分枝状PEI在不同细胞系转染效率和细胞毒性间的关系

发布时间：2018-01-26 19:53

本文关键词： PEI 基因转染细胞毒性荧光素酶内吞作用 caveolin-1　出处：《新乡医学院》2015年硕士论文　论文类型：学位论文

【摘要】：背景基因治疗能将外源正常基因导入靶细胞并使之有效表达,从而达到治疗目的,是一种新兴的治疗方法。聚乙烯亚胺(polyethylenimine,PEI)是一种高效的非病毒性基因转染载体,其枝状复合物尤其是25 kDa的分支状PEI有较高的基因转染效率,它在基因传递领域的应用非常广泛。同时PEI对细胞也有毒副作用,它的毒性与相对分子质量(Mr)有关,不同Mr或异构体的PEI,在体内介导基因转染的效率和细胞毒性截然不同。本课题将在不同的细胞系中研究其转染效率和毒性间的关系,并尝试探讨其分子机制。目的本研究拟确定PEI对不同细胞的转染效率和细胞毒性之间的内在联系,同时优化PEI基因转染的条件,确定转染时机及最优用量,并分析PEI在BMSC细胞中可能的分子机制。方法1.PEI在不同细胞系中基因转染效率和细胞毒性之间的关系不同哺乳动物细胞系293T、Hela和BMSC细胞培养后,悬浮转染pEGFP质粒与PEI的复合物,分别孵育24 h和48 h,利用荧光细胞计数法分析转染效率；悬浮转染pLuc(luciferase基因来自Renilla)质粒与PEI的复合物,利用分光光度计分析各细胞的荧光素酶活性；采用MTT比色法分析PEI对293T、Hela和BMSC细胞的毒性,Western印迹检测细胞毒性标记物一水解的(cleaved) PARP和水解的caspase-3,进而得出转染效率和毒性之间的相关性。2.PEI介导的BMSC细胞转染可能的分子机制构建caveolin-1过表达质粒,经PEI转染BMSC细胞,Western印迹法检测其对转染效率和毒性的影响。结果1.PEI对293T、Hela和BMSC3种细胞的细胞毒性和转染效率呈正相关,即转染效率越高,PEI的细胞毒性亦越高：①荧光素酶活性检测结果显示PEI:DNA(μg:μg)的比率为2 (4μg PEI:2 μg DNA)时,转染效率最高。②不同时间点转染的结果表明转染时间之间的差异不显著。③ pEGFP表达质粒转染293T、Hela和BMSC细胞的转染效率结果是：92.1土4.5%,29.2+3.4%和21.5±2.1%。④荧光素酶报告基因转染293T、Hela和BMSC细胞后荧光素酶活性分别是：2.873×108、3.35×107、1.94×107RLU/mg protein。⑤MTT法结果显示PEI转染pLuc DNA后死细胞百分比分别是66.1±2.1%,55.9+2.5%和45.1%±6.7%,这与Western印迹结果一致,即PEI的细胞毒性：293THelaBMSC细胞。2.Caveolin-1过表达时可提高对BMSC细胞的转染效率和毒性。结论1.PEI在各种细胞系的转染效率与PEI的毒性呈正相关性。2.Caveolin-1通过胞吞作用参与PEI介导的细胞转染,可以PEI作为基因载体用于基因治疗。
[Abstract]:Background Gene therapy can transfer exogenous normal genes into target cells and express them effectively. It is a new treatment method, polyethylene imine polyethylenimine. Pei) is a highly efficient non-viral gene transfection vector, and its dendritic complex, especially the 25 kDa branched PEI, has higher gene transfection efficiency. It is widely used in the field of gene transfer, and PEI also has toxic side effects on cells. Its toxicity is related to the relative molecular weight (Mr), different Mr or PEI of isomers. The efficiency of gene transfection in vivo is very different from that of cytotoxicity. The relationship between transfection efficiency and cytotoxicity will be studied in different cell lines. Objective to determine the relationship between transfection efficiency and cytotoxicity of PEI on different cells and optimize the conditions of PEI gene transfection. The optimal dosage and timing of transfection were determined. The possible molecular mechanism of PEI in BMSC cells was analyzed. 1. The relationship between gene transfection efficiency and cytotoxicity of PEI in different mammalian cell lines 293T. After Hela and BMSC cells were cultured, the complexes of pEGFP plasmid and PEI were transfected in suspension and incubated for 24 h and 48 h, respectively. The transfection efficiency was analyzed by fluorescent cell counting method. The pLuc(luciferase gene was transfected from the complex of Renilla) plasmid and PEI. The luciferase activity of each cell was analyzed by spectrophotometer. The toxicity of PEI to 293T Hela and BMSC cells was analyzed by MTT colorimetry. Western blot was used to detect the cytotoxic marker, hydrolyzed cleaved) PARP and hydrolyzed caspase-3. The relationship between transfection efficiency and toxicity. 2. The possible molecular mechanism of PEI-mediated transfection of BMSC cells to construct caveolin-1 overexpression plasmid. The effect of PEI on transfection efficiency and toxicity of BMSC cells was detected by Western blot. The cytotoxicity of Hela and BMSC3 cells was positively correlated with the transfection efficiency, that is, the higher the transfection efficiency was. The higher the cytotoxicity of PEI was, the higher the luciferase activity of 1: 1 was, when the ratio of PEI: DNA (渭 g: 渭 g) was 2 渭 g PEI:2 渭 g. The results of transfection at different time points showed that the difference of transfection time was not significant. 3. 3 pEGFP expression plasmid was transfected into 293T. The transfection efficiency of Hela and BMSC cells was 29.2 3.4% and 21.5 卤2.1.4 luciferase reporter gene was transfected into 293T. The luciferase activity of Hela and BMSC cells was 2.873 脳 10 ~ (8) and 3.35 脳 10 ~ (7) respectively. The results of 1.94 脳 10 ~ (7) RLU / mg protein.5MTT assay showed that the percentage of dead cells transfected with pLuc DNA by PEI was 66.1 卤2.1%. 55.9 2.5% and 45.1% 卤6.7, which was consistent with Western blotting. The cytotoxicity of PEI:. The overexpression of Caveolin-1 in 293THelaBMSC cells can increase the transfection efficiency and toxicity of BMSC cells. The toxicity of ei was positively correlated. 2. Caveolin-1 participated in PEI mediated cell transfection through cytosolic effect. PEI can be used as gene vector for gene therapy.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R450

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