【摘要】：目的 用不同濃度的曲尼司特處理體外培養(yǎng)的人視網(wǎng)膜色素上皮細胞（hRPE-19細胞株），觀察其對RPE細胞增殖的影響。 方法 1.體外培養(yǎng)人視網(wǎng)膜色素上皮細胞（hRPE-19細胞株）3-5代后，在倒置顯微鏡下、結(jié)合HE染色觀察細胞形態(tài)，并用細胞角蛋白CK18對細胞進行鑒定。 2.不同濃度的曲尼司特0（對照組）、12.5、25、50、100mg/l在作用hRPE-19細胞12h、24h、48h后，用四甲基偶氮唑藍(MTT)比色法檢測各組hRPE-19細胞的生長抑制率。 3.分別采用蛋白質(zhì)印跡法（Western Blot）和免疫組化法，觀察不同濃度的曲尼司特作用細胞24h后其表達轉(zhuǎn)化生成因子β1（TGF-β1）和血小板衍化生長因子受體A（PDGFR-A）蛋白的情況。 結(jié)果 1.體外培養(yǎng)的hRPE-19細胞為貼壁生長的不規(guī)則多角形細胞，細胞質(zhì)內(nèi)含少量的色素顆粒。細胞角蛋白CK18處理后細胞質(zhì)為棕黃色陽性。 2. MTT檢測結(jié)果示作用相同時間時，隨著曲尼司特濃度的增加，其對hRPE-19細胞的抑制率也漸增大，在一定范圍內(nèi)具有劑量依賴性，差異有統(tǒng)計學意義（P＜0.05)。 3.蛋白質(zhì)印跡法（Western Blot）顯示在作用細胞24h時，隨著曲尼司特濃度的增加，其對應條帶的灰度值逐漸減小，表達TGF-β1和PDGFR-A蛋白量漸少，差異有統(tǒng)計學意義（P＜0.05)。 4.免疫組化結(jié)果說明TGF-β1蛋白定位于hRPE-19細胞漿，，PDGFR-A蛋白定位于hRPE-19胞膜和胞漿，它們的表達量都隨曲尼司特濃度的增加而減少，差異有統(tǒng)計學意義（P＜0.05)。 結(jié)論 曲尼司特可以抑制人視網(wǎng)膜色素上皮細胞的增殖，并且在一定范圍內(nèi)具有劑量依賴性，差異有統(tǒng)計學意義（P＜0.05)，其抑制增殖的機制可能與其下調(diào)TGF-β1和PDGFR-A蛋白表達有關(guān)。
[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.
【學位授予單位】：遼寧醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R774

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