慢病毒载体介导增强型绿色荧光蛋白两种途径体内转染大鼠角膜基质细胞的实验研究

发布时间：2018-05-17 15:48

本文选题：慢病毒 + 角膜基质　；参考：《重庆医科大学》2011年硕士论文

【摘要】：目的:观察以慢病毒作为载体,介导增强型绿色荧光蛋白(lenti-EGFP)这一报告基因,通过两种方式体内转染大鼠角膜基质细胞的有效性和安全性,探索一种简便,有效且安全的转染载体及途径,为角膜相关疾病的基因治疗提供实验基础。方法:Lenti-EGFP通过两种不同方法体内转染大鼠角膜基质细胞,途径1:将Lenti-EGFP病毒液通过微量进样器直接注射进角膜基质;途径2:刮除角膜上皮,基质层敷贴浸带有等量lenti-EGFP病毒液的棉片。转染后定期裂隙灯检查,观察角膜绿色荧光的表达及外眼情况,以3d、7d、26d、48d四个不同时间点收集角膜,固定后切片,进行HE染色,观察角膜各层细胞的大体形态;共聚焦显微镜观察拍照,了解角膜荧光表达的强度、持续时间及分布情况;电镜观察基质细胞超微结构的变化;细胞凋亡检测,了解试验组与对照组角膜细胞凋亡情况。结果:1.裂隙灯古兰光下见转染lenti-EGFP的活体大鼠角膜成蓝绿色,对照组角膜未见荧光表达。2.HE染色显示各组别大鼠角膜的各层细胞形态未发生明显异常改变,电镜见实验组角膜基质细胞的超微结构未发生变异。3.共聚焦显微镜下观察见:转染lenti-EGFP的角膜基质细胞2d后增强型绿色荧光蛋白(EGFP)开始表达,7d时荧光表达到高峰,全层角膜均可见荧光表达,26d时表达明显减弱,48d时仅是上皮有微弱表达;空质粒组在第3d和7d时角膜上皮及基质层间可见微弱荧光,基质细胞中无荧光表达,26d和48d时角膜已无荧光表达;对照组的全层角膜在各时间段均未见荧光表达。4.Tunel检测发现实验组的角膜上皮及基质细胞凋亡率较正常组高,但与对照组比较差异无统计学意义(P0.05)。结论:Lenti-EGFP通过上述两种体内转染途径均可安全有效的转染活体大鼠角膜基质细胞。在转染初期,注射法的EGFP基因表达更强,随着时间的延长两种方式的荧光强度趋于相同,但基质注射途径仍略强于刮除上皮敷贴棉片这种方式。两种技术操作简便、基因转染快速,且转染效率较高、安全性好,为各种原因导致的角膜病变,尤其是对抑制准分子激光术后角膜Haze形成及板层角膜移植术后的排斥反应等转基因治疗提供了新思路。
[Abstract]:Objective: to investigate the efficacy and safety of lenti-EGFP gene mediated by lentivirus in rat corneal stromal cells. Effective and safe transfection vectors and pathways provide experimental basis for gene therapy of corneal related diseases. Methods Twenty Lenti-EGFP methods were used to transfect rat corneal stromal cells in vivo. Route 1: Lenti-EGFP virus solution was injected directly into corneal stroma through microsampler, and route 2: corneal epithelium was scraped and stromal layer was coated with cotton flakes containing the same amount of lenti-EGFP virus solution. After transfection with slit lamp, the expression of green fluorescence in cornea and the condition of external eye were observed. The cornea were collected at 4 different time points (3 days, 7 days, 26 days, 48 days), fixed sections were stained with HE, and the gross morphology of the cells in each layer of cornea was observed. The intensity, duration and distribution of fluorescence expression in cornea were observed by confocal microscope. Ultrastructural changes of stromal cells were observed by electron microscope. Apoptosis was detected in experimental group and control group. The result is 1: 1. Under slit lamp Gulan light, the cornea of living rats transfected with lenti-EGFP became blue-green, while that of control group showed no fluorescence expression .2.HE staining showed that the morphology of all layers of cornea in each group did not change abnormally. The ultrastructure of corneal stromal cells in the experimental group was not changed. Under confocal microscope, the expression of enhanced green fluorescent protein (EGFP) reached its peak at 7 days after transfection of lenti-EGFP into corneal stromal cells. Fluorescence expression was observed in the whole cornea at day 26, and the expression was weakly expressed in the epithelium at the 48d, and between the epithelium and the stroma on the 3rd and 7th day in the blank plasmid group, and there was weak fluorescence between the epithelium and the stromal layer in the blank plasmid group. No fluorescent expression was found in the stromal cells at day 26 and day 48, but no fluorescence expression was found in the whole cornea of the control group. 4. Tunel analysis showed that the apoptosis rate of corneal epithelium and stromal cells in the experimental group was higher than that in the normal group. But there was no significant difference between the control group and the control group (P 0.05). ConclusionTwo percent Lenti-EGFP can be used to transfect rat corneal stromal cells safely and effectively through the two in vivo transfection pathways. In the early stage of transfection, the EGFP gene expression was stronger, and the fluorescence intensity of the two methods tended to be the same with time, but the matrix injection pathway was still slightly stronger than that of scraping epithelium coated cotton flakes. The two techniques are easy to operate, rapid gene transfection, high transfection efficiency and good safety. In particular, it provides a new way to inhibit the formation of corneal Haze and the rejection of lamellar keratoplasty after excimer laser surgery.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R779.63

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