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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 2 4h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p5 3、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、30 0 μmol/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 (10 0 μmol/L、30 0 μmol/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~ 2 0 0bp) ;(4 )沙丁胺醇或 8 Br cAMP组p5 3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性率用 10 0、30 0μmol沙丁胺醇或 10 0 μmol 8 Br cAMP处理组与对照组比较差异有显著性 (q分别为 2 4 0 4、5 8 47、2 7 2 8,P均 <0 0 0 0 1)。但cromakalim组和先用普萘洛
Objective To study the effect of salbutamol (brand name: salbutamol) on inducing apoptosis of cultured human bronchial smooth muscle cells. Methods Human tracheal smooth muscle cells were isolated and cultured in Dulbecco ’s modified Eagle’ s medium (DMEM) containing 10% fetal bovine serum. Cells from passage 4-6 were used in the experiment. Cells were cultured for 24 or 48 hours with salbutamol or cromakalim or Br Camp, and morphological changes were observed under light and electron microscopes. Agarose electrophoresis analysis of DNA fragments. Streptavidin peroxidase (SP) immunohistochemical staining was used to monitor the changes of p53, Bcl2 and Bax gene expression. The percentage of apoptotic cells was determined by TUNEL technique. Results (1) Salbutamol or 8 Br cAMP decreased the number of viable cells. At 48h, the number of viable cells was the lowest at 30 μmol / L; (2) Morphological characteristics of apoptosis were observed in human bronchial smooth muscle cells incubated with salbutamol (100 μmol / L, 30 μmol / L) for 48h Cell shrinkage and chromatin condensation); (3) agarose gel electrophoresis showed that the DNA fragment was a typical ladder-like band (about 180 ~ 200 bp); (4) salbutamol or 8 Br cAMP group p5 3 or Bax gene expression was significantly higher than that of the control group, but the gene expression in BCl 2 group was lower than that in the control group. (5) TUNEL showed that the positive rate of apoptosis in human bronchial smooth muscle cells was treated with salbutamol or 10 0 μmol 8 Br cAMP The difference between the two groups was significant (q = 24.0 45.8 47,2 7 2 8, P <0 0 01). But cromakalim group and first with propranolol