纤维连接蛋白对POAG小梁网细胞增殖、黏附、迁移及对整合素α5β1、α4β1表达的影响

发布时间：2018-03-02 00:23

本文关键词： POAG小梁网细胞纤维连接蛋白整合素α5β1 整合素α4β1 免疫细胞化学　出处：《福建医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的探讨体外培养原发性开角型青光眼小梁网细胞的方法及其生物学特性,在此基础上研究纤维连接蛋白(fibronectin, FN)对POAG小梁网细胞增殖、黏附、迁移能力以及表达整合素α5β1和α4β1(integrinα5β1,α4β1)的影响。方法采用原发性开角型青光眼患者小梁切除术中取得带小梁网的巩膜组织块进行体外培养。运用透射电镜,免疫细胞化学等方法对细胞进行鉴定,并观察其生物学特性。采用CCK-8法和Transwell小室法检测体外培养的传3代小梁网细胞经浓度为0μg/ml(对照组),5μg/ml,10μg/ml,20μg/ml,40μg/ml,100μg/ml的FN处理24小时后,观察其细胞增殖、黏附、迁移能力以及表达整合素α5β1以及α4β1的表达量的变化。结果1.组织块培养10天左右,可见细胞从其边缘向外生长。经鉴定为小梁网细胞,免疫细胞化学示纤维连接蛋白、层粘连蛋白、神经元烯醇化酶表达阳性。电镜透视可见细胞表面的微绒毛,胞浆的溶酶体、吞噬小泡等超微结构。 2.应用CCK-8法和Transwell法检测经浓度为5μg/ml,10μg/ml,20μg/ml,40μg/ml,100μg/ml的FN处理的实验组细胞吸光度的比值增殖分别0.45±0.016,0.52±0.015,0.65±0.011,0.78±0.012及1.15±0.014,各实验组与对照组0.37±0.018相比,P0.05,均有显著性差异,黏附分别为0.38±0.021,0.43±0.015,0.52±0.0210.78±0.025,0.85±0.014及1.19±0.019各实验组与对照组0.38±0.021相比,P0.05,均有显著性差异。迁移分别为0.59±0.005,0.62±0.006,0.68±0.002,0.78±0.003及0.89±0.001各实验组与对照组0.57±0.006(P0.05),均有显著性差异。 3.免疫细胞化学染色法检测经浓度分别为5μg/ml,10μg/ml,20μg/ml,40μg/ml,100μg/ml的FN处理,实验组细胞整合素α5β1表达分别为0.242±0.015,0.254±0.018,0.261±0.005,0.267±0.014,0.365±0.019,与对照组0.239±0.011相比,P0.05均有显著差异。阳性面积比值分别为实验组0.1428±0.013,0.1946±0.018,0.2219±0.016,0.2679±0.011,0.17993±0.019,与对照组0.1252±0.012相比,P0.05均有显著差异。整合素α4β1表达分别为0.232±0.005,0.245±0.003,0.263±0.005,0.276±0.004,与对照组0.231±0.001相比,P0.05均有显著差异。阳性面积比值分别为实验组0.1218±0.013,0.1346±0.016,0.1419±0.012,0.1679±0.011,0.1993±0.019,对照组0.1202±0.012,P0.05均有显著差异。结论1.采用组织块培养法能成功体外培养原发性开角型青光眼人眼小梁网细胞,并鉴定其生物特性。 2.原发性开角型青光眼小梁网细胞表达整合素α5β1以及α4β1。 3.FN能促进体外培养原发性开角型青光眼小梁网细胞增殖、黏附、迁移能力,并在一定范围内呈现剂量依赖性。 4.FN能增加体外培养原发性开角型青光眼小梁网细胞整合素α5β1以及α4β1的表达,并在一定范围内呈现剂量依赖性。
[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 伪 5 尾 1 and 伪 4 尾 1 integrin 伪 5 尾 1, 伪 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 伪 5 尾 1 and 伪 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 卤0.0160.52 卤0.0150.65 卤0.0110.78 卤0.012 and 1.15 卤0.014, respectively, and there was significant difference between the experimental group and the control group (0.37 卤0.018), the ratio of the absorbance of the experimental group was 0.37 卤0.018, and the ratio of proliferation was 0.45 卤0.0160.52 卤0.0150.65 卤0.0110.78 卤0.012 and 1.15 卤0.014, respectively. There were significant differences in adhesion (0.38 卤0.021) 0.43 卤0.015 (0.52 卤0.0210.78 卤0.025) 0.85 卤0.014 and 1.19 卤0.019 between the experimental group and the control group (0.38 卤0.021), the migration was 0.59 卤0.0050.62 卤0.0060.68 卤0.0020.78 卤0.003 and 0.89 卤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 伪 5 尾 1 in the experimental group was 0.242 卤0.015, 0.254 卤0.018, 0.261 卤0.005, 0.267 卤0.014, 0.365 卤0.019, and 0.239 卤0.011, respectively, and the positive area ratio was 0.1428 卤0.0130.2219 卤0.0180.2219 卤0.0160.2679 卤0.0110.17993 卤0.01919, and 0.1252 卤0.012, respectively. The expression of integrin 伪 4 尾 1 was 0.232 卤0.005, 0.245 卤0.003, 0.263 卤0.005N, 0.276 卤0.004, respectively, which was significantly different from that of the control group (0.231 卤0.001). The positive area ratio was 0.1218 卤0.01346 卤0.01346 卤0.0160.1419 卤0.0120.1679 卤0.0110.1993 卤0.019in the experimental group, and 0.1202 卤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 伪 5 尾 1 and 伪 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 伪 5 尾 1 and 伪 4 尾 1 in trabecular reticulum cells of primary open-angle glaucoma in a dose-dependent manner.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R775

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