本文選題：早產(chǎn)兒視網(wǎng)膜病變 + 小干擾RNA　； 參考：《廣州醫(yī)學(xué)院》2012年碩士論文

【摘要】：目的探討缺氧誘導(dǎo)因子-1α(hypoxia-inducible factor-1α，HIF-1α)在早產(chǎn)兒視網(wǎng)膜病變（retinopathy of prematurity，ROP）發(fā)病中的作用，為ROP的基因治療尋找新的靶點(diǎn)。 方法將化學(xué)合成小分子干擾RNA(small interference RNA,SiRNA)用脂質(zhì)體介導(dǎo)法轉(zhuǎn)染大鼠視網(wǎng)膜血管內(nèi)皮細(xì)胞，轉(zhuǎn)染的細(xì)胞在含化學(xué)缺氧劑——二氯化鈷（CoCl2）的培養(yǎng)基中培養(yǎng)8h；應(yīng)用熒光定量聚合酶鏈反應(yīng)和Western印跡技術(shù)檢測(cè)轉(zhuǎn)染后HIF-1αmRNA和蛋白的表達(dá)量，應(yīng)用四甲基偶氮唑藍(lán)比色法對(duì)細(xì)胞增殖活性進(jìn)行檢測(cè)分析。正態(tài)分布計(jì)量資料用均數(shù)±標(biāo)準(zhǔn)差（x±s）表示，比較2組間是否存在差異采用t檢驗(yàn)。 結(jié)果成功將siRNA轉(zhuǎn)染大鼠視網(wǎng)膜血管內(nèi)皮細(xì)胞，熒光定量聚合酶鏈反應(yīng)技術(shù)檢測(cè)顯示，4條siRNA均能不同程度抑制HIF-1α的轉(zhuǎn)錄表達(dá)，siRNA1、siRNA2和siRNA4組HIF-1α mRNA分別降至空白對(duì)照組的16.20%、20.34%和30.49%，與陰性對(duì)照組（83.34%）之間差異有統(tǒng)計(jì)學(xué)意義（t分別為16.786、8.953和4.087，P均＜0.05）。Western印跡技術(shù)顯示，siRNA1和siRNA2轉(zhuǎn)染的大鼠視網(wǎng)膜血管內(nèi)皮細(xì)胞HIF-1α蛋白表達(dá)水平分別為0.496、0.654，明顯低于空白對(duì)照組（3.402）和陰性對(duì)照組（3.510），差異有統(tǒng)計(jì)學(xué)意義（t分別為6.861、2.893、4.567和5.072，，P均＜0.05）。siRNA1和siRNA2組細(xì)胞增殖抑制率分別為49.50%和67.41%，明顯高于陰性對(duì)照組（15.70%），差異有統(tǒng)計(jì)學(xué)意義（t分別為2.786和6.904，P＜0.05）。 結(jié)論化學(xué)合成的HIF-1α siRNA能有效抑制大鼠視網(wǎng)膜血管內(nèi)皮細(xì)胞在缺氧條件下HIF-1α基因及蛋白的表達(dá)，從而降低細(xì)胞增殖活性。
[Abstract]:Objective to investigate the role of hypoxia-inducible factor-1 偽 (HIF-1 偽) in the pathogenesis of retinopathy of preterm infant retinopathy (ROP) and to find a new target for gene therapy of ROP. Methods the chemically synthesized small interfering RNA(small interference RNAs siRNAs were transfected into rat retinal vascular endothelial cells by liposome mediated transfection. The transfected cells were cultured for 8 hours in a medium containing chemical anoxic agent Cobalt dichloride (CoCl2). The expression of HIF-1 偽 mRNA and protein was detected by fluorescence quantitative polymerase chain reaction (FQ-PCR) and Western blot. Cell proliferation activity was determined by tetramethyl azolium blue colorimetry. The measurement data of normal distribution were expressed by mean 鹵standard deviation (x 鹵s). T test was used to compare the differences between the two groups. Results siRNA was successfully transfected into rat retinal vascular endothelial cells. Fluorescence quantitative polymerase chain reaction (FQ-PCR) analysis showed that all four siRNA could inhibit the transcription expression of HIF-1 偽 in different degree. The HIF-1 偽 mRNA in siRNA4 group and siRNA4 group decreased to 16.20% and 30.49% of the blank control group, respectively, and there was significant difference between the four siRNA strips and the negative control group (83.34%). The expression levels of HIF-1 偽 protein in rat retinal vascular endothelial cells transfected with siRNA1 and siRNA2 were significantly lower than those in control group (P < 0.05).Western) and negative control group (P < 0.05).Western blotting, respectively), and the difference was statistically significant. The inhibitory rates of cell proliferation in the 0.05).siRNA1 and siRNA2 groups were 49.50% and 67.41%, respectively, which were significantly higher than those in the negative control group (P < 0.05). Conclusion chemically synthesized HIF-1 偽 siRNA can effectively inhibit the expression of HIF-1 偽 gene and protein in rat retinal vascular endothelial cells (RVEC) under hypoxia.
【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R722.6

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