrhythmia induced in Cx43 group was lower than that in DMEM/F12 group and EGFP group.Conclusion:Transplantation of allogeneic BMSC attenuated ventricular remodeling, improved cardiac function, and had no significant impact on induced arrhythmia. Furthermore, heart function after myocardial infarction could be improved significantly. and the rate of arrhythmia induced could be reduced significantly in Cx43 group transplanted BMSC which over-expressed Cx43. This effect may result from the overexpression of Cx43 gene which can improve gap junction remodeling, and increase electro-mechanical coupling between BMSC and the host myocardial cells.更多还原