HIF-1a siRNA对糖尿病大鼠视网膜VEGF抑制的研究

发布时间：2018-04-21 11:11

本文选题：糖尿病视网膜病变 + 视网膜新生血管　；参考：《天津医科大学》2010年硕士论文

【摘要】：目的在血管生成过程中,血管内皮生长因子(vascular endothelial growth factor, VEGF)被认为是一个最主要的调节因子。VEGF是血管内皮细胞的特异性有丝分裂原,能够促进内皮细胞的分裂增殖、迁移,增加血管的通透性。VEGF的表达受到多种因素的调节,其中组织的缺氧/缺血最为重要,VEGF的高表达主要是通过缺氧诱导因子-1α (hypoxia inducible factor-1α, HIF-1α)诱导的。抑制其表达,可以减少视网膜VEGF的表达,进而减少新生血管(neovascularization,NV)的形成。本研究采用RNA干扰技术,构建HIF-1α siRNA重组质粒,以链尿佐菌素(streptozotocin, STZ)诱导的大鼠糖尿病性视网膜病变(diabetic retinopathy, DR)模型为研究对象,探讨HIF-1α siRNA重组质粒对大鼠视网膜组织中VEGF表达的影响,评价其在糖尿病性视网膜新生血管(retinal neovascularization, RNV)疾病中的治疗作用。方法本研究包括2部分： 1.本研究采用随机对照研究。以pSilencer2.1-U6neo为质粒载体,构建针对大鼠的HIF-1α siRNA重组质粒,酶切、测序鉴定。体外培养恒河猴脉络膜视网膜血管内皮细胞(RF/6A),脂质体LipofectamineTM2000介导分别转染空载体质粒和HIF-1α siRNA重组质粒。采用Western blot检测VEGF蛋白表达的变化。 2.尾静脉注射STZ诱导建立大鼠DR模型,将大鼠随机分为糖尿病视网膜病变组(DR组)、空载体组和基因治疗组(HIF-1α siRNA转染组),正常对照组和DR组大鼠不做处理,空载体组和基因治疗组分别以脂质体LipofectamineTM2000介导玻璃体腔内注射pSilencer2.1-U6空载体质粒和HIF-la siRNA重组质粒。采用HE染色、FITC-dextran荧光造影视网膜铺片观察视网膜血管形态的变化,Real-time RT-PCR检测视网膜中VEGF mRNA的表达,免疫组织化学法检测视网膜VEGF蛋白的表达。采用SPSS13.0统计软件,组间数据比较采用单因素方差分析和LSD-t检验进行统计学分析,P0.05为差异有统计学意义。结果 1.将合成的DNA序列退火后克隆到质粒载体上,经酶切、测序鉴定确定为所需序列。体外培养RF/6A细胞,转染HIF-1α siRNA重组质粒24h后,Western blot显示基因治疗组细胞内仅见极微弱的VEGF蛋白表达,而对照组VEGF蛋白表达相对明显,差异具有统计学意义(P0.05),蛋白抑制率为54.9%。 2.荧光造影视网膜铺片显示正常对照组视网膜血管从视盘发出,向四周放射状均匀分布。糖尿病大鼠视网膜可见大量微动脉瘤,血管呈串珠样改变,血管周围可见高荧光渗漏及大片无灌注区。实时荧光定量RT-PCR (Real-time RT-PCR)显示视网膜VEGF mRNA:正常对照组仅见弱的VEGF mRNA表达,而DR组表达明显上调,空载体组与DR组比较差异无统计学意义(P0.05),基因治疗组较DR组明显减弱(P0.05),VEGF mRNA抑制率24h、48h、72h、1w时分别为：32.8%、43.6%、47.7%、50.9%。HE切片显示：正常对照组大鼠视网膜各层细胞排列整齐,细胞形态基本正常。糖尿病大鼠视网膜各层细胞结构排列紊乱,节细胞、周细胞等各层细胞数减少,视网膜内血管扩张,内界膜不完整,血管内皮细胞增殖,新生血管芽、新生血管簇呈垂直状突破内界膜生长。免疫组织化学染色显示,VEGF阳性表达为细胞浆出现棕黄的颗粒,主要定位于神经节细胞层,少量位于神经纤维层、内核层及色素上皮层。正常对照组VEGF蛋白呈弱阳性表达,而DR对照组和空载体组表达明显增强,基因治疗组较DR组和空载体组表达明显减少,差异有统计学意义(P0.05)。VEGF蛋白抑制率24h、48h、72h、1w时分别为：27.4%、40.6%、47.5%、64.5%。结论 1.成功构建针对大鼠的HIF-1α siRNA重组质粒。脂质体介导HIF-1α siRNA重组质粒能有效抑制恒河猴脉络膜视网膜血管内皮细胞VEGF蛋白的表达。 2.成功构建糖尿病大鼠早期新生血管模型。采用脂质体介导,玻璃体腔内注射HIF-1α siRNA重组质粒能有效抑制糖尿病大鼠视网膜中VEGF mRNA及蛋白的表达。以HIF-1α为靶点的基因治疗有可能成为有效抑制糖尿病性RNV的新手段。
[Abstract]:objective
In the process of angiogenesis, vascular endothelial growth factor (VEGF) is considered to be the most important regulatory factor,.VEGF is a specific mitogen of vascular endothelial cells, which can promote the proliferation of endothelial cells, migration, and the expression of the permeability of the vascular permeability.VEGF is regulated by a variety of factors. Hypoxia / ischemia is the most important part of the tissue, and the high expression of VEGF is mainly induced by the hypoxia inducible factor -1 alpha (hypoxia inducible factor-1 alpha, HIF-1 a). Inhibition of its expression can reduce the expression of VEGF in the retina, and then reduce the formation of neovascularization, NV. This study uses RNA interference technique to construct HIF-1 alpha s. IRNA recombinant plasmid, with streptozotocin (STZ) induced diabetic retinopathy (diabetic retinopathy, DR) model, was used to investigate the effect of HIF-1 a siRNA recombinant plasmid on the expression of VEGF in the retinal tissue of rats, and to evaluate it in the diabetic retina neovascularization (retinal neovascularization, R). NV) the therapeutic effect of the disease.
Method
This study consists of 2 parts:
1. the study adopted a randomized controlled study. Using pSilencer2.1-U6neo as plasmid vector, the recombinant plasmid of HIF-1 alpha siRNA for rats was constructed, enzyme digestion and sequencing identification. In vitro culture of Ganges RIver monkey choroidal retinal vascular endothelial cells (RF/6A), liposome LipofectamineTM2000 mediated separation transfection empty vector plasmids and HIF-1 alpha siRNA recombinant plasmids. The changes in the expression of VEGF protein were detected by Western blot.
