本文關(guān)鍵詞： PEI 基因轉(zhuǎn)染 細胞毒性 熒光素酶 內(nèi)吞作用 caveolin-1　出處：《新鄉(xiāng)醫(yī)學院》2015年碩士論文　論文類型：學位論文

【摘要】：背景基因治療能將外源正�；�?qū)氚屑毎⑹怪行П磉_,從而達到治療目的,是一種新興的治療方法。聚乙烯亞胺(polyethylenimine,PEI)是一種高效的非病毒性基因轉(zhuǎn)染載體,其枝狀復合物尤其是25 kDa的分支狀PEI有較高的基因轉(zhuǎn)染效率,它在基因傳遞領(lǐng)域的應用非常廣泛。同時PEI對細胞也有毒副作用,它的毒性與相對分子質(zhì)量(Mr)有關(guān),不同Mr或異構(gòu)體的PEI,在體內(nèi)介導基因轉(zhuǎn)染的效率和細胞毒性截然不同。本課題將在不同的細胞系中研究其轉(zhuǎn)染效率和毒性間的關(guān)系,并嘗試探討其分子機制。目的本研究擬確定PEI對不同細胞的轉(zhuǎn)染效率和細胞毒性之間的內(nèi)在聯(lián)系,同時優(yōu)化PEI基因轉(zhuǎn)染的條件,確定轉(zhuǎn)染時機及最優(yōu)用量,并分析PEI在BMSC細胞中可能的分子機制。方法1.PEI在不同細胞系中基因轉(zhuǎn)染效率和細胞毒性之間的關(guān)系不同哺乳動物細胞系293T、Hela和BMSC細胞培養(yǎng)后,懸浮轉(zhuǎn)染pEGFP質(zhì)粒與PEI的復合物,分別孵育24 h和48 h,利用熒光細胞計數(shù)法分析轉(zhuǎn)染效率；懸浮轉(zhuǎn)染pLuc(luciferase基因來自Renilla)質(zhì)粒與PEI的復合物,利用分光光度計分析各細胞的熒光素酶活性；采用MTT比色法分析PEI對293T、Hela和BMSC細胞的毒性,Western印跡檢測細胞毒性標記物一水解的(cleaved) PARP和水解的caspase-3,進而得出轉(zhuǎn)染效率和毒性之間的相關(guān)性。2.PEI介導的BMSC細胞轉(zhuǎn)染可能的分子機制構(gòu)建caveolin-1過表達質(zhì)粒,經(jīng)PEI轉(zhuǎn)染BMSC細胞,Western印跡法檢測其對轉(zhuǎn)染效率和毒性的影響。結(jié)果1.PEI對293T、Hela和BMSC3種細胞的細胞毒性和轉(zhuǎn)染效率呈正相關(guān),即轉(zhuǎn)染效率越高,PEI的細胞毒性亦越高：①熒光素酶活性檢測結(jié)果顯示PEI:DNA(μg:μg)的比率為2 (4μg PEI:2 μg DNA)時,轉(zhuǎn)染效率最高。②不同時間點轉(zhuǎn)染的結(jié)果表明轉(zhuǎn)染時間之間的差異不顯著。③ pEGFP表達質(zhì)粒轉(zhuǎn)染293T、Hela和BMSC細胞的轉(zhuǎn)染效率結(jié)果是：92.1土4.5%,29.2+3.4%和21.5±2.1%。④熒光素酶報告基因轉(zhuǎn)染293T、Hela和BMSC細胞后熒光素酶活性分別是：2.873×108、3.35×107、1.94×107RLU/mg protein。⑤MTT法結(jié)果顯示PEI轉(zhuǎn)染pLuc DNA后死細胞百分比分別是66.1±2.1%,55.9+2.5%和45.1%±6.7%,這與Western印跡結(jié)果一致,即PEI的細胞毒性：293THelaBMSC細胞。2.Caveolin-1過表達時可提高對BMSC細胞的轉(zhuǎn)染效率和毒性。結(jié)論1.PEI在各種細胞系的轉(zhuǎn)染效率與PEI的毒性呈正相關(guān)性。2.Caveolin-1通過胞吞作用參與PEI介導的細胞轉(zhuǎn)染,可以PEI作為基因載體用于基因治療。
[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 鹵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 鹵2.1%. 55.9 2.5% and 45.1% 鹵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.
【學位授予單位】：新鄉(xiāng)醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R450

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