腺病毒与慢病毒载体转染离体兔角膜基质细胞的对比研究

发布时间：2018-03-12 11:45

  本文选题：腺病毒载体　切入点：慢病毒载体　出处：《重庆医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的:利用增强型绿色荧光蛋白(EGFP)为报告基因,观察比较腺病毒与慢病毒载体分别介导EGFP对体外培养的兔角膜基质细胞的转染效率及对细胞的安全性,探讨较优一种病毒载体作为角膜基因治疗载体的可行性,为后继采用该病毒载体介导目的基因转染治疗屈光术后角膜haze生成奠定基础。 方法:离体兔角膜基质细胞的原代、传代培养及鉴定;实验分为转染组和对照组,转染组用慢病毒载体携带增强型绿色荧光蛋白报告基因(LV-EGFP)与腺病毒载体携带增强型绿色荧光蛋白基因(AV- EGFP)分别感染离体兔角膜基质细胞(RCSCs),对照组则加入空白培养液,在不同感染复数(MOI)及感染后不同的时间段在倒置荧光显微镜下观察EGFP的表达情况,流式细胞技术(FCM)检测转染效率;RT-PCR检测转染组和对照组EGFP的mRNA表达情况;光镜及电镜技术观察转染组细胞形态及超微结构的变化;MTT比色法检测两种病毒载体对角膜基质细胞活性的影响。 结果: 1. AV-EGFP感染基质细胞后从24-48小时开始就可见明显的绿色荧光,起始时间早于LV-EGFP转染组。随着MOI值增大,两转染组转染效率均增高。转染后第3天,慢病毒转染组在MOI=1000时转染效率可达61%,而腺病毒转染组转染效率为40%。在同一MOI值下,慢病毒较腺病毒载体转染效率更高,差异有统计学意义(P0.05)。2.RT-PCR检测两转染组均有EGFP的mRNA表达,而对照组无表达。AV-EGFP与LV-EGFP转染组相比,AV-EGFP转染组的EGFP mRNA表达偏低,差异有统计学意义(P0.05)。3.电镜结果显示AV-EGFP转染组在MOI=104时及LV-EGFP转染组在MOI=1000时转染组细胞出现细胞凋亡,而慢病毒在MOI=500时,转染组细胞超微结构未见明显异常。4.慢病毒载体在MOI≤500时,腺病毒载体在MOI≤1000时,两种病毒对细胞活性的影响,转染组分别与对照组比较,差异无统计学意义。慢病毒载体在MOI≥1000,腺病毒载体在MOI≥104转染组与对照组比较,差异有统计学意义(P0.05),病毒载体抑制细胞的活性,细胞存活率下降。且当MOI=104时,慢病毒载体转染组与腺病毒载体转染组比较差异无统计学意义(P0.05),两病毒载体对细胞活性影响无差别,均导致细胞活性下降。 结论:1.腺病毒与慢病毒载体均可有效转染离体兔角膜基质细胞,以慢病毒载体转染效率更高。2.腺病毒最适感染复数为1000,其转染效率在第三天可达40%,慢病毒最适感染复数为500,其转染效率在第三天可达50%。3.腺病毒载体用于离体兔角膜基质细胞转染的安全浓度范围应在MOI≤1000,慢病毒载体用于离体兔角膜基质细胞转染的安全浓度范围应在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，

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