线粒体ATP敏感钾通道对高氧诱导人肺泡上皮细胞凋亡的保护作用

发布时间：2018-02-24 08:37

本文关键词： 高氧肺损伤 A549 二氮嗪线粒体ATP敏感钾通道凋亡 Omi/HtrA2　出处：《泸州医学院》2011年硕士论文　论文类型：学位论文

【摘要】：目的：探讨线粒体ATP敏感钾通道(mitochondrial ATP-sensitive potassium channel,mitoKATP)对高氧诱导人肺泡上皮细胞凋亡的保护作用。方法：以人肺泡上皮A549细胞为研究对象,体外传代培养A549细胞系。以3L/min的900ml/L氧气和50ml/L二氧化碳高纯混合气作为攻击因素处理A549细胞。实验将A549细胞共分为三组：对照组(n=8)、高氧组(n=8),二氮嗪组(n=8)。对照组：加入含100mL/L胎牛血清培养液常规培养；高氧组：加入等量含100mL/L胎牛血清培养液,换液后,通入3L/min的90%氧气和5%二氧化碳高纯混合气10min,密闭培养；二氮嗪组：用终浓度为100μmol/L的等量二氮嗪培养液预处理24h后,再用高氧诱导A549细胞,密闭培养12、24、48h收集细胞指标检测如下指标：倒置相差显微镜下观察A-549细胞的形态学改变并照相；CCK-8法检测A549细胞活力；流式细胞仪检测细胞凋亡；免疫组化法检测细胞内Omi/HtrA2的表达；激光共聚焦显微镜检测线粒体膜电位变化；透射电镜法检测细胞超微结构的变化。结果：(1)倒置显微镜下：对照组细胞贴壁良好,呈铺路石样,为扁圆的多角形,细胞排列紧密,连接成片,数量多,悬浮细胞少,分裂相多。高氧组细胞受损明显,形态改变,由典型的扁圆多角形细胞变为圆形或椭圆形,细胞间空隙加大,连接松散,活细胞数量明显降低,悬浮细胞明显增多；二氮嗪组细胞损伤状况明显得到改善,细胞生长状况明显好转,形态明显变好,较高氧组活细胞数量增加,悬浮细胞数减少,但未恢复到对照组水平；(2)与对照组(0.34±0.03)比,高氧组细胞活力(0.13±0.02)下降(P0.01)；与高氧组相比,二氮嗪组细胞活力(0.21±0.04)增加,但未恢复至对照组水平(P0.01)；(3)与对照组(0.40%±0.10%)比,高氧组凋亡率(6.54%±0.48%)明显增加(P0.01)；与高氧组相比,二氮嗪组凋亡率(2.36%±0.51%)明显降低,差异有显著性p0.01)。(4)与对照组(15.26±2.80)相比,高氧组A549细胞内Omi/HtrA2的表达(44.94±4.47)明显增加,差异有显著性(P0.01)；与高氧组相比,二氮嗪组Omi/HtrA2的表达(28.22±1.07)明显降低,差异有显著性(P0.01)；(5)激光共聚焦显微镜下：与对照组(1.92±0.11)相比,高氧组细胞线粒体膜红/绿荧光强度比值(1.00±0.08)明显降低,差异有显著性(P0.01)；与高氧组相比,二氮嗪组A549细胞线粒体膜红/绿荧光强度比值(1.57±0.08)增加,差异有统计学意义(P0.01)。(6)对照组A549细胞结构完整,胞核呈椭圆形,胞浆内可见大量多泡体,线粒体结构清晰,未见肿胀；高氧组胞核圆形,核仁明显,大量线粒体肿胀,电子密度减低,嵴减少；二氮嗪组可见细胞胞核小,圆形,胞质电子密度较高,线粒体呈空泡状,线粒体嵴减少。结论：二氮嗪可能通过稳定线粒体膜电位,减少Omi/HtrA2表达,从而减少A549细胞凋亡来明显减轻高氧诱导的人肺泡上皮细胞的损伤。
[Abstract]:Aim: to investigate the protective effect of mitochondrial ATP sensitive potassium channel (ATP) on apoptosis of human alveolar epithelial cells induced by hyperoxia. A549 cell line was subcultured in vitro. A549 cells were treated with 3L / min of 900ml / L oxygen and 50ml / L carbon dioxide high purity mixture as attack factors. A549 cells were divided into three groups: control group, hyperoxia group, diazazine group, and control group. Fetal bovine serum containing 100 mL / L fetal serum was cultured in routine culture medium. In the hyperoxia group, 100 mL / L fetal bovine serum was added into the culture medium, and then the mixture of 90% oxygen and 5% carbon dioxide of 3 L / min was added to the medium for 10 minutes, and the diazazine group was pretreated with the same amount of diazazine at the final concentration of 100 渭 mol/L for 24 hours. A549 cells were induced by hyperoxia, and the cell indexes were collected for 48 hours. The morphological changes of A-549 cells were observed under inverted phase contrast microscope, and the viability of A549 cells was detected by CCK-8 method, and apoptosis was detected by flow cytometry. Immunohistochemical method was used to detect the expression of Omi/HtrA2 in cells; laser confocal microscopy was used to detect the changes of mitochondrial membrane potential; transmission electron microscopy was used to detect the ultrastructure of cells. The cells in the hyperoxia group were obviously damaged and changed from typical round polygonal cells to round or elliptical cells. The gap between cells was enlarged, the connection was loose, the number of living cells was obviously decreased, the number of suspended cells was obviously increased, the condition of cell damage, cell growth and morphology of diazazine group were obviously improved. In hyperoxia group, the number of living cells increased, but the number of suspended cells decreased, but did not return to the level of control group (0.34 卤0.03). Compared with the control group, the cell viability of hyperoxia group decreased by 0.13 卤0.02, and that of diazazine group increased by 0.21 卤0.04 compared with that of hyperoxia group. The apoptotic rate of hyperoxia group (6.54% 卤0.48) was significantly higher than that of control group (P 0.01% 卤0.51%), and the apoptotic rate of diazazine group (2.36% 卤0.511) was significantly lower than that of hyperoxia group (P 0.01% 卤0.41%), compared with that of control group (15.26 卤2.80). The expression of Omi/HtrA2 in A549 cells in hyperoxia group (44.94 卤4.47) was significantly higher than that in hyperoxidised group (P 0.01 卤0.11), and the expression of Omi/HtrA2 in diazazine group was significantly lower than that in hyperoxide group (P 0.01 卤1.07). The ratio of red / green fluorescence intensity of mitochondria membrane in hyperoxia group (1.00 卤0.08) was significantly decreased, the difference was significant (P 0.01), and the ratio of red / green fluorescence intensity of A549 cells in diazazine group was 1.57 卤0.08, compared with that in hyperoxidised group (1.57 卤0.08). The difference was statistically significant (P 0.01. 0. 0. 6) the A549 cells in the control group had intact structure, oval nucleus, a large number of vesicular bodies in the cytoplasm, clear mitochondria structure and no swelling, while in the hyperoxia group, the nucleus was round, the nucleolus was obvious, and a large number of mitochondria were swollen. In the diazazine group, the nucleus of the cells was small, round, the cytoplasm electron density was high, the mitochondria were vacuolated, and the mitochondria cristae were decreased. Conclusion: diazazine may decrease the Omi/HtrA2 expression by stabilizing the mitochondrial membrane potential. Thus, the apoptosis of A549 cells was reduced to alleviate the injury of human alveolar epithelial cells induced by hyperoxia.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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