曲尼司特对体外培养人视网膜色素上皮细胞增殖的影响

发布时间：2018-10-12 21:35

【摘要】：目的用不同浓度的曲尼司特处理体外培养的人视网膜色素上皮细胞（hRPE-19细胞株），观察其对RPE细胞增殖的影响。方法 1.体外培养人视网膜色素上皮细胞（hRPE-19细胞株）3-5代后，在倒置显微镜下、结合HE染色观察细胞形态，并用细胞角蛋白CK18对细胞进行鉴定。 2.不同浓度的曲尼司特0（对照组）、12.5、25、50、100mg/l在作用hRPE-19细胞12h、24h、48h后，用四甲基偶氮唑蓝(MTT)比色法检测各组hRPE-19细胞的生长抑制率。 3.分别采用蛋白质印迹法（Western Blot）和免疫组化法，观察不同浓度的曲尼司特作用细胞24h后其表达转化生成因子β1（TGF-β1）和血小板衍化生长因子受体A（PDGFR-A）蛋白的情况。结果 1.体外培养的hRPE-19细胞为贴壁生长的不规则多角形细胞，细胞质内含少量的色素颗粒。细胞角蛋白CK18处理后细胞质为棕黄色阳性。 2. MTT检测结果示作用相同时间时，随着曲尼司特浓度的增加，其对hRPE-19细胞的抑制率也渐增大，在一定范围内具有剂量依赖性，差异有统计学意义（P＜0.05)。 3.蛋白质印迹法（Western Blot）显示在作用细胞24h时，随着曲尼司特浓度的增加，其对应条带的灰度值逐渐减小，表达TGF-β1和PDGFR-A蛋白量渐少，差异有统计学意义（P＜0.05)。 4.免疫组化结果说明TGF-β1蛋白定位于hRPE-19细胞浆，，PDGFR-A蛋白定位于hRPE-19胞膜和胞浆，它们的表达量都随曲尼司特浓度的增加而减少，差异有统计学意义（P＜0.05)。结论曲尼司特可以抑制人视网膜色素上皮细胞的增殖，并且在一定范围内具有剂量依赖性，差异有统计学意义（P＜0.05)，其抑制增殖的机制可能与其下调TGF-β1和PDGFR-A蛋白表达有关。
[Abstract]:Aim to observe the effect of tranilast on the proliferation of human retinal pigment epithelium (hRPE-19) cells cultured in vitro. Method 1. Human retinal pigment epithelial cells (hRPE-19 cells) were cultured in vitro for 3-5 generations. The morphology of the cells was observed under inverted microscope and HE staining. The cells were identified by cytokeratin CK18. 2. The growth inhibition rate of hRPE-19 cells was measured by (MTT) colorimetry with tetramethylazolyl blue (MTT) assay after 12h ~ 24h and 48h after treatment with different concentrations of tranilast 0 (control group), 12.5U 25U 50100mg / l, respectively. The growth inhibition rate of hRPE-19 cells in each group was measured by (MTT) colorimetry. The expression of transforming factor 尾 1 (TGF- 尾 1) and platelet-derived growth factor receptor A (PDGFR-A) were observed by Western blot (Western Blot) and immunohistochemistry respectively. Result 1. The hRPE-19 cells cultured in vitro were irregular polygonal cells which were adherent to the wall and contained a small amount of pigment granules in the cytoplasm. Cytokeratin CK18 treated cytoplasm was brownish yellow positive. 2. 2. The results of MTT showed that the inhibitory rate of tranilast on hRPE-19 cells gradually increased with the increase of tranilast concentration at the same time, in a dose-dependent manner, the difference was statistically significant (P < 0. 05). Western blotting (Western Blot) showed that with the increase of tranilast concentration, the gray value of the corresponding band gradually decreased, and the expression of TGF- 尾 1 and PDGFR-A protein decreased (P < 0. 05). The immunohistochemical results showed that TGF- 尾 1 protein was localized in the cytoplasm of hRPE-19, and PDGFR-A protein was located in the membrane and cytoplasm of hRPE-19. The expression of PDGFR-A 尾 1 protein decreased with the increase of tranilast concentration, and the difference was statistically significant (P < 0 05). Conclusion tranilast can inhibit the proliferation of human retinal pigment epithelial cells in a dose-dependent manner. The mechanism of inhibiting proliferation may be related to the down-regulation of TGF- 尾 1 and PDGFR-A protein expression.
【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R774

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