超声微泡造影剂促进重组腺相关病毒载体介导基因转染视网膜神经节细胞的体内实验

发布时间：2018-07-09 19:27

本文选题：超声微泡造影剂 + 重组腺相关病毒　；参考：《重庆医科大学》2010年硕士论文

【摘要】： 背景:已有报道,超声微泡造影剂能够增强质粒介导报告基因转染体内外视网膜神经节细胞的效率。目的:探讨超声微泡造影剂能否提高重组腺相关病毒(rAAV2)介导增强型绿色荧光蛋白(EGFP)基因在体内转染视网膜神经节细胞(RGCs)的效率,并初步探讨其安全性。设计、时间及地点:基因形态学观察实验,于2008-03/09在重庆医科大学超声影像学研究所实验室完成。方法:75只SD大鼠随机分为五组:A组(15只):玻璃体腔注射磷酸盐缓冲液(PBS);B组(15只):玻璃体腔注射rAAV2-EGFP溶液;C组(15只):玻璃体腔注射rAAV2-EGFP溶液后立即用超声辐照眼球;D组(15只):玻璃体腔注射rAAV2-EGFP和微泡造影剂的混悬液;E组(15只):玻璃体腔注射rAAV2-EGFP和微泡造影剂的混悬液后立即用超声辐照眼球。玻璃体腔住射后第21天,用3%荧光金逆行标记RGCs。术后第28天,取出眼球,制作视网膜铺片、视网膜冰冻切片及视网膜病理切片。主要观察指标:在激光共聚焦显微镜下观察视网膜冰冻切片并计算EGFP基因在RGCs中表达的平均荧光密度;在激光共聚焦显微镜下观察视网膜铺片并计算EGFP基因在RGCs的转染率及在RGCs表达的平均荧光密度;在视网膜铺片上进行RGCs计数及观察视网膜病理切片以判断视网膜的损伤情况。结果: 荧光金成功标记RGCs,在B、C、D、E四组均可观察到RGCs中有EGFP表达。其中,B、C、D、E组RGCs转染率分别是12.75±1.33%、15.78±0.31%、17.54±2.01%、20.10±0.74%,经统计分析,差异有统计学意义(P0.05)。在视网膜冰冻切片及铺片中计算各组平均荧光密度可知,E组平均荧光密度最强,均明显高于B、C、D组,统计学上具有显著差异性(P0.01)。A、B、C、D、E五组RGCs计数经统计学分析,差异无统计学意义(P0.05)。B、C、D、E四组视网膜各层组织结构与正常大鼠视网膜各层组织结构无明显差异。结论:在一定条件下,超声微泡造影剂能够在体内安全、有效地提高rAAV2介导EGFP基因转染RGCs的效率。
[Abstract]:Background: it has been reported that ultrasound microbubble contrast agent can enhance the efficiency of plasmid mediated reporter gene transfection into retinal ganglion cells in vivo and in vitro. Aim: to investigate whether ultrasound microbubble contrast agent can improve the efficiency of recombinant adeno-associated virus (rAAV2) -mediated enhanced green fluorescent protein (EGFP) gene transfection into retinal ganglion cells (RGCs) and its safety. Design, time and place: gene morphological observation experiment, 2008-03 / 09, Laboratory of Ultrasonic Imaging Institute, Chongqing Medical University. Methods Seventy-five Sprague-Dawley rats were randomly divided into five groups: group A (n = 15): group B (n = 15) treated with phosphate buffer solution (PBS); group C (n = 15) treated with rAAV2-EGFP solution: group D (n = 15): group D was irradiated by ultrasound immediately after injection of rAAV2-EGFP solution into vitreous cavity. (15 rats): the suspension of rAAV2-EGFP and microbubble contrast agent was injected into vitreous cavity (n = 15): the suspension of rAAV2-EGFP and microbubble contrast agent was injected into vitreous cavity and the eyeball was irradiated with ultrasound immediately after injection. RGCs were retrograde labeled with 3% fluorescent gold on the 21st day after vitreous cavity irradiation. On the 28th day after operation, the eyeball was taken out, retinal slices were made, retinal frozen sections and retinal pathological sections were made. Main outcome measures: the frozen sections of retina were observed under laser confocal microscope and the average fluorescence density of EGFP gene expression in RGCs was calculated. The transfection efficiency of EGFP gene in RGCs and the average fluorescence density of EGFP gene expression in RGCs were calculated under laser confocal microscope, and the RGCs count and retinal pathological sections were observed to judge the damage of retina. Results: the expression of EGFP in RGCs was observed in four groups. The transfection efficiency of RGCs was 12.75 卤1.33 and 15.78 卤0.31 and 17.54 卤2.01and 20.10 卤0.74, respectively. The difference was statistically significant (P0.05). The mean fluorescence density of group E was higher than that of group B (P 0.01), and the mean fluorescence density of group E was significantly higher than that of group B (P 0.01). The count of RGCs in group C (P 0.01) was significantly higher than that in group C (P 0.01). The RGCs count in group E was significantly higher than that in group B (P 0.01). There was no significant difference between the four groups (P0.05). There was no significant difference in the tissue structure of each layer of retina between the four groups and the normal rats. Conclusion: under certain conditions, ultrasound microbubble contrast agent is safe in vivo and can effectively improve the efficiency of EGFP gene transfection into RGCs mediated by rAAV2.
【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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