摘要
目的:观察抑喘中药雄黄对哮喘豚鼠肺泡灌洗液中嗜酸细胞的作用,并分析量效及时效性。方法:实验于2005-03/2006-07在广州医学院中心实验室进行。①采用卵白蛋白制备哮喘豚鼠模型并激发哮喘,提取肺泡灌洗液并分离其中的嗜酸细胞。②将嗜酸细胞悬浮生长于含10%小牛血清的RPMI 1640培养液中,嗜酸细胞初始浓度均为2×109L-1。一组雄黄以1.5625mg/L终浓度分别加入细胞培养液中,培养3,6,12,24,48h。以不加雄黄为对照。另一组将雄黄终浓度调整为0.05,0.1,0.19,0.39,0.78,1.56,3.19mg/L,培养6h。以不加雄黄为对照。③Wright's染色、透射电子显微镜观察嗜酸细胞的形态学变化。流式细胞仪检测观察嗜酸细胞凋亡率。免疫组织化学染色观察嗜酸细胞bcl-2基因表达。结果:①嗜酸细胞与雄黄作用6h后,Wright's染色、透射电子显微镜观察到嗜酸细胞核固缩,染色体边集现象,核膜表面凹凸不平,部分嗜酸细胞出现了细胞核碎裂,有少量的凋亡小体形成。②1.5625mg/L雄黄在培养基中分别培养3,6,12,24,48h后,流式细胞检测可见嗜酸细胞凋亡率随着雄黄作用时间的延长而先升高,于24h达最高,凋亡率为26.21%,而后下降,各不同时间组嗜酸细胞凋亡率与对照组相比,差异均有显著性。将嗜酸细胞与不同终浓度雄黄在培养基中分别培养6h后,流式细胞检测可见嗜酸细胞凋亡率随着雄黄浓度的增加而升高,各不同浓度组嗜酸细胞凋亡率与不加雄黄的对照组相比,差异亦均有显著性。③经雄黄处理的嗜酸细胞bcl-2蛋白表达较对照组低。结论:雄黄促进哮喘豚鼠肺泡灌洗液中嗜酸细胞凋亡,随着雄黄作用时间的延长和浓度增加这种作用更明显。bcl-2基因参与了嗜酸细胞凋亡的过程。
AIM： To observe the realgar induced the apoptosis of eosinophils from bronchoalveolar lavage fluid in asthmatic guinea pigs, and to analyze the dose-effect and time-effect. METHODS： The experiment was conducted in the Central Laboratory of Guangzhou Medical College from March 2005 to July 2006.①The asthmatic guinea pigs were prepared with ovalbumin and the eosinophils were separated from bronchoalveolar lavage fluid.②Eosinophils were cultured in RPMI 1640 culture solution containing 10% calf serum. The incipient concentration of eosinophils was 2×10^9 L^-1. A group of realgar with the final concentration of 1.5625 mg/L was added in the cell culture fluid for 3, 6, 12, 24 and 48 hours. Those without realgar were as control. Final concentration of realgar in another group was adjusted to 0.05,0.1,0.19,0.39,0.78,1.56,3.19 mg/L for 6 hours. Those without realgar were as control.③The morphology of apoptosis of eosinophils was observed by Wright＇s staining and transmission electron microscope. The rate of apoptosis of eosinophils was assayed by the flow cytometry. The expression of bcl-2 gene in eosinophils was shown by immunohistochemistry staining. RESULTS： ①Six hours after interaction between eosinophils and realgar, Wright＇s staining and transmission electron microscope showed that karyopyknosis of eosinophils, chromosome margination appeared. The surface of karyolemma was uneven. Partial eosinophils had nuclear fragmentation and a small quantity of apoptotic body. ②After 1.5625 mg/L realgar culturing for 3, 6, 12, 24 and 48 hours, the apoptotic rate of eosinophils increased with the prolongation of realgar intervention, and reached the peak with the apoptotic rate of 26.21%, and then decreased. There were significant differences of apoptotic rate between different time groups and control group. After 6 hours treatment of realgar of different final concentration, the apoptotic rate of the eosinophils increased with increasing realgar concentration. There were significant differences of apoptoti
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第29期5769-5772,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
广州市教育局科技项目(1007)
广东省中医药局科研课题(103072)~~
