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  本文關(guān)鍵詞： opticin 視網(wǎng)膜色素上皮細(xì)胞 血管內(nèi)皮生長因子　出處：《浙江大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：摘要目的：探討缺氧血管內(nèi)皮生長因子對視網(wǎng)膜色素上皮細(xì)胞opticin分泌的影響及內(nèi)在機(jī)制。 方法：原代培養(yǎng)人眼視網(wǎng)膜膜色素上皮(retinal pigment epithelium, RPE)細(xì)胞,取生長良好的第3-6代細(xì)胞接種于含DMEM培養(yǎng)液的六孔板中,于缺氧條件下或加入不同濃度血管內(nèi)皮生長因子(vascular endothelial growth factor, VEGF) (lng/ml、10ng/ml、50ng/ml、100ng/ml)進(jìn)行培養(yǎng),RIPA裂解液裂解細(xì)胞提取總蛋白并收集細(xì)胞培養(yǎng)液。逆轉(zhuǎn)錄PCR檢測缺氧后RPE細(xì)胞中opticin、VEGF mRNA的表達(dá)。蛋白印跡法檢測細(xì)胞內(nèi)和細(xì)胞培養(yǎng)液中的opticin的含量。明膠酶譜法用于檢測細(xì)胞培養(yǎng)液中的明膠酶活性。各組數(shù)據(jù)采用單因素方差分析。 結(jié)果：逆轉(zhuǎn)錄PCR顯示缺氧后RPE細(xì)胞opticin mRNA表達(dá)未受影響,而VEGF mRNA表達(dá)升高。蛋白印跡法顯示缺氧后RPE細(xì)胞內(nèi)opticin的含量與對照組無顯著差異,而在RPE細(xì)胞培養(yǎng)液中缺氧組opticin含量明顯下降。不同濃度的外源性的VEGF作用同樣不影響RPE細(xì)胞內(nèi)opticin的表達(dá),差異無統(tǒng)計(jì)學(xué)意義(F=2.29,P=0.095),而在VEGF添加組RPE細(xì)胞培養(yǎng)液中opticin含量較正常組明顯下降(F=107.28,P0.001)。明膠酶譜結(jié)果顯示,在與基質(zhì)金屬蛋白酶(matrix metalloproteinases, MMP)2型相對應(yīng)的位置出現(xiàn)明顯的消化條帶,且酶的活性隨VEGF濃度的增加而遞增。低濃度EDTA可抑制由VEGF引起的RPE細(xì)胞培養(yǎng)液中opticin含量的減少。 結(jié)論：缺氧以及VEGF作用可影響RPE細(xì)胞的opticin分泌,可能與增多的MMP-2對opticin的酶解作用有關(guān)。
[Abstract]:Objective: To investigate the effect of hypoxia vascular endothelial growth factor on the secretion of opticin in retinal pigment epithelial cells (RPE) and its intrinsic mechanism.
Methods: primary cultured human retinal pigment epithelial membrane (retinal pigment epithelium, RPE) cells, the 3-6 generation cells were well grown in 6-well plate containing DMEM culture liquid, in anoxic conditions or with different concentrations of vascular endothelial growth factor (vascular endothelial, growth factor, VEGF) (lng/ml, 10ng/ml, 50ng/ml, 100ng/ml) were cultured in RIPA cell lysate protein extraction and cell culture medium was collected. Opticin RT PCR detection after hypoxia in RPE cells, the expression of VEGF mRNA. Western blot was used to detect the content in the cell culture medium and opticin medium. For the activity of gelatinase zymography to detect cell culture. All the data were analyzed by one-way ANOVA.
Results: PCR showed that RPE cells opticin reverse transcriptase mRNA expression was not affected after hypoxia, while VEGF increased the expression of mRNA. Western blot shows that hypoxia in RPE cells after opticin content had no significant difference with control group, hypoxia group decreased significantly and the cultivation of the content of opticin in the solution in RPE cells. The effects of different concentrations of exogenous VEGF also RPE did not affect the expression of opticin in cells, the difference was not statistically significant (F=2.29, P=0.095), and the addition of VEGF RPE cells cultured in opticin were significantly decreased than that of normal group (F=107.28, P0.001). The results show that in gelatin zymography, and matrix metalloproteinases (matrix, metalloproteinases, MMP) position corresponding to type 2 the emergence of a distinct band of digestive enzyme activity, and increased with the increase of VEGF concentration. Low concentration of EDTA could be inhibited by VEGF induced RPE cells decreased the content of opticin in the solution.
Conclusion: the effect of hypoxia and VEGF on the secretion of opticin in RPE cells may be related to the enzymatic hydrolysis of opticin by the increase of MMP-2.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R774.1

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本文編號：1484203