牵拉力对人小梁网细胞活性及房水流出调节功能影响的实验研究

Effect of stretch on human trabecular cell viability and function regulation of aqueous humor outflow

目的观察强机械牵拉力作用下,人小梁网细胞活性及房水流出调节相关基因表达的改变。设计实验研究。研究对象人小梁网细胞。方法依据牵拉力作用时间将原代培养的小梁网细胞分为0 h对照、12 h、24 h三组。采用FLEXCELL力学加载设备对体外培养的人小梁网细胞施加最大延伸比例为22%的机械牵拉力。用流式细胞学的方法检测各组小梁细胞的凋亡比率,采用蛋白印迹法检测细胞存活相关蛋白的表达,用定量聚合酶链式反应(qPCR)检测与房水流出调节相关的15个基因在牵张力作用下的表达量,并比较实验组与对照组间的差异。主要指标小梁网细胞凋亡率、整合素-Fak信号通路相关分子、房水流出调节相关的15个基因的表达量。结果在牵拉力作用后,对照组、12 h组、24 h组的凋亡率分别为0.71%±0.18%、0.77%±0.19%、0.87%±0.31%(P=0.7298);三组磷酸化Akt蛋白的相对表达量分别为1.00、2.17±0.06、1.69±0.07(P<0.01);整合素信号通路中的磷酸化Fak的相对表达量分别为1.00、1.85±0.09、1.31±0.08(P<0.01)。此外,参与调节房水流出的基质金属蛋白酶1、2、3、7、9、10、11、12、14基因表达均升高(P均<0.01);基质金属蛋白酶抑制剂1、2、3基因的表达均降低(P均<0.01);白介素6基因相对表达量分别为1.00、1.28±0.06、1.47±0.06(P<0.01)。结论高强度的机械牵拉能够增加小梁网细胞的活性,增强房水流出调节相关因子的表达,这可能是schlemm管成形术降低眼压的机制之一。

Objective Observing the changes of human trabecular cell viability and expression of genes involved in aqueous humor outflow regulation under the strong mechanical stretch. Design Experimental study. Participants Human trabecular meshwork cells(HTMCs). Methods The HTMCs were divided into three groups: 0 h control, 12 h and 24 h according to the stretch time. Cultured HTMCs were subjected cyclic stretch on the Flexcell Strain system at a level of 22% increment in surface area for 12, 24 hours. The AV-PI method was used to detect the apoptosis rate of HTMCs. Western-blotting was used to detect the expression of signal pathway protein related to survive, and qPCR was used to detect the expression of the genes related to the regulation of aqueous outflow. Main Outcome Measures The apoptosis rate of HTMCs, the expression of integrin-Fak-Akt cell signaling pathway, the expressions of 15 genes related to aqueous humor outflow regulation. Results Compared the HTMCs undergoing the mechanical stretch (0, 12, 24 h), the apoptosis rate was 0.71%±0.18%, 0.77%±0.19%, 0.87%±0.31%, respectively (P=0.7298). The phosphorylated Akt was 1.00, 2.17±0.06, 1.69±0.07, respectively (P<0.01). The phosphorylated Fak397 was 1.00, 1.85±0.09, 1.31±0.08, respectively (P<0.01). At the same time, the expression of matrix metalloproteinases (1, 2, 3, 7, 9, 10, 11, 12, 14) increased (all P<0.01). The expression of metallopeptidase inhibitor (1, 2, 3) decreased (all P<0.01). And the expression of IL-6 was 1.00, 1.28±0.06, 1.47±0.06, respectively(P<0.01). Conclusion High strength mechanical stretch can increase the vitality of trabecular meshwork cells and promote the secretion of aqueous outflow regulation related factors. This may be one of the mechanisms of canaloplasty to reduce the intraocular pressure.