慢病毒介导HCN2基因转染骨髓间充质干细胞的实验研究

发布时间：2018-01-13 03:12

本文关键词：慢病毒介导HCN2基因转染骨髓间充质干细胞的实验研究　出处：《泸州医学院》2011年硕士论文　论文类型：学位论文

【摘要】：目的：探讨通过超极化激活的环核苷酸门控的阳离子通道2 (hyperpolarization-activated cyclic nucleotide-gated cation channel, HCN2)基因稳定转染大鼠骨髓间充质干细胞(bone mesenchymal stem cells, BMSCs)的可行性。探求HCN2转染的最佳感染复数(multiplication of infection, MOI),为进一步研究起搏电流的电生理学特性提供前期实验基础。方法：1.骨髓间充质干细胞的分离、培养和鉴定：采用改良全骨髓贴壁培养的方法分离纯化BMSCs,培养于含20%FBS的DMEM培养基,并传代培养；观察培养细胞的生长状态；通过CD34、CD4、CD45、CD90免疫组织化学和流式细胞技术进行细胞鉴定。应用体外成骨诱导,观察BMSCs向成骨细胞分化的功能特性。2.对购得的HCN2质粒进行扩增和纯化,对纯化质粒行蛋白电泳和基因测序,鉴定HCN2基因。3.重组慢病毒(Feline immunodeficiency virus,FIV)载体HCN2的构建及其鉴定：将鉴定正确的重组慢病毒进行扩增、纯化和滴度测定。以增强型绿色荧光蛋白(enhanced green fluorescent protein, EGFP)基因为信号基因,确定最佳感染复数；观察FIV-EGFP转染BMSCs表达强度随时间的变化情况；流式细胞检测FIV-HCN2转染BMSCs的细胞活力的变化。结果：1.改良全骨髓贴壁分离法法获得了高纯度(90%)和高活力(90%)的BMSCs,经形态学观察和免疫组织化学检测,显示所培养细胞呈梭形、纺锤形和多角形,免疫化学染色CD44、CD90阳性,CD34阴性,符合BMSCs的形态和表面标记；流式细胞仪分析,CD44、CD90阳性细胞分别占99.5%、90.9%。体外成骨诱导BMSCs,茜红素染色可见大量钙结节后,说明BMSCs具有向成骨细胞分化能力。2. HCN2的质粒扩增方法可靠,扩增的质粒经双酶切证实基因片段大小正常,目的基因测序结果证实与GeneBank上基本一致。3.重组慢病毒HCN2的构建。使用包装病毒上清液做慢病毒滴度检测未见荧光；用上清液感染BMSCs,24h、48h、72h也未检测出有意义的绿色荧光。结论：1.采用改良全骨髓贴壁法,可分离获得较高纯度、高活力的BMSCs。BMSCs体外增殖速度快,可短期内达到实验所需要细胞数量。2.含有HCN2的质粒扩增方法可靠,扩增的质粒经双酶切证实基因片段大小正常,目的基因测序结果证实与GeneBank上基本一致。3.重组慢病毒HCN2转染干细胞有待进一步研究。
[Abstract]:Objective: to investigate the cationic channel 2 (cationic channel 2) activated by hyperpolarization-activated cyclic nucleotides (cationic acid). Hyperpolarization-activated cyclic nucleotide-gated cation channel. HCN2) gene was stably transfected into rat bone marrow mesenchymal stem cells (mesenchymal stem cells). To find out the best infection number of HCN2 transfection, multiplex of infection, moi). In order to further study the electrophysiological characteristics of pacemaker current, we provide a preliminary experimental basis. Methods: 1. Isolation of bone marrow mesenchymal stem cells. Culture and identification: BMSCs were isolated and purified by modified whole bone marrow adherent culture method and cultured on DMEM medium containing 20s. The growth state of cultured cells was observed. The cells were identified by immunohistochemistry and flow cytometry (FCM). Osteogenesis was induced by osteogenesis in vitro. To observe the functional characteristics of BMSCs differentiation into osteoblasts. 2. To amplify and purify the acquired HCN2 plasmid, and to sequence the purified plasmid by line protein electrophoresis and gene sequencing. HCN2 gene. 3.Recombinant lentivirus Feline immunodeficiency virus. Construction and identification of HCN2 vector: the correct recombinant lentivirus was amplified. Purification and titer determination. Enhanced green fluorescent protein (EGFP) gene was used as signal gene. Determine the optimal complex number of infections; The expression intensity of FIV-EGFP transfected BMSCs was observed with time. Flow cytometry was used to detect the changes of cell viability in BMSCs transfected with FIV-HCN2. Results: 1. The modified whole bone marrow adherent separation method obtained high purity and high activity. BMSCs. Morphological observation and immunohistochemical examination showed that the cultured cells were fusiform, spindle-shaped and polygonal, and immunocytochemical staining was positive for CD44 + CD90 and negative for CD34. Conformed to the morphology and surface marker of BMSCs; Flow cytometry analysis showed that CD44 + CD90 positive cells accounted for 99. 5% and 99. 9% respectively. A large number of calcium nodules were observed after osteogenesis induced by BMSCs in vitro. The results showed that BMSCs had the ability to differentiate into osteoblasts. 2. The method of plasmid amplification of HCN2 was reliable, and the amplified plasmid was confirmed to be normal by double enzyme digestion. Objective the result of gene sequencing confirmed that the construction of recombinant lentivirus HCN2 was basically consistent with that on GeneBank. No fluorescence was found in the detection of lentivirus titer using the supernatant of packaging virus. Significant green fluorescence could not be detected after infection with supernatant BMSCs1 for 24 h or 48 h. Conclusion: 1. High purity can be obtained by modified whole bone marrow adherent method. BMSCs.BMSCs with high activity can proliferate rapidly in vitro and can reach the number of cells needed in the experiment in a short time. The method of plasmid amplification containing HCN2 is reliable. The amplified plasmid was confirmed to be of normal size by double enzyme digestion. Objective Gene sequencing confirmed that it was basically the same as that on GeneBank. The recombinant lentivirus HCN2 transfected stem cells needed to be further studied.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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