Myocilin蛋白对青光眼小梁网细胞FN表达和黏附能力的抑制作用

Inhibitory effect of myocilin on fibronectin expression and adhesion in trabecular meshwork cells of glaucoma

目的:分析不同浓度重组myocilin蛋白对体外培养的原发性开角型青光眼(primary open angle glaucoma,POAG)患者小梁网细胞纤维连接蛋白(fibronectin,FN)表达及对黏附功能的影响。方法:体外培养POAG患者小梁网细胞,分别加入含重组myocilin蛋白终浓度为0(对照组)、0.5、1、1.5、2、2.5μg/mL的无血清培养基,运用Western blot、ELISA法分别检测小梁网细胞内和细胞培养液中FN的表达水平;并运用CCK-8法检测myocilin蛋白对POAG小梁网细胞黏附的影响。结果:Western blot检测结果:FN/β-actin比值分别为34.8±0.6、33.4±1.0、28.9±0.8、21.6±0.9、15.9±1.1、11.9±0.8;0.5μg/mL组与对照组相比,差异无统计学意义(P=0.092);1.0、1.5、2、2.5μg/mL组与对照组比较,差异有统计学意义(F=346.131,P<0.05)。ELISA法检测各组细胞培养液中FN浓度为:0.4654±0.0039、0.4596±0.0032、0.4216±0.0037、0.4214±0.0039、0.4043±0.0039、0.3806±0.0071μg/mL;0.5μg/mL组与对照组相比,差异无统计学意义(P=0.120);1.0、1.5、2、2.5μg/mL组与对照组比较,差异有统计学意义(F=176.054,P<0.05)。运用CCK-8法检测各组细胞吸光度均值分别为1.98140.1624、1.8848±0.0267、1.4895±0.0916、1.4120±0.1087、1.3392±0.1391、1.0310±0.0639;0.5μg/mL组与对照组相比,差异无统计学意义(P=0.300);1、1.5、2、2.5μg/mL组与对照组相比,差异有统计学意义(F=177.818,P<0.05)。结论:Myocilin蛋白能抑制POAG小梁网细胞FN的表达,并呈现一定的剂量依赖性;myocilin蛋白可降低POAG小梁网细胞的黏附能力。

AIM:To investigate the effect of myocilin on the expression of fibronectin(FN) and the adhesion function in trabecular meshwork cells of primary open angle glaucoma(POAG) in vitro.METHODS:The concentration of recombinant myocilin protein was 0(control group),0.5,1,1.5,2 and2.5μ g/mL,respectively.The expression of FN in the cells and the cell culture medium were detected by Western blot and ELISA.The effect of myocilin protein on the adhesion of cultured POAG trabecular meshwork cells was detected by CCK-8 method.RESULTS:Western blot showed that the ratio of FN/β-actin was 34.8±0.6,33.4±1.0,28.9±0.8,21.6±0.9,15.9±1.1 and 11.9 ± 0.8;that of 0.5μ g/mL group was not significantly different compared with that of control group(P=0.092);that of 1.0,1.5,2 and 2.5μ g/mL group were significantly different compared with that of the control group(F=346.131,P<0.05).The concentration of FN in the cell culture medium was 0.4654 ± 0.0039,0.4596 ±0.0032,0.4216±0.0037,0.4214 ±0.0039,0.4043 ±0.0039,0.3806±0.0071μ g/mL by ELISA;difference between that of0.5μ g/mL group and that of control group was not statistically significant(P=0.120);the difference between of 1,1.5,2,2.5μ g/mL group and the control group were statistically significant(F = 176.054,P<0.05).The mean values of the absorbance values of each group were1.9814±0.1624,1.8848±0.0267,1.4895±0.0916,1.4120±0.1087,1.3392 ± 0.1391,1.0310 ± 0.0639 through CCK-8 method;that of 0.5μ g/mL group was not significantly different compared with that of control group(P=0.300);the difference between of 1,1.5,2,2.5μ g/mL group and the control group were statistically significant(F =177.818,P<0.05). CONCLUSION:Myocilin protein can inhibit the fibronectin expression in POAG trabecular meshwork cells,showing a dose dependent manner.Myocilin protein can reduce the adhesion of POAG trabecular meshwork cells.