构建ANG-shRNA慢病毒载体及其对糖尿病大鼠眼底病变的影响

发布时间：2018-04-12 14:40

本文选题：血管生成素 + 慢病毒载体　；参考：《华北理工大学》2017年硕士论文

【摘要】：目的通过链脲佐菌素(STZ)建立1型糖尿病大鼠模型,并构建ANG-shRNA慢病毒载体,干扰糖尿病大鼠视网膜ANG mRNA的表达,揭示ANG在糖尿病眼底病变过程中的作用。方法构建ANG-shRNA慢病毒载体,作用于大鼠肝癌细胞(RH-35)及糖尿病大鼠视网膜,筛选干扰效率最高的shRNA序列,干扰糖尿病大鼠视网膜ANG mRNA的表达。采用Evans蓝活体灌注视网膜铺片,PAS染色法及电镜观察糖尿病大鼠视网膜血管和视网膜细胞超微结构的变化;采用实时荧光定量PCR(q PCR)和Western blot检测ANG、VEGF和相关因子Bcl-2、Bax、Hsp27相对表达量的变化。结果1通过体外和体内实验的综合筛选出ANG-shRNA-2为干扰效率最高的序列。2利用STZ成功建立了1型糖尿病大鼠模型,成模率为92.9%。3通过Evans蓝活体灌注视网膜铺片观察到干扰组大鼠,血管走形趋于正常,渗漏和渗出斑的现象有所减轻。4 PAS染色发现,干扰组大鼠内界膜光滑平整,各层细胞数量明显增加。5电镜观察干扰组血管管腔较规则完整,视杆细胞细胞核染色质分布均匀,线粒体肿胀空泡变性的情况减轻,膜盘形态趋于正常,排列整齐。6 q PCR和Western blot检测各组ANG、VEGF、Bcl-2、Bax及Hsp27相对表达情况发现,干扰组ANG、VEGF和Bax的相对表达量明显低于NC组,且随着病程的延长,除空白对照组外,其它各组ANG、VEGF和Bax表达量逐渐增加,差异均具有统计学意义(P0.05);Bcl-2在干扰组的表达量高于NC组,Hsp27的表达量在模型对照组、NC组和干扰组的表达量均增加,除空白对照组外,其他各组Bcl-2和Hsp27的相对表达量随着病程的延长,逐渐增加,且差异均具有统计学意义(P0.05)。结论通过构建ANG-shRNA慢病毒载体,作用于1型糖尿病大鼠视网膜,降低视网膜中ANG mRNA的表达,对糖尿病视眼底病变具有一定的抑制作用。
[Abstract]:Objective to establish a rat model of type 1 diabetes mellitus by streptozotocin (STZ) and construct ANG-shRNA lentivirus vector to interfere the expression of ANG mRNA in retina of diabetic rats and reveal the role of ANG in the process of diabetic fundus disease.Methods ANG-shRNA lentivirus vector was constructed to act on rat hepatoma cell line RH-35) and the retina of diabetic rats. The shRNA sequence with the highest interference efficiency was screened and the expression of ANG mRNA in the retina of diabetic rats was interfered with.The ultrastructural changes of retinal blood vessels and retinal cells in diabetic rats were observed by Evans blue perfusion in vivo with pas staining and electron microscope, and the relative expression of Evans and Bcl-2Bax-Hsp27 were detected by real-time fluorescence quantitative PCR(q and Western blot.The relative expression of VEGF and Bax in the interference group was significantly lower than that in the NC group, and with the prolongation of the course of disease, the expression of VEGF and Bax in the other groups increased gradually except the blank control group.It increased gradually, and the difference was statistically significant (P 0.05).Conclusion ANG-shRNA lentivirus vector can inhibit the expression of ANG mRNA in retina of type 1 diabetic rats.
【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R771.3;R-332

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