本文關(guān)鍵詞： POAG小梁網(wǎng)細胞 纖維連接蛋白 整合素α5β1 整合素α4β1 免疫細胞化學　出處：《福建醫(yī)科大學》2010年碩士論文　論文類型：學位論文

【摘要】： 目的探討體外培養(yǎng)原發(fā)性開角型青光眼小梁網(wǎng)細胞的方法及其生物學特性,在此基礎上研究纖維連接蛋白(fibronectin, FN)對POAG小梁網(wǎng)細胞增殖、黏附、遷移能力以及表達整合素α5β1和α4β1(integrinα5β1,α4β1)的影響。 方法采用原發(fā)性開角型青光眼患者小梁切除術(shù)中取得帶小梁網(wǎng)的鞏膜組織塊進行體外培養(yǎng)。運用透射電鏡,免疫細胞化學等方法對細胞進行鑒定,并觀察其生物學特性。采用CCK-8法和Transwell小室法檢測體外培養(yǎng)的傳3代小梁網(wǎng)細胞經(jīng)濃度為0μg/ml(對照組),5μg/ml,10μg/ml,20μg/ml,40μg/ml,100μg/ml的FN處理24小時后,觀察其細胞增殖、黏附、遷移能力以及表達整合素α5β1以及α4β1的表達量的變化。 結(jié)果1.組織塊培養(yǎng)10天左右,可見細胞從其邊緣向外生長。經(jīng)鑒定為小梁網(wǎng)細胞,免疫細胞化學示纖維連接蛋白、層粘連蛋白、神經(jīng)元烯醇化酶表達陽性。電鏡透視可見細胞表面的微絨毛,胞漿的溶酶體、吞噬小泡等超微結(jié)構(gòu)。 2.應用CCK-8法和Transwell法檢測經(jīng)濃度為5μg/ml,10μg/ml,20μg/ml,40μg/ml,100μg/ml的FN處理的實驗組細胞吸光度的比值增殖分別0.45±0.016,0.52±0.015,0.65±0.011,0.78±0.012及1.15±0.014,各實驗組與對照組0.37±0.018相比,P0.05,均有顯著性差異,黏附分別為0.38±0.021,0.43±0.015,0.52±0.0210.78±0.025,0.85±0.014及1.19±0.019各實驗組與對照組0.38±0.021相比,P0.05,均有顯著性差異。遷移分別為0.59±0.005,0.62±0.006,0.68±0.002,0.78±0.003及0.89±0.001各實驗組與對照組0.57±0.006(P0.05),均有顯著性差異。 3.免疫細胞化學染色法檢測經(jīng)濃度分別為5μg/ml,10μg/ml,20μg/ml,40μg/ml,100μg/ml的FN處理,實驗組細胞整合素α5β1表達分別為0.242±0.015,0.254±0.018,0.261±0.005,0.267±0.014,0.365±0.019,與對照組0.239±0.011相比,P0.05均有顯著差異。陽性面積比值分別為實驗組0.1428±0.013,0.1946±0.018,0.2219±0.016,0.2679±0.011,0.17993±0.019,與對照組0.1252±0.012相比,P0.05均有顯著差異。 整合素α4β1表達分別為0.232±0.005,0.245±0.003,0.263±0.005,0.276±0.004,與對照組0.231±0.001相比,P0.05均有顯著差異。陽性面積比值分別為實驗組0.1218±0.013,0.1346±0.016,0.1419±0.012,0.1679±0.011,0.1993±0.019,對照組0.1202±0.012,P0.05均有顯著差異。 結(jié)論1.采用組織塊培養(yǎng)法能成功體外培養(yǎng)原發(fā)性開角型青光眼人眼小梁網(wǎng)細胞,并鑒定其生物特性。 2.原發(fā)性開角型青光眼小梁網(wǎng)細胞表達整合素α5β1以及α4β1。 3.FN能促進體外培養(yǎng)原發(fā)性開角型青光眼小梁網(wǎng)細胞增殖、黏附、遷移能力,并在一定范圍內(nèi)呈現(xiàn)劑量依賴性。 4.FN能增加體外培養(yǎng)原發(fā)性開角型青光眼小梁網(wǎng)細胞整合素α5β1以及α4β1的表達,并在一定范圍內(nèi)呈現(xiàn)劑量依賴性。
[Abstract]:Objective to investigate the methods and biological characteristics of trabecular reticulum cells cultured in vitro for primary open-angle glaucoma, and to study the proliferation and adhesion of fibronectin (FNN) to POAG trabecular reticulum cells. Migration and expression of integrin 偽 5 尾 1 and 偽 4 尾 1 integrin 偽 5 尾 1, 偽 4 尾 1. Methods the scleral tissue with trabecular meshwork was obtained from trabeculectomy in patients with primary open angle glaucoma in vitro. The cells were identified by transmission electron microscopy and immunocytochemistry. CCK-8 assay and Transwell chamber assay were used to detect the proliferation and adhesion of trabecular meshwork reticulum cells cultured in vitro after treated with 10 渭 g / ml 10 渭 g / ml 10 渭 g / ml 10 渭 g / ml of 10 渭 g / ml FN for 24 hours. Migration ability and expression of integrin 偽 5 尾 1 and 偽 4 尾 1. Results 1. After about 10 days of tissue mass culture, the cells grew from the edge to the outside. They were identified as trabecular meshwork cells. Immunocytochemistry showed fibronectin, laminin, and laminin. 