缺氧以及血管内皮生长因子对视网膜色素上皮细胞opticin表达的影响

发布时间：2018-02-02 09:36

本文关键词： opticin 视网膜色素上皮细胞血管内皮生长因子　出处：《浙江大学》2011年硕士论文　论文类型：学位论文

【摘要】：摘要目的：探讨缺氧血管内皮生长因子对视网膜色素上皮细胞opticin分泌的影响及内在机制。方法：原代培养人眼视网膜膜色素上皮(retinal pigment epithelium, RPE)细胞,取生长良好的第3-6代细胞接种于含DMEM培养液的六孔板中,于缺氧条件下或加入不同浓度血管内皮生长因子(vascular endothelial growth factor, VEGF) (lng/ml、10ng/ml、50ng/ml、100ng/ml)进行培养,RIPA裂解液裂解细胞提取总蛋白并收集细胞培养液。逆转录PCR检测缺氧后RPE细胞中opticin、VEGF mRNA的表达。蛋白印迹法检测细胞内和细胞培养液中的opticin的含量。明胶酶谱法用于检测细胞培养液中的明胶酶活性。各组数据采用单因素方差分析。结果：逆转录PCR显示缺氧后RPE细胞opticin mRNA表达未受影响,而VEGF mRNA表达升高。蛋白印迹法显示缺氧后RPE细胞内opticin的含量与对照组无显著差异,而在RPE细胞培养液中缺氧组opticin含量明显下降。不同浓度的外源性的VEGF作用同样不影响RPE细胞内opticin的表达,差异无统计学意义(F=2.29,P=0.095),而在VEGF添加组RPE细胞培养液中opticin含量较正常组明显下降(F=107.28,P0.001)。明胶酶谱结果显示,在与基质金属蛋白酶(matrix metalloproteinases, MMP)2型相对应的位置出现明显的消化条带,且酶的活性随VEGF浓度的增加而递增。低浓度EDTA可抑制由VEGF引起的RPE细胞培养液中opticin含量的减少。结论：缺氧以及VEGF作用可影响RPE细胞的opticin分泌,可能与增多的MMP-2对opticin的酶解作用有关。
[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.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R774.1

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