本文選題：腺病毒載體　切入點：慢病毒載體　出處：《重慶醫(yī)科大學》2010年碩士論文　論文類型：學位論文

【摘要】： 目的:利用增強型綠色熒光蛋白(EGFP)為報告基因,觀察比較腺病毒與慢病毒載體分別介導EGFP對體外培養(yǎng)的兔角膜基質細胞的轉染效率及對細胞的安全性,探討較優(yōu)一種病毒載體作為角膜基因治療載體的可行性,為后繼采用該病毒載體介導目的基因轉染治療屈光術后角膜haze生成奠定基礎。 方法:離體兔角膜基質細胞的原代、傳代培養(yǎng)及鑒定;實驗分為轉染組和對照組,轉染組用慢病毒載體攜帶增強型綠色熒光蛋白報告基因(LV-EGFP)與腺病毒載體攜帶增強型綠色熒光蛋白基因(AV- EGFP)分別感染離體兔角膜基質細胞(RCSCs),對照組則加入空白培養(yǎng)液,在不同感染復數(shù)(MOI)及感染后不同的時間段在倒置熒光顯微鏡下觀察EGFP的表達情況,流式細胞技術(FCM)檢測轉染效率;RT-PCR檢測轉染組和對照組EGFP的mRNA表達情況;光鏡及電鏡技術觀察轉染組細胞形態(tài)及超微結構的變化;MTT比色法檢測兩種病毒載體對角膜基質細胞活性的影響。 結果: 1. AV-EGFP感染基質細胞后從24-48小時開始就可見明顯的綠色熒光,起始時間早于LV-EGFP轉染組。隨著MOI值增大,兩轉染組轉染效率均增高。轉染后第3天,慢病毒轉染組在MOI=1000時轉染效率可達61%,而腺病毒轉染組轉染效率為40%。在同一MOI值下,慢病毒較腺病毒載體轉染效率更高,差異有統(tǒng)計學意義(P0.05)。2.RT-PCR檢測兩轉染組均有EGFP的mRNA表達,而對照組無表達。AV-EGFP與LV-EGFP轉染組相比,AV-EGFP轉染組的EGFP mRNA表達偏低,差異有統(tǒng)計學意義(P0.05)。3.電鏡結果顯示AV-EGFP轉染組在MOI=104時及LV-EGFP轉染組在MOI=1000時轉染組細胞出現(xiàn)細胞凋亡,而慢病毒在MOI=500時,轉染組細胞超微結構未見明顯異常。4.慢病毒載體在MOI≤500時,腺病毒載體在MOI≤1000時,兩種病毒對細胞活性的影響,轉染組分別與對照組比較,差異無統(tǒng)計學意義。慢病毒載體在MOI≥1000,腺病毒載體在MOI≥104轉染組與對照組比較,差異有統(tǒng)計學意義(P0.05),病毒載體抑制細胞的活性,細胞存活率下降。且當MOI=104時,慢病毒載體轉染組與腺病毒載體轉染組比較差異無統(tǒng)計學意義(P0.05),兩病毒載體對細胞活性影響無差別,均導致細胞活性下降。 結論:1.腺病毒與慢病毒載體均可有效轉染離體兔角膜基質細胞,以慢病毒載體轉染效率更高。2.腺病毒最適感染復數(shù)為1000,其轉染效率在第三天可達40%,慢病毒最適感染復數(shù)為500,其轉染效率在第三天可達50%。3.腺病毒載體用于離體兔角膜基質細胞轉染的安全濃度范圍應在MOI≤1000,慢病毒載體用于離體兔角膜基質細胞轉染的安全濃度范圍應在MOI≤500。
[Abstract]:Aim: to compare the transfection efficiency and safety of EGFP mediated by adenovirus and lentivirus on rabbit corneal stromal cells in vitro using enhanced green fluorescent protein (EGFP) as a reporter gene. To explore the feasibility of using a better virus vector as a corneal gene therapy vector, and to lay a foundation for the subsequent application of the virus vector mediated target gene transfection in the treatment of corneal haze after refractive surgery. Methods: the primary passage culture and identification of rabbit corneal stromal cells in vitro were divided into two groups: transfection group and control group. In transfection group, lentivirus vector carrying enhanced green fluorescent protein reporter gene (LV-EGFP) and adenovirus vector carrying enhanced green fluorescent protein gene (AV-EGFP) were used to infect rabbit corneal stromal cells in vitro, while blank culture medium was added to control group. The expression of EGFP was observed under inverted fluorescence microscope in different infection groups and different time periods after infection. Flow cytometry was used to detect the transfection efficiency and RT-PCR was used to detect the mRNA expression of EGFP in the transfected group and control group. The morphological and ultrastructural changes of the cells in the transfected group were observed by light microscopy and electron microscopy. MTT colorimetric assay was used to detect the effects of two viral vectors on the activity of corneal stromal cells. Results: 1. Green fluorescence was observed from 24 to 48 hours after AV-EGFP infection in stromal cells, and the onset time was earlier than that of LV-EGFP transfection group. With the increase of MOI value, the transfection efficiency of the two groups increased, and the transfection efficiency of the two groups increased 3 days after transfection. The transfection efficiency of lentivirus group was 61g at MOI = 1000, while that of adenovirus transfection group was 400.The transfection efficiency of lentivirus was higher than that of adenovirus vector at the same MOI value, and the difference was statistically significant (P 0.05). 2. The mRNA expression of EGFP was detected by RT-PCR in both groups. However, the expression of EGFP mRNA in the control group was lower than that in the LV-EGFP transfection group, and the difference was statistically significant (P 0.05) .3. the electron microscopic results showed that the cells in the AV-EGFP transfection group and LV-EGFP transfection group were apoptotic at MOI = 1000, while the lentivirus expression in the MOI=500 group was higher than that in the AV-EGFP transfection group. The effect of lentivirus vector on cell activity at MOI 鈮，

本文編號：1601415