沙丁胺醇在诱导培养人气管平滑肌细胞凋亡中的作用

目的 研究沙丁胺醇（商品名：舒喘灵）对于诱导培养人气管平滑肌细胞凋亡的作用。 方法 分离人气管平滑肌细胞并且在含有10％胎牛血清的达尔伯克改良伊格尔（DMEM）培养基中培养,4－6代的细胞用于实验。细胞用沙丁胺醇或色满卡林（cromakalim)或8－溴－环磷酸腺苷（Br-Camp)培养24h或48h ,光镜和电镜观察形态学改变。琼脂糖电泳分析DNA碎片。链亲和素－过氧化物酶（SP）免疫组化染色监测p53、Bcl－2和Bax基因表达的改变。通过破碎DNA的原位末端标记技术（TUNEL）检测凋亡细胞的百分数。结果 （1）沙丁胺醇或8－Br-cAMP降低存活细胞的数目。在48h、300μmol/L的浓度,存活的细胞数量最低;（2）人气管平滑肌细胞用沙丁胺醇（100μmol/L、300μmol/L)孵化48h显示出凋亡的形态学特征（细胞皱缩、染色质浓聚）;（3）琼脂糖电泳显示,核酸DNA断裂片呈典型的梯状条带（大约180－200bp);（4）沙丁胺醇或8－Br-cAMP组p53或Bax基因表达显著高于对照组,但Bcl－2组基因表达低于对照组;（5）TUNEL显示,人气管平滑肌细胞的凋亡阳必用100、300μmol沙丁胺醇或100μmol8-Br-cAMP处理组与对照组比较差异有显著性（q分别为24．04、58．47、27．28,P均＜0．001）。但cromakalim组和先用普萘洛尔（商品名：心得安）后与沙丁胺醇组与对照组比较,差异无显著性（q分别为0．12、0．53;P分别为0．932、0．717）,不影响凋亡细胞的基本水平。结论 （1）在体外,沙丁胺醇以时间和浓度依赖的方式诱导人气管平滑肌细胞凋亡。（2）cAMP蛋白激酶A通路对于沙丁胺醇诱导的凋亡是必须的和充分的。

Role of salbutamol in inducing apoptosis of cultured human airway smooth muscle cells

OBJECTIVE: To study the effect of salbutamol on inducing apoptosis of cultured human airway smooth muscle cells (ASMCs). METHODS: Human ASMCs were isolated and cultured in DMEM containing 10% fetal bovine serum. Cells of Passes 4 approximately 6 were used in experiments. The cells were cultured with salbutamol, cromakalim, 8-Br-cAMP for 24 or 48 hours. Morphological changes were observed by light microscopy and electron microscopy. DNA fragmentation was analyzed by agarose gels. SP immunohistological staining method was performed to detect the changes of expressions of p53, Bcl-2 and Bax gene. The percentage of apoptosis cell was detected by situ end labeling technique (TUNEL) of fragmental DNA. RESULTS: (1) Salbutamol or 8-Br-cAMP decreased the number of viable cells. At 48 hour, at 300 micromol/L, number of viable cells was the lowest. (2) Human ASMCs incubated with salbutamol (100 micromol/L or 300 micromol/L) for 48 h revealed morphological features of apoptosis (cellular shrinkage, condensation of chromatin). (3) Agarose gel electrophoresis showed a characteristic "ladder" of DNA bands representing integer multiples of the internucleosomal DNA length (about 180 approximately 200 bp). (4) The expression of p53 or Bax gene in salbutamol or 8-Br- cAMP group was significantly higher than that in control group, but the expression of BCl-2 gene was lower than that in control group. (5) The TUNEL indicated: apoptotic positive rate of human ASMCs was significantly different following 100 micromol, 300 micromol salbutamol or 100 micromol 8-Br-cAMP treatment (q = 24.04, 58.47, 27.78 respectively, P < 0.0001), but cromakalim, propranolol before salbutamol not affected basal level of apoptotic cell (q = 0.12, 0.52 respectively, P = 0.932, 0.717 respectively), as compared with the controls. CONCLUSIONS: (1) Salbutamol induced apoptosis in human ASMCs in vitro in time and concentration dependent manner. (2) A cAMP-protein kinase A pathway is necessary and sufficient for salbutamol induced apoptosis. (3) beta(2) adrenergic agonist or cAMP analogue may prevent airway remodeling in asthma.