2. The expression of neuronal enolase was positive. The ultrastructure of cell surface microvilli, cytoplasmic lysosomes and phagocytic vesicles were observed by electron microscopy. 2. CCK-8 and Transwell were used to detect the ratio of absorbance proliferation of the experimental group treated with 10 渭 g / ml 10 渭 g / ml 10 渭 g / ml FN of 40 渭 g / ml of 40 渭 g / ml FN, respectively. The ratio of proliferation of the cells in the experimental group was 0.45 鹵0.0160.52 鹵0.0150.65 鹵0.0110.78 鹵0.012 and 1.15 鹵0.014, respectively, and there was significant difference between the experimental group and the control group (0.37 鹵0.018), the ratio of the absorbance of the experimental group was 0.37 鹵0.018, and the ratio of proliferation was 0.45 鹵0.0160.52 鹵0.0150.65 鹵0.0110.78 鹵0.012 and 1.15 鹵0.014, respectively. There were significant differences in adhesion (0.38 鹵0.021) 0.43 鹵0.015 (0.52 鹵0.0210.78 鹵0.025) 0.85 鹵0.014 and 1.19 鹵0.019 between the experimental group and the control group (0.38 鹵0.021), the migration was 0.59 鹵0.0050.62 鹵0.0060.68 鹵0.0020.78 鹵0.003 and 0.89 鹵0.001 respectively. 3. Immunocytochemical staining was used to detect FN treated with 10 渭 g / ml 10 渭 g / ml 10 渭 g / ml or 40 渭 g / ml 40 渭 g / ml / ml 100 渭 g / ml FN, respectively. The expression of integrin 偽 5 尾 1 in the experimental group was 0.242 鹵0.015, 0.254 鹵0.018, 0.261 鹵0.005, 0.267 鹵0.014, 0.365 鹵0.019, and 0.239 鹵0.011, respectively, and the positive area ratio was 0.1428 鹵0.0130.2219 鹵0.0180.2219 鹵0.0160.2679 鹵0.0110.17993 鹵0.01919, and 0.1252 鹵0.012, respectively. The expression of integrin 偽 4 尾 1 was 0.232 鹵0.005, 0.245 鹵0.003, 0.263 鹵0.005N, 0.276 鹵0.004, respectively, which was significantly different from that of the control group (0.231 鹵0.001). The positive area ratio was 0.1218 鹵0.01346 鹵0.01346 鹵0.0160.1419 鹵0.0120.1679 鹵0.0110.1993 鹵0.019in the experimental group, and 0.1202 鹵0.0122in the control group. Conclusion 1. Human trabecular meshwork cells of primary open angle glaucoma can be successfully cultured in vitro by tissue mass culture and their biological characteristics are identified. 2. 2.The expression of integrin 偽 5 尾 1 and 偽 4 尾 1 in trabecular reticulum cells of primary open-angle glaucoma. 3. FN could promote the proliferation, adhesion and migration of trabecular meshwork cells cultured in vitro in a dose-dependent manner. 4. FN increased the expression of integrin 偽 5 尾 1 and 偽 4 尾 1 in trabecular reticulum cells of primary open-angle glaucoma in a dose-dependent manner.
【學位授予單位】：福建醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R775

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1 劉馳;付榮嶸;;纖維連接蛋白對人晶狀體上皮細胞系的增殖、黏附和移行的影響[J];國際眼科雜志;2009年08期

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