Rho相关激酶抑制剂thiazovivin对人角膜内皮细胞形态和功能影响的研究

Effect of a new ROCK inhibitor thiazovivin on the morphology and function of human corneal ;endothelial cells

目的：探讨N-芐基-2-（嘧啶-4-基氨基）（Thiazovivin）对人角膜内皮细胞（HCEC）形态和功能的影响。方法实验研究。培养原代HCEC，采用光镜观察细胞形态及神经元特异性烯醇化酶（NSE）的免疫荧光实验进行鉴定。以N-钙黏着蛋白（N-cadherin）和钠钾泵（Na＋/K＋-ATPase）为指标，通过免疫荧光实验考察添加不同浓度（0、2、4、6μmol/L）Thiazovivin作用不同时间（24及48 h）对原代HCEC形态和离子泵功能的影响，以期筛选出改善HCEC形态和离子泵功能效果最佳的Thiazovivin的浓度及作用时间。最后通过免疫荧光实验和免疫印迹实验考察Thiazovivin对Rho相关激酶（ROCK）表达的影响。结果培养的原代HCEC的NSE染色均匀，荧光强度明显，细胞呈六边形紧密排列。原代HCEC在0、2、4、6μmol/L的Thiazovivin分别作用24 h后，Na＋/K＋-ATPase抗体染色后的平均吸光度（A）值分别为1.27±0.08、3.72±0.17、21.07±4.67、3.69±0.34，4μmol/L的Thiazovivin的Na＋/K＋-ATPase免疫荧光表达最强，其A值最大；通过N-cadherin免疫荧光染色，发现HCEC在4μmol/L Thiazovivin作用24 h后，细胞轮廓清晰，细胞结构完整。而4μmol/L的Thiazovivin作用时间延长至48 h，通过N-cadherin和Na＋/K＋-ATPase免疫荧光染色，发现与其作用24 h相比，荧光强度无明显变化，但细胞排列比24 h时略整齐。此外，Thiazovivin能减弱ROCK免疫荧光染色的荧光强度及下调ROCK蛋白表达。结论 Thiazovivin对原代HCEC的形态、连接和离子泵功能具有保护作用，4μmol/L Thiazovivin作用24 h对细胞形态、连接及离子泵功能的改善效果最佳，且Thiazovivin可显著抑制ROCK蛋白表达。（中华眼科杂志，2016，52：686-692）

Objective To evaluate the effect of thiazovivin, a novel ROCK inhibitor, on the morphology and function of human corneal endothelial cells （HCECs）. Methods 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. Results 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, 6μmol/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 （4μmol/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.