本文關(guān)鍵詞： 糖尿病視網(wǎng)膜病變 缺氧誘導(dǎo)因子-1α 小干擾RNA 視網(wǎng)膜新生血管 基因治療　出處：《天津醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】：目的 以pSilencer2.1-U6neo為質(zhì)粒載體,構(gòu)建缺氧誘導(dǎo)因子-1α (HIF-1α)特異性小干擾RNA (siRNA),研究其對糖尿病大鼠視網(wǎng)膜HIF-1α表達(dá)的影響。觀察玻璃體腔注射HIF-1α siRNA后不同時間糖尿病大鼠視網(wǎng)膜表達(dá)HIF-1α的變化情況。 方法 1.構(gòu)建HIF-1α特異性siRNA重組質(zhì)粒。SD大鼠共100只,隨機(jī)分成正常組(A)組(21只)和糖尿病組(79只)。鏈脲佐菌素(STZ)尾靜脈注射制作糖尿病大鼠模型。飼養(yǎng)18周后,FITC-Dextran熒光造影視網(wǎng)膜鋪片證實(shí)DR模型成功后,糖尿病大鼠又隨機(jī)分成對照(B)組、空載體(C)組和基因治療(D)組。向空載體組玻璃體腔注射pSilencer空載體質(zhì)粒,基因治療組玻璃體腔注射HIF-1α特異性siRNA重組質(zhì)粒。根據(jù)玻璃體腔注射后24小時、48小時和72小時,A、 B、C、D各組隨機(jī)分成三個組。 2.分別于注藥后24小時、48小時、72小時后處死相應(yīng)組動物,摘除眼球,應(yīng)用組織病理學(xué)觀察不同時間點(diǎn)各組大鼠視網(wǎng)膜的病理改變。采用實(shí)時熒光RT-PCR法檢測各組視網(wǎng)膜HIF-1α mRNA水平的差異；免疫組化SP法觀察視網(wǎng)膜各層HIF-1蛋白的染色情況。Image-Pro Plus6.0圖像處理分析軟件分析各組染色陽性區(qū)的平均累積光密度(integrated optic density,IOD)。 3.運(yùn)用SPSS11.5統(tǒng)計(jì)軟件,經(jīng)正態(tài)性檢驗(yàn)及方差齊性檢驗(yàn),對同一組間不同時間,同一時間不同組間數(shù)據(jù)進(jìn)行分析,采用單因素方差分析,并用SNK檢驗(yàn)進(jìn)行組間比較,P0.05差異具有統(tǒng)計(jì)學(xué)意義。 結(jié)果 1.STZ誘導(dǎo)的糖尿病大鼠高血糖狀態(tài)持久穩(wěn)定,表現(xiàn)為明顯的“三多一少”典型癥狀。在各時間點(diǎn),正常組與糖尿病組大鼠體重、血糖均有統(tǒng)計(jì)學(xué)差異(P0.05)。 2. FITC-Dextran熒光造影視網(wǎng)膜鋪片顯示糖尿病組視網(wǎng)膜血管形態(tài)和分布發(fā)生明顯改變,可見微血管瘤及異常吻合的新生血管團(tuán)、高熒光素滲漏和無灌注區(qū),證實(shí)DR模型制作成功。正常大鼠視網(wǎng)膜血管走行良好,視網(wǎng)膜淺層大血管及深層血管網(wǎng)均清晰可見,未見視網(wǎng)膜微血管瘤形成和視網(wǎng)膜新生血管形成。 3.實(shí)時熒光RT-PCR結(jié)果顯示,同一時間點(diǎn)C組和B組視網(wǎng)膜HIF-1α mRNA表達(dá)較A組明顯上調(diào),D組視網(wǎng)膜組織中HIF-1α mRNA較B組、C組下調(diào),差異具有統(tǒng)計(jì)學(xué)意義(P0.05),B組和C組表達(dá)差異無統(tǒng)計(jì)學(xué)意義(P0.05)。D組不同時間點(diǎn)視網(wǎng)膜HIF-1α mRNA的表達(dá)差異具有統(tǒng)計(jì)學(xué)差異(F=9.437,P=0.002)。進(jìn)一步進(jìn)行組間比較,玻璃體腔注射HIF-1α siRNA48小時及72小時后視網(wǎng)膜HIF-1α mRNA表達(dá)較注射24小時后明顯減少(P1=0.003,P2=0.001),注射72小時后較注射48小時后視網(wǎng)膜HIF-1αamRNA表達(dá)差異不具有統(tǒng)計(jì)學(xué)意義(P=0.749)。HIF-1α siRNA干擾24小時、48小時和72小時的抑制效率分別為32.59%、46.92%和52.28%。 