Myocilin蛋白对人眼小梁网细胞FN表达及细胞迁移与粘附的影响

发布时间：2018-05-15 18:23

  本文选题：细胞培养 + 小梁网细胞　； 参考：《福建医科大学》2012年硕士论文

【摘要】：目的在体外培养人眼小梁网细胞的基础上，研究myocilin蛋白对人眼小梁网细胞纤维连接蛋白(fibronectin，FN)表达以及对小梁网细胞迁移、粘附功能的影响，探索myocilin蛋白与原发性开角型青光眼发病的关系。 方法采用组织块培养法体外培养正常人眼小梁网细胞，并运用倒置显微镜观察、透射电镜和免疫细胞化学等方法对细胞进行鉴定。取传三代的小梁网细胞分别加入含重组myocilin蛋白终浓度为0ug/m（l对照组）、0.5ug/ml、1ug/ml、1.5ug/ml、2ug/ml、2.5ug/ml的无血清培养基，继续培养24小时后，运用Western blot、Elisa法分别检测小梁网细胞内及细胞培养液中FN的表达水平；运用Transwell小室法、CCK-8法检测myocilin蛋白对小梁网细胞迁移、粘附的影响。 结果1.成功建立人眼小梁网细胞体外培养体系，经鉴定确定为人眼小梁网细胞。 2. Western blot检测结果显示，在一定范围内，随myocilin蛋白浓度的增高小梁网细胞内FN表达呈下降趋势。3. Elisa法检测经浓度为0ug/ml、0.5ug/ml、1ug/ml、1.5ug/ml、2ug/ml、2.5ug/ml myocilin蛋白干预的各组细胞培养液中FN浓度为：0.5817±0.0959ug/ml、0.5572±0.0443ug/ml、0.5868±0.0735ug/ml、0.5015±0.0472ug/ml、0.4263±0.0426ug/ml、0.2838±0.0972ug/ml。1.5ug/ml、2ug/ml、2.5ug/ml myocilin蛋白组与对照组的差异有统计学意义(P0.05)，，且此三组实验组组间比较存在显著性差异。4.Transwell小室法检测结果显示，实验组细胞迁移数较对照组少，且实验组与对照组的差异有统计学意义(P0.05）。5.运用CCK-8法检测经浓度为0ug/ml、0.5ug/ml、1ug/ml、1.5ug/ml、2ug/ml、2.5ug/mlmyocilin蛋白处理的各组细胞吸光度值的均值分别为0.1614±0.0061、0.1529±0.0139、0.1273±0.0172、0.1271±0.0230、0.1165±0.0084、0.1103±0.0045。随myocilin蛋白浓度的增高各组细胞吸光度值呈现下降趋势，各实验组与对照组相比差异有统计学意义（P0.05）。 结论1.myocilin蛋白能抑制小梁网细胞纤维连接蛋白的表达，并呈现一定的剂量依耐性；2.myocilin蛋白可降低小梁网细胞的迁移和粘附能力。
[Abstract]:Objective to study the effects of myocilin protein on the expression of fibronectin FN and the migration and adhesion of human trabecular meshwork cells in vitro. To explore the relationship between myocilin protein and primary open angle glaucoma. Methods normal human trabecular meshwork cells were cultured in vitro by tissue mass culture, and the cells were identified by inverted microscope, transmission electron microscope and immunocytochemistry. The third generation trabecular meshwork cells were added to the serum-free medium containing the final concentration of recombinant myocilin protein as the 0ug/m(l control group (0ug/m(l control group). After 24 hours of culture, the expression of FN in trabecular meshwork cells and cell culture medium was detected by Western blottimerassay. The effect of myocilin protein on the migration and adhesion of trabecular meshwork cells was detected by Transwell chamber assay and CCK-8 method. Result 1. The in vitro culture system of human trabecular meshwork cells was successfully established and identified as human trabecular meshwork cells. 2. The results of Western blot showed that the expression of FN in trabecular meshwork cells decreased with the increase of myocilin protein concentration in a certain range. Elisa娉曟��娴嬬粡娴撳害涓

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