携带重组人胰岛素基因的慢病毒载体转染人脐带间充质干细胞的实验研究

发布时间：2018-07-07 20:45

  本文选题：Insulin/EGFP基因 + 转染　； 参考：《兰州大学》2010年硕士论文

【摘要】： 目的：1,探讨人脐带间充质干细胞(human umbilical cord mesenchymal stem cells hUCMSCs)体外分离、培养、纯化条件,并对hUCMSCs的生物学特性进行研究；2,构建含人重组胰岛素(insulin)与增强型绿色荧光蛋白(EGFP)基因慢病毒表达载体pLenti6.3-insulin-IRES-EGFP,并进行慢病毒颗粒的包装,纯化,浓缩及滴度测定；3,以GFP作为报告基因,筛选慢病毒转染人脐带间充质干细胞最适MOI值,为进一步进行携带外源基因的转染探索条件；4,研究携带重组人胰岛素慢病毒颗粒转染人脐带间充质干细胞后,基因及蛋白水平的表达情况,为进一步进行体内回植提供研究基础。 方法：1,将剔除动脉和静脉的新鲜人脐带组织剪成小块培养(1mm×1mm×1mm),培养1-2周后得到贴壁细胞,倒置相差显微镜下观察细胞形态,细胞计数绘制细胞生长曲线；应用流式细胞仪鉴定细胞表面标志；化学染色检测体外诱导成脂和成骨分化的能力；2,应用PCR方法扩增获得insulin基因序列,在目的基因上游加上BamHI,下游加上AscI两个酶切位点；将PCR产物进行T载体克隆,转化入感受态DH5α细胞中,通过筛选获得重组质粒pMD18T-insulin,测序分析选定序列正确的克隆提取质粒；用限制性内切酶分别酶切pMD18T-insulin和pLenti6.3-IRES-EGFP质粒,将insulin基因片段和pLenti6.3-IRES-EGFP载体连接,转化入感受态DH5α细胞中,通过筛选获得慢病毒表达载体pLenti6.3-insulin-IRES-EGFP,并进行测序。中量抽提慢病毒载体,将慢病毒表达载体转染293T细胞,包装病毒,纯化及浓缩并测定病毒滴度。3,以EGFP作为报告基因,分别以MOI值0,1,3,5,7,10,15,20转染细胞,观察48小时,72小时,96小时,荧光表达情况,筛选慢病毒转染人脐带间充质干细胞最适转染时间及MOI值；4,转染人脐带间充质干细胞,应用qPCR及RT-PCR方法检测重组人胰岛素基因表达情况,应用westen bloting方法检测胰岛素蛋白表达情况。 结果：1,体外培养一周,细胞从组织块中爬出；细胞传代培养至第10代,无明显形态及增值能力改变；细胞阳性表达CD44, CD106,而CD34, HLA-DR表达阴性。体外诱导实验证实,hUCMSCs具有成脂及成骨分化的能力。2,聚合酶链反应获得长度347bp带有BamHI和AscI两个酶切位点序列的目的基因,经与克隆载体pMD18-T载体和慢病毒表达载体pLenti6.3-IRES-EGFP连接,构建慢病毒表达载体pLenti6.3-insulin-IRES-EGFP。成功包装病毒,纯化及浓缩并测定病毒滴度。3,重组慢病毒载体具有较高的转染效率,当转染复数(MOI)值是10时,转染效率最高可达到90%。应用qPCR及RT-PCR方法检测重组人胰岛素基因表达情况,证实基因水平的表达量,转染组为阳性,未转染组为阴性。应用westen bloting方法检测胰岛素蛋白表达情况,证实转染组细胞及上清表达阳性；未转染组表达均为阴性。 结论：1,hUCMSCs能在体外培养、扩增并具骨髓间充质干细胞相似的生物学特性；2,成功构建慢病毒表达载体pLenti6.3-insulin-IRES-EGFP并包装病毒颗粒纯化及浓缩并测定病毒滴度；3,筛选出最适MOI值为10,携带重组人胰岛素基因慢病毒颗粒转染hUCMSCs后,其在基因及蛋白水平均阳性表达胰岛素。
[Abstract]:Objective: 1, to study the isolation, culture and purification of human umbilical cord mesenchymal stem cells (human umbilical cord mesenchymal stem cells hUCMSCs) in vitro, and to study the biological characteristics of hUCMSCs; 2, to construct a lentivirus expression vector containing recombinant human recombinant insulin (insulin) and enhanced green fluorescein (EGFP) gene. -EGFP, and carry out the packaging, purification, concentration and titer determination of lentivirus particles; 3, using GFP as the reporter gene, screening the optimal MOI value of lentivirus transfected human umbilical cord mesenchymal stem cells to further carry out transfection conditions with exogenous gene, and 4, study the transfection of human umbilical cord mesenchymal stem cells with recombinant human insulin lentivirus particles. The expression level of gene and protein will provide a basis for further research on in vivo replantation.
Methods: 1, the fresh human umbilical cord tissues removed from the arteries and veins were cut into small mass culture (1mm x 1mm x 1mm). After 1-2 weeks of culture, the adherent cells were obtained. The cell morphology was observed under the inverted phase contrast microscope and the cell growth curve was plotted by the cell count; the cell surface markers were identified by the flow cytometry; the chemical staining was used to detect the formation of lipid and formation in vitro. The ability of bone differentiation: 2, the insulin gene sequence was amplified by PCR method, the upstream of the target gene was added with BamHI, and the downstream