高压力培养下角膜内皮细胞凋亡的启动机制  

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

作　　者：梁玲玲[1] 袁进 幸正茂[3] 廖洪斐[4] 

机构地区：[1]南昌大学医学院,江西新视界眼科医院,江西省南昌市330006 [2]中山眼科中心,广东省广州市510060 [3]南昌爱尔眼科医院,江西省南昌市330006 [4]南昌大学附属眼科医院,江西省南昌市330006

出　　处：《眼科新进展》2017年第10期910-913,共4页Recent Advances in Ophthalmology

基　　金：国家自然科学基金资助(编号:30801263)~~

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50mm Hg(1 k Pa=7.5 mm Hg)压力条件下,培养1 h、2 h、24 h,另设正常压力(15 mm Hg)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 mm Hg,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 mm Hg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 mm Hg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 mm Hg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 mm Hg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 mm Hg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 mm Hg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PObjective To investigate the initiation pathway of corneal endothelial cell apoptosis induced by high-pression. Methotls Primary rabbit corneal endothelial cells were identified by immunohistochemistry and cultured under high pressure 50 mm- Hg （ 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 l0-6 mol · L-1 anti-Caspase 8 and anti-Caspase 9 for lh, 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 de- termined by immunofluorescence staining in all groups. Results The expression lev- els 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 be- tween 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 signifi- cant difference （all P 〈 0.01 ） ,and the difference approached statistical significance be- tween 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, respec- tively, which was significantly lower than that in the control group （0.727 ± 0.021 ）, and the

关 键 词：Caspase-9抑制剂 高压力 角膜内皮细胞 凋亡 

分 类 号：R772.2[医药卫生—眼科]