HIV-1来源的慢病毒载体介导绿色荧光蛋白基因转染血管内皮祖细胞的实验研究
发布时间：2019-03-19 09:56
【摘要】： 目的：探索人脐带血血管内皮祖细胞(EPCs)分离、培养、扩增和鉴定的方法；探讨HIV-1来源的慢病毒载体介导绿色荧光蛋白(GFP)基因转染血管内皮祖细胞的可行性和方法。方法：采用密度梯度离心法和贴壁细胞分离法相结合,分离和筛选出人脐带血内皮祖细胞,用EGM-2培养基培养、扩增,采用细胞免疫荧光染色和流式细胞仪检测内皮祖细胞表面标记CD133、CD34、VEGFR-2的方法鉴定实验培养的内皮祖细胞。取生长活跃的EPCs,以HIV-1来源的慢病毒为载体,以绿色荧光蛋白(GFP)基因为目的基因转染EPCs。感染复数MOI分别为：1：10,1：50,1：100,MTT法检测不同病毒滴度时细胞增殖情况,计算转染率；取1：50转染组与未转染的空白组进行对照,于转染后1-10天观察两组细胞增殖情况。用t检验、卡方检验、方差分析等方法对实验结果进行统计学分析,P0.05有统计学意义。结果：单个核细胞经EGM-2培养基培养一周后,即分化成CD133、KDR和CD34阳性的EPCs。1:10转染和1：50转染后细胞增殖无明显影响,细胞显绿色荧光。1：10比率转染后48小时绿色荧光细胞比率仅(35.2±2.75)%；1：50比率转染后48小时荧光显微镜下观察转染率为(87.4±1.89)%,转染率高于1：10组(P0.05)。1：50转染后的细胞继续培养并与未转染组对照,显示两组细胞生长曲线无明显差异(P0.05)。1：100比率转染后,细胞的增殖处于停滞状态,镜下可见细胞坏死形成碎片。结论：1、采用密度梯度离心法和贴壁细胞分离法相结合,经EGM-2培养基培养扩增可从脐带血获得较高纯度和较大数量的EPCs;2、采用HIV-1来源的慢病毒载体介导绿色荧光蛋白基因转染标记血管内皮祖细胞是可行的；以MOI=1：50进行转染,对细胞生长影响小,转染效率高。
[Abstract]:Objective: To explore the methods of isolation, culture, amplification and identification of human umbilical cord blood vessel endothelial progenitor cells (EPCs), and to explore the feasibility and method of the lentiviral vector-mediated green fluorescent protein (GFP) gene from HIV-1. Methods: The human umbilical cord blood endothelial progenitor cells were isolated and screened by density gradient centrifugation and adherent cell separation. The endothelial progenitor cells were cultured and amplified with EGM-2 medium, and the surface markers of the endothelial progenitor cells were detected by immunofluorescence staining and flow cytometry. VEGFR-2 was used to identify the cultured endothelial progenitor cells. EPCs were transfected with the lentivirus of HIV-1, and the EPCs were transfected with the green fluorescent protein (GFP) gene as the target gene. The proliferation of cells was detected by MTT method in 1:10,1:50,1:100 and MTT method, and the proliferation of two groups was observed 1-10 days after transfection. The results of the experiment were statistically analyzed by t-test, chi-square test and variance analysis. Results: One week after the single core cell was cultured with EGM-2 medium, the cells were differentiated into CD133, KDR and CD34-positive EPCs.1:10 was transfected and the cell proliferation was not significantly affected after 1:50 transfection. The ratio of green fluorescent cells was only 35.2 (2.75)% after 1:10 ratio transfection. The transfection rate was 87.4% 1.89% and the transfection rate was higher than that of 1:10 (P0.05). The proliferation of the cells was in a dead state, and the cells were seen to form fragments under the microscope. Conclusion:1. High-purity and large number of EPCs can be obtained from umbilical cord blood by the combination of density gradient centrifugation and adherent cell separation. Transfection of the green fluorescent protein gene with a lentiviral vector from the HIV-1 source is feasible; the transfection is performed with MOI = 1:50, the effect on the growth of the cells is small, and the transfection efficiency is high.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346
本文编号：2443419
[Abstract]:Objective: To explore the methods of isolation, culture, amplification and identification of human umbilical cord blood vessel endothelial progenitor cells (EPCs), and to explore the feasibility and method of the lentiviral vector-mediated green fluorescent protein (GFP) gene from HIV-1. Methods: The human umbilical cord blood endothelial progenitor cells were isolated and screened by density gradient centrifugation and adherent cell separation. The endothelial progenitor cells were cultured and amplified with EGM-2 medium, and the surface markers of the endothelial progenitor cells were detected by immunofluorescence staining and flow cytometry. VEGFR-2 was used to identify the cultured endothelial progenitor cells. EPCs were transfected with the lentivirus of HIV-1, and the EPCs were transfected with the green fluorescent protein (GFP) gene as the target gene. The proliferation of cells was detected by MTT method in 1:10,1:50,1:100 and MTT method, and the proliferation of two groups was observed 1-10 days after transfection. The results of the experiment were statistically analyzed by t-test, chi-square test and variance analysis. Results: One week after the single core cell was cultured with EGM-2 medium, the cells were differentiated into CD133, KDR and CD34-positive EPCs.1:10 was transfected and the cell proliferation was not significantly affected after 1:50 transfection. The ratio of green fluorescent cells was only 35.2 (2.75)% after 1:10 ratio transfection. The transfection rate was 87.4% 1.89% and the transfection rate was higher than that of 1:10 (P0.05). The proliferation of the cells was in a dead state, and the cells were seen to form fragments under the microscope. Conclusion:1. High-purity and large number of EPCs can be obtained from umbilical cord blood by the combination of density gradient centrifugation and adherent cell separation. Transfection of the green fluorescent protein gene with a lentiviral vector from the HIV-1 source is feasible; the transfection is performed with MOI = 1:50, the effect on the growth of the cells is small, and the transfection efficiency is high.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346
【参考文献】
相关期刊论文 前1条
1 李传勇;李晓强;姜坤;孟庆友;于小滨;雷锋锐;;绿色荧光蛋白基因体外转染骨髓源性血管内皮祖细胞[J];中国组织工程研究与临床康复;2009年14期
，本文编号：2443419
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2443419.html
最近更新
教材专著
