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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:

37卷

期数:

2017年10期

页码:

910-913

栏目:

实验研究

出版日期:

2017-10-05

文章信息/Info

Title:

Priming mechanism for the apoptosis of corneal endothelial cells induced by high pressure

作者:

梁玲玲; 袁进; 幸正茂; 廖洪斐

330006　江西省南昌市，南昌大学医学院、江西新视界眼科医院（梁玲玲）；510060　广东省广州市，中山眼科中心（袁进）；330006　江西省南昌市，南昌爱尔眼科医院（幸正茂）；330006　江西省南昌市，南昌大学附属眼科医院（廖洪斐）

Author(s):

LIANG Ling-Ling; YUAN Jin; XING Zheng-Mao; LIAO Hong-Fei

Medical College of Nanchang University & Xinshijie Eye Hospital（LIANG Ling-Ling），Nanchang 330006，Jiangxi Province，China；Zhongshan Eye Center（YUAN Jin），Guangzhou 510060，Guangdong Province，China；Nanchang Aier Eye Hospital（XING Zheng-Mao），Nanchang 330006，Jiangxi Province，China；The Affiliated Ophthalmology Hospital of Nanchang University（LIAO Hong-Fei），Nanchang 330006，Jiangxi Province，China

关键词:

Caspase-9抑制剂; 高压力; 角膜内皮细胞; 凋亡

Keywords:

anti-Caspase 9; high pressure; corneal endothelial cells; apoptosis

分类号:

R772

DOI:

10.13389/j.cnki.rao.2017.0231

文献标志码:

A

摘要:

目的　探讨高压力培养下角膜内皮细胞凋亡的启动机制。方法　原代兔角膜内皮细胞经免疫组织化学鉴定后，在50 mmHg(1 kPa=7.5 mmHg)压力条件下，培养1 h、2 h、24 h，另设正常压力（15 mmHg）作为正常压力组，培养24 h。第一代兔角膜内皮细胞融合达70%~80%后，细胞在加压前1 h分别使用浓度均为10^-6 mol·L^-1抗Caspase-8和抗Caspase-9预处理，然后置于压力装置中，压力设定为50 mmHg，培养24 h后检测蛋白Bcl-2和P53表达，并且以无抑制剂处理的50 mmHg压力培养的细胞为对照组。各组培养的细胞均经Western blot检测Bcl-2和P53的表达。免疫荧光染色检测兔角膜内皮细胞胞浆细胞色素C的含量。结果　50 mmHg压力组角膜内皮细胞，加压1 h、2 h、24 h P53的表达量分别为0.651±0.007、0.805±0.006、0.839±0.011，较正常压力组（0.033±0.004）升高，差异均有统计学意义（均为P＜0.01）；50 mmHg压力组随着加压时间的延长，角膜内皮细胞中P53 蛋白表达量逐渐增加，各时间两两比较，差异均有统计学意义（均为P＜0.01）。50 mmHg压力组中加压1 h、2 h、24 h Bcl-2的表达量分别为0.590±0.009、0.724±0.005、0.314±0.016，较正常压力组（0.081±0.013）反应性升高，差异均有统计学意义（均为P＜0.01）；50 mmHg压力组各时间两两比较差异均有统计学意义（均为P＜0.01）。实验发现正常压力组培养24 h后，细胞核呈蓝色，胞浆中无细胞色素C释放；50 mmHg压力组培养1 h可见部分细胞胞浆呈红色，提示细胞色素C释放进入到胞浆内，随着加压时间延长荧光强度增加，加压24 h见胞浆内出现广泛红色强荧光。抗Caspase-9和抗Caspase-8预处理组的角膜内皮细胞P53表达量分别为0.535±0.007、0.703±0.010，较对照组（0.727±0.021）下调，差异均有统计学意义（均为P＜0.01）；抗Caspase-9和抗Caspase-8预处理组中Bcl-2的表达量呈抑制性下降，分别为0.312±0.003、0.442±0.011，较对照组 （0.501±0.011）下降，差异均有统计学意义（均为P＜0.01）。抗Caspase-9预处理组的角膜内皮细胞P53和Bcl-2表达量较抗Caspase-8预处理组下降，差异亦均有统计学意义（均为P＜0.01），表明抗Caspase-9对高压下角膜内皮细胞凋亡的抑制作用更显著。结论　Caspase-9抑制剂可有效阻断高压力对角膜内皮细胞的促凋亡作用，高压力对角膜内皮细胞的损伤主要是触发了线粒体细胞色素C的释放，激活了Caspase-9参与的内源性酶联反应性凋亡途径。

Abstract:

Objective　To investigate the initiation pathway of corneal endothelial cell apoptosis induced by high-pression.Methods　Primary rabbit corneal endothelial cells were identified by immunohistochemistry and cultured under high pressure 50 mmHg (1 kPa=7.5 mmHg) for 1 h，2 h，24 h,respectively，while cells cultured under the normal pressure 15 mmHg served as the normal pression group.In addition，the first generation of rabbits corneal endothelial cells with 70% to 80% fusion were pretreated with 10^-6 mol·L^-1 anti-Caspase 8 and anti-Caspase 9 for 1h，followed by 50 mmHg pression for the treatment of the cells；while cells cultured with no inhibitor in the same pression served as the control group.Then the expression of P53 and Bcl-2 protein was detected by Western blot，and cytochrome C in rabbit corneal endothelial cells was determined by immunofluorescence staining in all groups.Results　The expression levels of P53 in the 50 mmHg group were 0.651±0.007，0.805±0.006 and 0.839±0.011 after 1 h，2 h，24 h high-pression respectively，which were significantly higher than those in the normal pressure group (0.033±0.004)，and the difference approached statistical significance (all P<0.01).The expression of P53 protein in corneal endothelial cells gradually increased as time went on，and the difference was statistically significant between each two time-points (all P<0.01).Moreover，the expression of Bcl-2 in the 50 mmHg pressure group was 0.590±0.009，0.724±0.005 and 0.34±0.016，respectively，which was higher than that in the normal pressure group (0.081±0.013)，with significant difference (all P<0.01)，and the difference approached statistical significance between each two time points in this group (all P<0.01).The expression level of P53 in anti-Caspase 9 and anti-Caspase 8 group was 0.535±0.007 and 0.703±0.010，respectively，which was significantly lower than that in the control group (0.727±0.021)，and the difference was statistically significant (all P<0.01).The expression of Bcl2 was 0.312±0.003 and 0.442±0.011，respectively，which were significantly lower than that in the control group (0.501±0.011)，with statistical difference (P<0.01).Finally，the expression of P53 and Bcl-2 in anti-Caspase 9 group was lower than that of anti-Caspase 8 group (P<0.01)，indicating that anti-Caspase 9 had more enhanced inhibitory effect on the apoptosis of corneal endothelial cells than anti-Caspase 8.Conclusion　Anti-Caspase 9 inhibitor could effectively block the corneal endothelial cell apoptosis induced by high pressure.And the damage from high pressure on corneal endothelial cells mainly triggers the release of cytochrome C from chondriosome to activate the endogenous enzyme linked apoptotic pathway in which Caspase 9 involves.

备注/Memo

备注/Memo:

国家自然科学基金资助（编号：30801263）

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更新日期/Last Update: 2017-10-24