The rat model of DR was induced by 2. caudal intravenous injection of STZ, and the rats were randomly divided into diabetic retinopathy group (group DR), no-load group and gene therapy group (HIF-1 alpha siRNA transfection group), normal control group and DR group did not do the treatment. The no-load group and gene therapy group were injected with pSile in the vitreous cavity of liposome LipofectamineTM2000, respectively. Ncer2.1-U6 empty vector plasmids and HIF-la siRNA recombinant plasmids. HE staining was used to observe retinal vascular morphologic changes. Real-time RT-PCR was used to detect the expression of VEGF mRNA in retina. Immunohistochemical staining was used to detect the expression of retina VEGF protein. The data were compared by SPSS13.0 statistical software. Univariate analysis of variance and LSD-t test were used for statistical analysis. P0.05 was statistically significant.
Result
1. the synthesized DNA sequence was cloned to plasmid carrier and identified as required sequence by enzyme digestion and sequencing. RF/6A cells were cultured in vitro. After transfection of HIF-1 alpha siRNA recombinant plasmid 24h, Western blot showed that only a very weak VEGF protein expression was found in the cells of the gene therapy group, but the expression of VEGF protein in the group was relatively obvious, and the difference was statistically significant. Study significance (P0.05), protein inhibition rate was 54.9%.
The retinal vasculature of 2. fluorograph showed that the retinal vessels from the normal control group were emitted from the optic disc and distributed evenly around the retina. A large number of microaneurysms were seen in the retina of diabetic rats. The blood vessels showed beads like changes, high fluorescence leakage and large perfusion area around the blood vessels. Real time fluorescence quantitative RT-PCR (Real-time RT-PCR) showed the retina. The expression of VEGF mRNA: in the normal control group was only weak VEGF mRNA expression, but the expression of DR group was obviously up. There was no significant difference between the DR group and the DR group (P0.05), and the gene therapy group was significantly lower than the DR group (P0.05). The VEGF mRNA inhibition rate was 32.8%, 43.6%, 47.7%, respectively. The cells of each layer were arranged regularly and the cell morphology was basically normal. The cell structure of each layer of retina of diabetic rats was arranged in disorder, the number of cells in each layer of ganglion cells, pericytes and other layers decreased, the endoretinal vascular dilatation, the incompleteness of the inner boundary membrane, the proliferation of vascular endothelial cells, the new vascular buds, and the growth of the new vascular clusters in the vertical breakthroughs of the inner boundary membrane. The positive expression of VEGF was that the positive expression of the cytoplasm was brown yellow particles, mainly located in the ganglion cell layer, a small amount in the nerve fiber layer, the kernel layer and the pigment epithelium. The VEGF protein in the normal control group was weakly positive, but the expression of the DR control group and the empty body group was obviously stronger, and the gene therapy group was more expressed than the DR group and the empty body group. The difference was statistically significant (P0.05). The inhibition rate of.VEGF protein was 24h, 48h, 72h and 1W were 27.4%, 40.6%, 47.5%, 64.5%. respectively.
conclusion
1. the recombinant plasmid of HIF-1 alpha siRNA in rats was successfully constructed. The recombinant plasmid mediated by liposome mediated HIF-1 alpha siRNA could effectively inhibit the expression of VEGF protein in the choroidal retinal vascular endothelial cells of Ganges RIver monkey.
2. successfully constructed the early neovascularization model of diabetic rats. Using liposome mediated, intravitreal injection of HIF-1 alpha siRNA recombinant plasmid can effectively inhibit the expression of VEGF mRNA and protein in the retina of diabetic rats. Gene therapy with HIF-1 alpha as a target may be a new part of effective inhibition of diabetic RNV.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R587.2;R774.1

【参考文献】