Wu Q N, Ling Y Z, Ouyang C, Xie L J, Huang T
Department of ophthalmology, Sun Yat-sen University, Zhongshan Ophthalmic Centre, Guangzhou 510060, China.
Zhonghua Yan Ke Za Zhi. 2016 Sep 11;52(9):686-92. doi: 10.3760/cma.j.issn.0412-4081.2016.09.010.
To evaluate the effect of thiazovivin, a novel ROCK inhibitor, on the morphology and function of human corneal endothelial cells(HCECs).
The primary HCECs were identified by light microscopy and immunofluorescence staining of neuron-specific enolase. To screen the optimal concentration and action time of thiazovivin for maintaining the morphology and function of primary HCECs, Na (+)/K (+)-ATPase and N-cadherin were chosen as indicators, and the morphology and function of HCECs in various concentrations(0 μmol/L, 2 μmol/L, 4 μmol/L, and 6 μmol/L)for different durations(24 h and 48 h)were examined by immunofluorescence experiments. The effect of thiazovivin on the expression of ROCK was investigated by immunofluorescence and Western blot.
The primary HCECs cultured were hexagonal, closely packed, homogeneously and obviously stained by neuron-specific enolase. The immunofluorescence staining of Na(+)/K(+)-ATPase showed that when the primary HCECs cultured with various concentrations of thiazovivin(0, 2, 4, 6 μmol/L)for 24 h, the fluorescence were obvious, and the average absorbance values(A)were 1.27±0.08, 3.72±0.17, 21.07±4.67, 3.69±0.34, respectively. And the immunofluorescence staining of N-cadherin revealed that when the primary HCECs treated with 4 μmol/L thiazovivin for 24 h, the cell boundary was clear and the structure of the cells was intact. While the treating time of thiazovivin(4 μmol/L)on HCECs extended to 48 h, the immunofluorescence staining of Na(+)/K(+)-ATPase and N-cadherin showed that compared to HCECs treated with thiazovivin(4 μmol/L)for 24 h, the fluorescence intensity did not change significantly, but the cells arranged slightly untidy. In addition, the immunofluorescence staining of ROCK was weakened and the expression of ROCK was reduced by thiazovivin. Thiazovivin was effective for protecting the morphology and function of HCECs. An optimal improvement in the morphology, connection and function of HCECs was found when the primary HCECs were cultured with 4 μmol/L thiazovivin for 24 h. Moreover, the expression of ROCK protein could be significantly inhibited by thiazovivin. (Chin J Ophthalmol, 2016, 52: 686-692).
评估新型ROCK抑制剂噻唑维因对人角膜内皮细胞（HCECs）形态和功能的影响。
通过光学显微镜和神经元特异性烯醇化酶免疫荧光染色鉴定原代HCECs。为筛选噻唑维因维持原代HCECs形态和功能的最佳浓度及作用时间，选择Na(+)/K(+)-ATP酶和N-钙黏蛋白作为指标，通过免疫荧光实验检测不同浓度（0 μmol/L、2 μmol/L、4 μmol/L和6 μmol/L）的噻唑维因作用不同时间（24小时和48小时）时HCECs的形态和功能。通过免疫荧光和蛋白质印迹法研究噻唑维因对ROCK表达的影响。
培养的原代HCECs呈六边形，排列紧密，神经元特异性烯醇化酶染色均匀且明显。Na(+)/K(+)-ATP酶免疫荧光染色显示，原代HCECs用不同浓度（0、2、4、6 μmol/L）的噻唑维因培养24小时时，荧光明显，平均吸光度值（A）分别为1.27±0.08、3.72±0.17、21.07±4.67、3.69±0.34。N-钙黏蛋白免疫荧光染色显示，原代HCECs用4 μmol/L噻唑维因处理24小时时，细胞边界清晰，细胞结构完整。当噻唑维因（4 μmol/L）对HCECs的处理时间延长至48小时时，Na(+)/K(+)-ATP酶和N-钙黏蛋白免疫荧光染色显示，与用噻唑维因（4 μmol/L）处理24小时的HCECs相比，荧光强度无明显变化，但细胞排列稍显紊乱。此外，噻唑维因使ROCK免疫荧光染色减弱，ROCK表达降低。噻唑维因对保护HCECs的形态和功能有效。原代HCECs用4 μmol/L噻唑维因培养24小时时，HCECs的形态、连接和功能得到最佳改善。此外，噻唑维因可显著抑制ROCK蛋白的表达。（《中华眼科杂志》，2016年，52: 686 - 692）
