高压力培养下角膜内皮细胞凋亡的启动机制

发布时间：2019-05-07 19:30

【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P0.01);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P0.01)。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P0.01);50 mm Hg压力组各时间两两比较差异均有统计学意义(均为P0.01)。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P0.01);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P0.01)。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P0.01),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 mechanism of corneal endothelial cell apoptosis in high pressure culture. Methods the primary rabbit corneal endothelial cells were identified by immunohistochemistry and cultured under 50mm Hg (1 k Pa=7.5 mm Hg) pressure) for 1 h, 2 h, 24 h. The normal pressure (15 mm Hg) was used as the normal pressure group and cultured for 24 h. After the fusion of the first generation rabbit corneal endothelial cells reached 70% / 80%, the cells were pretreated with 10 ~ (- 6) mol 路L ~ (- 1) anti-Caspase-8 and anti-Caspase-9 at the concentration of 10 ~ (- 6) mol 路L ~ (- 1) and then placed in a pressure device. The pressure was set at 50 mm Hg, for 24 h to detect the expression of protein Bcl-2 and p53, and the control group was treated with 50 mm Hg pressured cells without inhibitor. The expression of Bcl-2 and p53 were detected by Western blot. The content of cytoplasmic cytochrome C (cytochrome C) in rabbit corneal endothelial cells was detected by immunofluorescence staining. Results the expression of p53 in 50 mm Hg pressure group was 0.651 卤0.007, 0.805 卤0.006,0.839 卤0.011, respectively, which was higher than that in normal pressure group (0.033 卤0.004). The difference was statistically significant (all P0.01). The expression of p53 protein in corneal endothelial cells increased gradually with the prolongation of the pressure time in 50 mm Hg pressure group (all P0.01). In 50 mm Hg pressure group, the expression of p53 protein increased at 1 h, 2 h, and in the 50 mm Hg pressure group, the expression of p53 protein increased gradually with the prolongation of the pressure time (all P0.01). The expression of Bcl-2 in 24 h group was 0.590 卤0.009,0.724 卤0.005,0.314 卤0.016, which was significantly higher than that in normal pressure group (0.081 卤0.013) (P0.01). There was significant difference in each time of 50 mm Hg pressure group (P 0.01). It was found that after 24 hours of culture in the normal pressure group, the nucleus was blue and no cytochrome C release was found in the cytoplasm. In the 50 mm Hg pressure group, the cytoplasm of some cells was red at 1 h, suggesting that cytochrome C release into the cytoplasm, the fluorescence intensity increased with the prolongation of the pressure time, and a wide range of red strong fluorescence appeared in the cytoplasm at 24 h under pressure. The expression of p53 in corneal endothelial cells of anti-Caspase-9 and anti-Caspase-8 pretreatment groups was 0.535 卤0.007 and 0.703 卤0.010, respectively, which was significantly lower than that of control group (0.727 卤0.021) (P 0.01). The expression of Bcl-2 decreased in anti-Caspase-9 and anti-Caspase-8 pretreatment groups (0.312 卤0.003 and 0.442 卤0.011, respectively), which was significantly lower than that in control group (0.501 卤0.011) (P 0.01). The expression of p53 and Bcl-2 in corneal endothelial cells of anti-Caspase-9 pretreatment group was lower than that of anti-Caspase-8 pretreatment group, and the difference was statistically significant (P 0.01). The results showed that anti-Caspase-9 could inhibit the apoptosis of corneal endothelial cells under high pressure. Conclusion Caspase-9 inhibitor can effectively block the apoptosis of corneal endothelial cells induced by high pressure. The injury of high pressure on corneal endothelial cells mainly triggers the release of mitochondrial cytochrome C. Activation of endogenous enzyme-linked reactive apoptosis pathway involved in Caspase-9.
【作者单位】：南昌大学医学院、江西新视界眼科医院;中山眼科中心;南昌爱尔眼科医院;南昌大学附属眼科医院;
【基金】：国家自然科学基金资助(编号:30801263)~~
【分类号】：R77

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