was added with the AscI two enzyme cutting sites, and the PCR product was cloned by T vector and transformed into the receptor DH5 alpha cells. The recombinant plasmid pMD18T-insulin was obtained by screening, and the correct cloning extract was sequenced and analyzed. PMD18T-insulin and pLenti6.3-IRES-EGFP plasmids were cut by restriction endonuclease, the insulin gene fragment and pLenti6.3-IRES-EGFP vector were linked and transformed into the receptive DH5 alpha cells. The Lentivirus Expression Vector pLenti6.3-insulin-IRES-EGFP was screened and sequenced. The lentivirus vector was extracted from the lentivirus vector, and the lentivirus vector was extracted. The vector transfected 293T cells, packed the virus, purified and condensed the virus titer.3, and transfected EGFP as the reporter gene, transfected cells with MOI value 0,1,3,5,7,10,15,20 respectively, observed 48 hours, 72 hours, 96 hours, fluorescence expression, screening the optimal transfection time and MOI value of human umbilical cord mesenchymal stem cells transfected by lentivirus; 4, transfection of human umbilical cord The expression of recombinant human insulin gene was detected by qPCR and RT-PCR, and the expression of Insulin protein was detected by westen bloting.
1, 1, in vitro culture, the cells crawled out of the tissue block, and the cells were cultured to tenth generations without obvious morphologic and value-added changes. The positive expression of CD44, CD106, and CD34, HLA-DR expression was negative. In vitro induction experiments confirmed that hUCMSCs has the ability of lipid and osteogenic differentiation.2, and the polymerase chain reaction obtained the length 347bp The target genes of the two enzyme cutting sites of BamHI and AscI were linked with the cloned carrier pMD18-T vector and the Lentivirus Expression Vector pLenti6.3-IRES-EGFP to construct the Lentivirus Expression Vector pLenti6.3-insulin-IRES-EGFP. to successfully package the virus, purify and concentrate and determine the virus titer.3. The recombinant lentivirus carrier has high transfection efficiency. The transfection complex (MOI) value was 10, the highest transfection efficiency could reach the 90%. application qPCR and RT-PCR method to detect the recombinant human insulin gene expression. The expression of gene level was confirmed, the transfection group was positive and the untransfected group was negative. The expression of Insulin protein was detected by westen bloting method, and the expression of cell and supernatant in the transfected group was confirmed. The expression of untransfected group was negative.
Conclusion: 1, hUCMSCs can be cultured in vitro, amplification and similar biological characteristics of bone marrow mesenchymal stem cells. 2, successful construction of Lentivirus Expression Vector pLenti6.3-insulin-IRES-EGFP and packaging virus particles to purify and concentrate and determine virus titer; 3, the optimum MOI value is 10, carrying recombinant human insulin gene lentivirus particles turn. After hUCMSCs staining, they expressed insulin at both gene and protein levels.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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