4. HIF-1α蛋白在正常大鼠視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層和外叢狀層有少量陽性染色細(xì)胞,糖尿病組染色陽性細(xì)胞數(shù)表達(dá)增多,主要位于視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層、內(nèi)叢狀層、內(nèi)核層、外叢狀層。同一時間點(diǎn)C組和B組視網(wǎng)膜HIF-1α染色陽性區(qū)的平均IOD較正常組明顯上調(diào)(P0.05),B組和C組表達(dá)差異無統(tǒng)計(jì)學(xué)意義(P0.05),D組視網(wǎng)膜組織中HIF-1α蛋白染色陽性區(qū)的平均IOD較B、C組下調(diào),差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。D組不同時間點(diǎn)視網(wǎng)膜HIF-1α蛋白的表達(dá)差異具有統(tǒng)計(jì)學(xué)差異(F=26.373,P=0.000)。進(jìn)一步進(jìn)行組間比較,玻璃體腔注射HIF-1α siRNA48小時及72小時后視網(wǎng)膜HIF-1a蛋白表達(dá)較24小時明顯減少(P1=0.000,P2=0.000),注射72小時后較注射48小時后視網(wǎng)膜HIF-1α蛋白表達(dá)差異不具有統(tǒng)計(jì)學(xué)意義(P=0.215)。 結(jié)論 本實(shí)驗(yàn)構(gòu)建HIF-1α特異性siRNA重組質(zhì)粒,將HIF-1α siRNA成功的轉(zhuǎn)染至大鼠視網(wǎng)膜,通過實(shí)時熒光RT-PCR和免疫組織化學(xué)染色方法觀察糖尿病大鼠視網(wǎng)膜HIF-1α的表達(dá),發(fā)現(xiàn)HIF-1α特異性siRNA降低了糖尿病大鼠視網(wǎng)膜HIF-1α的表達(dá),從而有望抑制其靶基因VEGF等促進(jìn)血管新生因子的表達(dá),抑制視網(wǎng)膜新生血管的形成,成為治療DR的一種新的途徑。
[Abstract]:objective
With pSilencer2.1-U6neo plasmid to construct hypoxia inducible factor -1 alpha (HIF-1 alpha) specific small interfering RNA (siRNA), to study its effect on retinal HIF-1 alpha in diabetic rats. To observe the expression of intravitreal injection of HIF-1 alpha siRNA at different time after the retina of diabetic rats of HIF-1 alpha changes.
Method
1. construction of HIF-1 alpha specific recombinant plasmid siRNA 100.SD rats were randomly divided into normal control group (A) group (21 rats) and diabetic group (79 rats). Streptozotocin (STZ) diabetic rat model was established by tail vein injection. After 18 weeks of feeding, FITC-Dextran fluorescence angiography was the DR model proved successful, and diabetic rats were randomly divided into control group (B) (C), empty vector group and gene therapy group (D). To empty vector group pSilencer intravitreal injection of empty plasmid, gene therapy group HIF-1 intravitreal injection of alpha specific siRNA recombinant plasmid. According to the 24 hours after the intravitreal injection, 48 hours and 72 hours, A, B, C, D were randomly divided into three groups.
