Lenti-EGFP转染离体兔角膜上皮细胞的实验研究

发布时间：2018-01-27 17:28

本文关键词： 慢病毒角膜上皮细胞有效性安全性细胞实验　出处：《重庆医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的观察慢病毒(lentivirus)载体用于角膜上皮细胞基因转染的有效性及安全性。方法角膜上皮细胞的原代及传代培养并应用免疫荧光技术做细胞鉴定。慢病毒载体介导的增强型绿色荧光蛋白(lenti-EGFP)以不同的感染复数(MOI=0,1,10,50,100,500)转染实验细胞,于转染后24、48、72、96h运用倒置荧光显微镜观察不同感染复数(MOI)下增强型绿色荧光蛋白(EGFP)的表达并计算细胞转染率,测定最佳转染剂量,即最适感染复数;RT-PCR方法检测EGFP基因的表达情况。lenti-EGFP以最适感染复数转染角膜上皮细胞,通过组织化学技术(HE染色、透射电子显微镜)观察正常角膜上皮细胞及转染细胞的形态及超微结构变化;流式细胞技术(FCM)检测慢病毒载体对角膜上皮细胞凋亡的影响。结果EGFP于转染48h即开始有表达,随着转染时间的延长其表达增强。MOI=1、10、50、100时,角膜上皮细胞转染率随着感染复数的增加而增加,各感染复数下的细胞转染率差异有统计学意义(P0.05),MOI在100与500时,转染率差异无统计学意义(P0.05),即最适感染复数为100。RT-PCR结果提示转染组细胞内EGFP基因有明确表达。当MOI=100时,角膜上皮细胞HE染色提示转染细胞形态规则,与正常角膜细胞形态一致;透射电子显微镜观察转染细胞超微结构与正常细胞无明显差别。流式细胞术检测转染组细胞凋亡率与正常组细胞无明显差别(P0.05)。结论lenti-EGFP能够有效、安全地转染离体兔角膜上皮细胞。
[Abstract]:Objective to observe the efficacy and safety of lentivirus vector in gene transfection of corneal epithelial cells. Methods the primary and passage culture of corneal epithelial cells was performed and identified by immunofluorescence. Lenti-EGFP mediated by lentivirus vector was expressed in different infective plural numbers (. MOI=0. After transfection, the experimental cells were transfected with 244872. At 96 h, the expression of enhanced green fluorescent protein (EGFP) under different infected complex moi was observed by inverted fluorescence microscope, and the transfection efficiency was calculated. The optimal transfection dose was determined, that is, the optimal complex number of infection. RT-PCR method was used to detect the expression of EGFP gene. Lenti-EGFP was transfected into corneal epithelial cells with the most suitable number of infections. The corneal epithelial cells were stained with HE by histochemical technique. The morphology and ultrastructure of normal corneal epithelial cells and transfected cells were observed by transmission electron microscope (TEM). Flow cytometry (FCM) was used to detect the effect of lentivirus vector on corneal epithelial cell apoptosis. Results the expression of EGFP began at 48h after transfection and increased with the extension of transfection time. The transfection efficiency of corneal epithelial cells increased with the increase of the complex number of infection. There was no significant difference in transfection efficiency (P 0.05), that is, the optimal complex number of infection was 100. RT-PCR results showed that the EGFP gene was expressed clearly in the transfected cells, when MOI = 100. The HE staining of corneal epithelial cells showed that the morphology of transfected cells was consistent with that of normal corneal cells. The ultrastructure of transfected cells was not different from that of normal cells by transmission electron microscope, but the apoptosis rate of transfected cells was not significantly different from that of normal cells by flow cytometry. Conclusion lenti-EGFP can effectively and safely transfect rabbit corneal epithelial cells in vitro.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R774.1

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