NO介导的小鼠胸腺细胞中线粒体的变化在细胞凋亡过程中的作用

The changes of the thymocyte mitochondria mediated by nitric oxide in thymocyte apoptosis

目的：研究一氧化氮（NO）介导的胸腺细胞凋亡中线粒体膜电位（△ψm）和心磷脂（CL）含量变化的特点.方法：以S-亚硝基-N-乙酰青霉胺（SNAP）作为NO的供体诱导胸腺细胞凋亡,以地塞米松（DEX）作为阳性对照药物;设空白对照组、SNAP组和DEX组3个实验组;经膜联蛋白Ⅴ（annexinⅤ mAb）和碘化丙啶（PI）染色后,用流式细胞术（FCM）检测细胞磷脂酰丝氨酸（PS）外翻;用3,3＇-二已基噁羰花青碘化物[DiOC6（3）]和PE-anti-annexin Ⅴ mAb检测凋亡中△ψm变化;用壬基吖啶橙（NAO）和PE-anti-annexin Ⅴ mAb检测凋亡中线粒体CL变化.结果：SNAP作用后6 h,胸腺细胞出现典型的细胞凋亡特征,多数annexin Ⅴ阳性的细胞出现皱缩.DEX组△ψm降低且未凋亡的细胞比例显著高于空白对照组（P＜0.01）;而SNAP组该群细胞所占比例与空白对照组比较,差异无统计学意义（P＞0.05）.各组中约40%～50%的DiOC6（3）阴性细胞同正常细胞的大小.SNAP组CL含量降低的凋亡细胞所占比例显著高于对照组（P＜0.01）,未见CL含量降低且未凋亡的细胞群.空白对照组和SNAP组中分别有（48.32±3.96）%、（43.64±4.90）%的细胞CL含量降低但大小同正常细胞.结论：NO介导的小鼠胸腺细胞的凋亡过程,依次为磷脂酰丝氨酸外翻、线粒体去极化、CL氧化及细胞皱缩.同DEX模型组相比较,NO介导的小鼠胸腺细胞线粒体的变化为凋亡过程中较晚期的变化.

AIM： To study the changes of mitochondrial potential （△ψm） and cardiolipin （CL） content during thymocyte apoptosis mediated by nitric oxide （NO）. METHODS： SNAP was used as NO＇s donor to induce thymocyte apoptosis in mice and dexamethasone （DEX） was used as positive control drug. Three experiment groups were set, which were blank control group, SNAP group and DEX group. The ectropion of cell phosphatidylserin （PS） was detected by flowcytometry after stained with FITC-anti-annexin Ⅴ mAb/PI. The changes of △ψm and CL content during cell apoptosis were detected by DiOC6 （3）/PE-anti-annexin Ⅴ mAb and NAO/PE-anti-annexin Ⅴ mAb, respectively. RESULTS： 6 hours after SNAP treatment, the thymocytes exhibited typical cell apoptotic characteristics and crenation appeared in most annexin Ⅴ-positive cells. In DEX group the rate of △ψm decreased and the non-apoptotic cells were significantly higher than those in of blank control group but there was no significant difference between SNAP and control groups. 40% -50% of the size of DiOC6 （3）-negative cells in each group was the same as normal cells. The percentage of the cells with reduced CL content in SNAP group was significantly higher than that in blank control group （ P 〈0.01 ）. The cells with reduced CL content and non-apoptosis were not found. In blank control group and SNAP group, both reduced CL content and normal-sized cells were （48.32 ± 3.96） % and （43.64 ± 4.90） %, respectively. CONCLUSION： The process of thymocyte apoptosis mediated by NO in mice is successively PS ectropion, mitochondrial depolarization, CL oxidation and cell crenation. As compared with DEX group, the mitochondrial change mediated by NO is a later event in cell apoptosis.