2. to 24 hours after injection, 48 hours, 72 hours after the corresponding group of animal, removal of the eye, histopathological observation on pathological changes of retina in rats at different time points. The difference by using real-time fluorescence RT-PCR method to detect the retinal HIF-1 alpha mRNA levels; immunohistochemical SP method was used to observe the retinal layers HIF-1 protein staining of.Image-Pro Plus6.0 image processing and analysis software were analyzed with positive staining area cumulative average optical density (integrated optic, density, IOD).
3., using SPSS11.5 statistical software, by normality test and homogeneity test of variance, the data of different groups at the same time and at the same time between different groups were analyzed. One-way ANOVA and SNK test were used to compare the data between groups. The difference of P0.05 was statistically significant.
Result
High blood glucose in diabetic rats induced by 1.STZ lasting stability, is obviously a little more than three typical symptoms. At each time point, the normal weight group and diabetic rats, there were differences in blood glucose (P0.05).
2. FITC-Dextran fluorescein angiography retinal flatmount showed retinal vascular morphology and distribution of diabetes group changed obviously, visible micro hemangioma and abnormal neovascularization anastomosis group, high fluorescein leakage and non perfusion area, confirmed that the DR model was established successfully. The retinal vessels in normal rats running well, the superficial layer of the retina vessels and deep vascular network visible, and retinal neovascularization no retinal microaneurysm.
3. real time RT-PCR results showed that at the same time C group and B group of retinal HIF-1 alpha mRNA expression compared with A group was significantly increased in the D group, the retinal tissue HIF-1 alpha mRNA compared with B group, C group decreased, the difference was statistically significant (P0.05), B group and C group was no statistically significant difference (P0.05) the differential expression of.D group at different time points of retinal HIF-1 alpha mRNA with statistical difference (F=9.437, P=0.002). Further comparison between groups, the expression of mRNA in retinal HIF-1 alpha was significantly less than 24 hours after the injection of intravitreal injection of HIF-1 alpha siRNA48 and 72 hours (P1=0.003, P2=0.001), 72 h after injection with injection of 48 hours after the retinal expression of HIF-1 alpha amRNA difference was not statistically significant (P=0.749).HIF-1 alpha siRNA interference for 24 hours, the inhibition efficiency of 48 hours and 72 hours were 32.59%, 46.92% and 52.28%.
There is a small amount of 4. HIF-1 protein positive staining cells in the retinal ganglion cell layer and outer plexiform layer of normal rats, diabetic group staining positive cells expression increased, mainly located in the retinal ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer. At the same time C group and B group of retinal HIF-1 alpha the average IOD positive staining area was significantly increased compared with normal group (P0.05), B group and C group was no statistically significant difference (P0.05), the average IOD positive area than B staining of HIF-1 protein in retinal tissue in the D group, C group decreased, the difference was statistically significant (P0.05) expression of.D group at different time points retinal HIF-1 alpha protein has a statistically significant difference (F=26.373, P=0.000). Further comparison between groups, the retinal expression of HIF-1a protein was significantly reduced by 24 hours compared with intravitreal injection of HIF-1 alpha siRNA48 and 72 hours (P1=0.000, P2=0.000), injection of 72 hours There was no significant difference in the expression of HIF-1 alpha protein in the retina after 48 hours after injection (P=0.215).
conclusion
The constructed HIF-1 alpha specific siRNA recombinant plasmid, HIF-1 alpha siRNA successfully transfected into rat retina, observe the expression of retinal HIF-1 alpha in diabetic rats by real-time RT-PCR and immunohistochemistry method, HIF-1 alpha specific siRNA decreased the expression of retinal HIF-1 alpha in diabetic rats, which is expected to inhibit the the target gene VEGF promotes the expression of angiogenic factors, inhibit retinal neovascularization, become a new approach to the treatment of DR.

【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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