本文選題：糖尿病視網(wǎng)膜病變 + 視網(wǎng)膜新生血管�。� 參考：《天津醫(yī)科大學(xué)》2010年碩士論文

【摘要】：目的 在血管生成過程中,血管內(nèi)皮生長因子(vascular endothelial growth factor, VEGF)被認為是一個最主要的調(diào)節(jié)因子。VEGF是血管內(nèi)皮細胞的特異性有絲分裂原,能夠促進內(nèi)皮細胞的分裂增殖、遷移,增加血管的通透性。VEGF的表達受到多種因素的調(diào)節(jié),其中組織的缺氧/缺血最為重要,VEGF的高表達主要是通過缺氧誘導(dǎo)因子-1α (hypoxia inducible factor-1α, HIF-1α)誘導(dǎo)的。抑制其表達,可以減少視網(wǎng)膜VEGF的表達,進而減少新生血管(neovascularization,NV)的形成。本研究采用RNA干擾技術(shù),構(gòu)建HIF-1α siRNA重組質(zhì)粒,以鏈尿佐菌素(streptozotocin, STZ)誘導(dǎo)的大鼠糖尿病性視網(wǎng)膜病變(diabetic retinopathy, DR)模型為研究對象,探討HIF-1α siRNA重組質(zhì)粒對大鼠視網(wǎng)膜組織中VEGF表達的影響,評價其在糖尿病性視網(wǎng)膜新生血管(retinal neovascularization, RNV)疾病中的治療作用。 方法 本研究包括2部分： 1.本研究采用隨機對照研究。以pSilencer2.1-U6neo為質(zhì)粒載體,構(gòu)建針對大鼠的HIF-1α siRNA重組質(zhì)粒,酶切、測序鑒定。體外培養(yǎng)恒河猴脈絡(luò)膜視網(wǎng)膜血管內(nèi)皮細胞(RF/6A),脂質(zhì)體LipofectamineTM2000介導(dǎo)分別轉(zhuǎn)染空載體質(zhì)粒和HIF-1α siRNA重組質(zhì)粒。采用Western blot檢測VEGF蛋白表達的變化。 2.尾靜脈注射STZ誘導(dǎo)建立大鼠DR模型,將大鼠隨機分為糖尿病視網(wǎng)膜病變組(DR組)、空載體組和基因治療組(HIF-1α siRNA轉(zhuǎn)染組),正常對照組和DR組大鼠不做處理,空載體組和基因治療組分別以脂質(zhì)體LipofectamineTM2000介導(dǎo)玻璃體腔內(nèi)注射pSilencer2.1-U6空載體質(zhì)粒和HIF-la siRNA重組質(zhì)粒。采用HE染色、FITC-dextran熒光造影視網(wǎng)膜鋪片觀察視網(wǎng)膜血管形態(tài)的變化,Real-time RT-PCR檢測視網(wǎng)膜中VEGF mRNA的表達,免疫組織化學(xué)法檢測視網(wǎng)膜VEGF蛋白的表達。采用SPSS13.0統(tǒng)計軟件,組間數(shù)據(jù)比較采用單因素方差分析和LSD-t檢驗進行統(tǒng)計學(xué)分析,P0.05為差異有統(tǒng)計學(xué)意義。 結(jié)果 1.將合成的DNA序列退火后克隆到質(zhì)粒載體上,經(jīng)酶切、測序鑒定確定為所需序列。體外培養(yǎng)RF/6A細胞,轉(zhuǎn)染HIF-1α siRNA重組質(zhì)粒24h后,Western blot顯示基因治療組細胞內(nèi)僅見極微弱的VEGF蛋白表達,而對照組VEGF蛋白表達相對明顯,差異具有統(tǒng)計學(xué)意義(P0.05),蛋白抑制率為54.9%。 2.熒光造影視網(wǎng)膜鋪片顯示正常對照組視網(wǎng)膜血管從視盤發(fā)出,向四周放射狀均勻分布。糖尿病大鼠視網(wǎng)膜可見大量微動脈瘤,血管呈串珠樣改變,血管周圍可見高熒光滲漏及大片無灌注區(qū)。實時熒光定量RT-PCR (Real-time RT-PCR)顯示視網(wǎng)膜VEGF mRNA:正常對照組僅見弱的VEGF mRNA表達,而DR組表達明顯上調(diào),空載體組與DR組比較差異無統(tǒng)計學(xué)意義(P0.05),基因治療組較DR組明顯減弱(P0.05),VEGF mRNA抑制率24h、48h、72h、1w時分別為：32.8%、43.6%、47.7%、50.9%。HE切片顯示：正常對照組大鼠視網(wǎng)膜各層細胞排列整齊,細胞形態(tài)基本正常。糖尿病大鼠視網(wǎng)膜各層細胞結(jié)構(gòu)排列紊亂,節(jié)細胞、周細胞等各層細胞數(shù)減少,視網(wǎng)膜內(nèi)血管擴張,內(nèi)界膜不完整,血管內(nèi)皮細胞增殖,新生血管芽、新生血管簇呈垂直狀突破內(nèi)界膜生長。免疫組織化學(xué)染色顯示,VEGF陽性表達為細胞漿出現(xiàn)棕黃的顆粒,主要定位于神經(jīng)節(jié)細胞層,少量位于神經(jīng)纖維層、內(nèi)核層及色素上皮層。正常對照組VEGF蛋白呈弱陽性表達,而DR對照組和空載體組表達明顯增強,基因治療組較DR組和空載體組表達明顯減少,差異有統(tǒng)計學(xué)意義(P0.05)。VEGF蛋白抑制率24h、48h、72h、1w時分別為：27.4%、40.6%、47.5%、64.5%。 結(jié)論 1.成功構(gòu)建針對大鼠的HIF-1α siRNA重組質(zhì)粒。脂質(zhì)體介導(dǎo)HIF-1α siRNA重組質(zhì)粒能有效抑制恒河猴脈絡(luò)膜視網(wǎng)膜血管內(nèi)皮細胞VEGF蛋白的表達。 2.成功構(gòu)建糖尿病大鼠早期新生血管模型。采用脂質(zhì)體介導(dǎo),玻璃體腔內(nèi)注射HIF-1α siRNA重組質(zhì)粒能有效抑制糖尿病大鼠視網(wǎng)膜中VEGF mRNA及蛋白的表達。以HIF-1α為靶點的基因治療有可能成為有效抑制糖尿病性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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R587.2;R774.1

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