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

【摘要】： 目的:利用增強(qiáng)型綠色熒光蛋白(EGFP)為報(bào)告基因,觀察比較腺病毒與慢病毒載體分別介導(dǎo)EGFP對(duì)體外培養(yǎng)的兔角膜基質(zhì)細(xì)胞的轉(zhuǎn)染效率及對(duì)細(xì)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(dǎo)目的基因轉(zhuǎn)染治療屈光術(shù)后角膜haze生成奠定基礎(chǔ)。 方法:離體兔角膜基質(zhì)細(xì)胞的原代、傳代培養(yǎng)及鑒定;實(shí)驗(yàn)分為轉(zhuǎn)染組和對(duì)照組,轉(zhuǎn)染組用慢病毒載體攜帶增強(qiáng)型綠色熒光蛋白報(bào)告基因(LV-EGFP)與腺病毒載體攜帶增強(qiáng)型綠色熒光蛋白基因(AV- EGFP)分別感染離體兔角膜基質(zhì)細(xì)胞(RCSCs),對(duì)照組則加入空白培養(yǎng)液,在不同感染復(fù)數(shù)(MOI)及感染后不同的時(shí)間段在倒置熒光顯微鏡下觀察EGFP的表達(dá)情況,流式細(xì)胞技術(shù)(FCM)檢測(cè)轉(zhuǎn)染效率;RT-PCR檢測(cè)轉(zhuǎn)染組和對(duì)照組EGFP的mRNA表達(dá)情況;光鏡及電鏡技術(shù)觀察轉(zhuǎn)染組細(xì)胞形態(tài)及超微結(jié)構(gòu)的變化;MTT比色法檢測(cè)兩種病毒載體對(duì)角膜基質(zhì)細(xì)胞活性的影響。 結(jié)果: 1. AV-EGFP感染基質(zhì)細(xì)胞后從24-48小時(shí)開始就可見明顯的綠色熒光,起始時(shí)間早于LV-EGFP轉(zhuǎn)染組。隨著MOI值增大,兩轉(zhuǎn)染組轉(zhuǎn)染效率均增高。轉(zhuǎn)染后第3天,慢病毒轉(zhuǎn)染組在MOI=1000時(shí)轉(zhuǎn)染效率可達(dá)61%,而腺病毒轉(zhuǎn)染組轉(zhuǎn)染效率為40%。在同一MOI值下,慢病毒較腺病毒載體轉(zhuǎn)染效率更高,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。2.RT-PCR檢測(cè)兩轉(zhuǎn)染組均有EGFP的mRNA表達(dá),而對(duì)照組無表達(dá)。AV-EGFP與LV-EGFP轉(zhuǎn)染組相比,AV-EGFP轉(zhuǎn)染組的EGFP mRNA表達(dá)偏低,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。3.電鏡結(jié)果顯示AV-EGFP轉(zhuǎn)染組在MOI=104時(shí)及LV-EGFP轉(zhuǎn)染組在MOI=1000時(shí)轉(zhuǎn)染組細(xì)胞出現(xiàn)細(xì)胞凋亡,而慢病毒在MOI=500時(shí),轉(zhuǎn)染組細(xì)胞超微結(jié)構(gòu)未見明顯異常。4.慢病毒載體在MOI≤500時(shí),腺病毒載體在MOI≤1000時(shí),兩種病毒對(duì)細(xì)胞活性的影響,轉(zhuǎn)染組分別與對(duì)照組比較,差異無統(tǒng)計(jì)學(xué)意義。慢病毒載體在MOI≥1000,腺病毒載體在MOI≥104轉(zhuǎn)染組與對(duì)照組比較,差異有統(tǒng)計(jì)學(xué)意義(P0.05),病毒載體抑制細(xì)胞的活性,細(xì)胞存活率下降。且當(dāng)MOI=104時(shí),慢病毒載體轉(zhuǎn)染組與腺病毒載體轉(zhuǎn)染組比較差異無統(tǒng)計(jì)學(xué)意義(P0.05),兩病毒載體對(duì)細(xì)胞活性影響無差別,均導(dǎo)致細(xì)胞活性下降。 結(jié)論:1.腺病毒與慢病毒載體均可有效轉(zhuǎn)染離體兔角膜基質(zhì)細(xì)胞,以慢病毒載體轉(zhuǎn)染效率更高。2.腺病毒最適感染復(fù)數(shù)為1000,其轉(zhuǎn)染效率在第三天可達(dá)40%,慢病毒最適感染復(fù)數(shù)為500,其轉(zhuǎn)染效率在第三天可達(dá)50%。3.腺病毒載體用于離體兔角膜基質(zhì)細(xì)胞轉(zhuǎn)染的安全濃度范圍應(yīng)在MOI≤1000,慢病毒載體用于離體兔角膜基質(zhì)細(xì)胞轉(zhuǎn)染的安全濃度范圍應(yīng)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(hào)：1601415