氨茶碱诱导体外培养的人气管平滑肌细胞凋亡

The efficacy of aminophylline on the apoptosis of human trachea smooth muscle cells in vitro

目的 为了研究氨茶碱是否及怎样诱导人气管平滑肌细胞 (TSMCs)凋亡。方法 分离人ASMCs,在含 10 %胎牛血清的DMEM中培养。第 4～ 6代细胞用于作实验。细胞用氨茶碱或 8 Br cAMP培养 2 4或 4 8h。用光镜和电镜观察形态学的改变。用琼脂糖电泳分析DNA片断。SP免疫组化染色检测 p5 3、bcl 2和bax基因表达的改变。凋亡细胞的百分率通过DNA片断的原位末端标记 (TUNEL)来检测。结果 ①氨茶碱或 8 Br cAMP以时间和浓度依赖的方式降低存活的细胞数 ;②电镜检测显示核皱缩、染色质浓聚和凋亡小体形成 ;③破碎DNA的琼脂糖电泳显示了典型的梯状条带 ;④凋亡组 p5 3或bax的基因表达高于对照组 ,但bcl 2的基因表达低于对照组 ;⑤氨茶碱或 8 Br cAMP组TUNEL的阳性率高于对照组 (P <0 0 1)。结论 ①氨茶碱诱导体外培养的人TSMCs凋亡 ;②氨茶碱诱导体外培养的人TSMCs凋亡也许部分通过cAMP PKA通路。

AIM To investigate weather and how aminophylline induces human trachea smooth muscle cells (TSMCs) to apoptosis. METHODS Human TSMCs were isolated and cultured in DMEM containing 10% fetal bovine serum. Passage 4～6 cell were used in experiment. The cells were cultured with aminophylline or 8 Br cAMP for 24 or 48 h. Light micros copy and electron microscopy were used to observe morphological change. DNA fragmentation was analyzed by agarose gels electrophoresis. SP Immunohistological staing method was performed to detect the changes of expressions of p53,bcl 2 and bax gene. The apoptosis cell percentage were detected by situ end labeling technique(TUNEL) of fragmental DNA. RESULTS ①Aminophylline or 8 Br cAMP decreased the number of viable cells in time and concentration dependent manner; ② Electron microscopic examination showed nuclear contraction, chromatin condensation and apoptotic bodies formation in aminophylline group or 8 Br cAMP group; ③ Agarose gel electrophoresis of fragmented DNA showed a ladder like pattern; ④ The expression of p53 or bax gene in apoptosis group was significantly higher than in control group, but the expression of bcl 2 gene was lower than in control group; ⑤ The positive rate of TUNEL in aminophylline or 8 Br cAMP group was significantly higher than in control group ( P <0 01). CONCLUSION ① Aminophylline induces apoptosis in cultured human TSMCs; ②Apoptosis of cultured human ASMCs induced by aminophylline may play a role partly through cAMP PKA pathways.