凋亡抑制基因Bcl-2表达与支气管哮喘关系的研究

A study on the relationship of apoptosis inhabit gene Bcl-2 and Asthma

目的支气管哮喘的发病机制非常复杂,至今尚未完全阐明。目前认为细胞凋亡与支气管哮喘发病相关。Bcl-2基因是近年来广泛重视的一种凋亡抑制基因,可抑制多种细胞凋亡,与多种疾病的发生存在密切的关系。本研究测定支气管哮喘患者外周血白细胞Bcl-2基因mRNA的表达量及其血清中蛋白的含量,研究Bcl-2在支气管哮喘中的表达及其相互关系,初步探讨Bcl-2基因在支气管哮喘的发生、发展中的生物学意义。方法20例支气管哮喘中重度发作患者为实验组,20例无支气管哮喘、变态反应性疾病的健康人为对照组。采取抗凝静脉血标本5ml,立即用Dextron-PBS分离出血白细胞,采用TristarTM试剂按试剂盒说明提取总RNA,-80°超低温冰箱保存,采用TaKaRa试剂盒检测Bcl-2基因mRNA表达量。两步法:第一步:用ExScriptTM反转录酶,对Bcl-2mRNA反转录成cDNA;第二步:用Premix Ex TaqTM酶对反转录后的cDNA进行扩增,10倍梯度稀释cDNA模板,分别为100、10-1、10-2、10-3、10-4倍梯度。用美国PE公司5700型实时荧光定量PCR仪进行检测,以GAPDH基因为对照基因,用仪器附带SDS软件自动算出Bcl-2基因与对照基因比值,确定Bcl-2基因的相对含量。同时抽取抗凝静脉血2ml,伊红染色,行外周血嗜酸性粒细胞计数。抽取不抗凝静脉血2ml,分离血清,采用澳大利亚Bender Medsystems公司人Bcl-2蛋白检测试剂盒,按试剂盒说明操作,通过酶联免疫吸附法(ELISA)检测Bcl-2蛋白含量。应用SAS6.12计算机软件对实验结果进行统计学分析。结果支气管哮喘患者外周血嗜酸性粒细胞计数较正常对照组比较有非常显著性增加(P<0.01),20例支气管哮喘患者外周血细胞Bcl-2基因mRNA表达量高于正常对照组,两组有非常显著性差异(P<0.01),外周血清Bcl-2蛋白含量与正常对照组差异无显著性(P>0.05)。结论本研究证实支气管哮喘患者外周血嗜酸性粒细胞显著增多。支气管哮喘患者外周血细胞Bcl-2基因mRNA表达显著增多,揭示Bcl-2基因在支气管哮喘发病中具有一定的作用和意义,Bcl-2基因与支气管哮喘密切相关。而外周血清中Bcl-2蛋白含量与支气管哮喘的发病无直接相关。

Objective Pathogenesis on incidence and prevention of asthma howe of asthma is yet not clear although lots of global researches doing, Pathogenesis of asthma is complicated which related to many factors such as environment, behavior, genetics, psychology and immunity. Currently apoptosis is thought in association with asthma, Bcl-2 gene is a kind of gene which can restrain apoptosis of many kinds of cells and is studied by many researchers. Bcl-2 gene not only plays an important role in adjustment of apoptosis, but also has close relation with development of lots of diseases. There are lots of researches on relation of Bcl-2 gene and tumor, while few reports on relation of Bcl-2 gene and asthma were published. This study measured the amounts of Bcl-2 gene in white blood cell （WBC） and related proteins to investigate the relations of Bcl-2 gene and asthma at genetic level, and the biological role of Bcl-2 gene was explored in development of asthma, so as to provide basis for developing new treatment methods such as gene treatment. Methods In total 20 cases of asthma patients seeking treatment in Hainan Province People＇s Hospital from 2004 to 2005 were selected as subjects, and 20 cases of healthy people without asthma or allergy diseases as control.-5 ml vein anti-agglutination blood was drawn from each subject and control, and WBC were isolated soon after drawing the blood using Dextron-PBS. TristarTM kits were used to extract RNA, and RNA was stored at a temperature of-80℃. TaKaRa kits were used to measure the amounts of mRNA delivered by Bcl-2 gene. The following steps were： first, ExScriptTM reverse transcriptase was used to reverse Bcl-2 mRNA to cDNA; second, Premix Ex TaqTM enzyme to amplify the cDNA. cDNA template was diluted by 10 grades, namely 100,10-1, 10-2,10-3, 10-4 times. Upper index of Bcl-2 was provided by Shanghai Biologic Engineering Technolo- gy Company： 5, GGT GCC ACC TGT GGT CCA CCT-3, lower index： 5, CTT CAC ＂TTG TGG CCC A- GA TAG G-3. A 5700 RT-PCR machine made by PE company in USA was used to test, with GAPDH gene as control. The ratio of Bcl-2 and control was provided by SDS software to learn the relative amount of Bcl-2 gene. 2 ml vein anti-agglutination blood was drawn and stained by eosin. Under microscopic, the acidophil cell was numbered. 2 ml of vein blood was drawn and serum was separated to measure the amount of Bcl-2 gene by ELISA method using protein test kit manufactured by Bender Medsystems company in Austalia. SAS software was used to do the analysis. Results the amount of blood eosinophils is significantly higher for asthma than for control（ P〈0.01 ）. Amount of mRNA delivered by Bcl-2 gene is significantly higher in asthma than in control （P〈0.01）. The amount of Bcl-2 related protein in serum is similar both groups （P〉0.05）. Conclusion Asthma is a kind of chronic ＇inflammation feature by high peripheral blood eosinophils and high airway eosinophils . This study showed that amount of peripheral blood eosinophils is significantly higher in asthma patients. High amount of mRNA delivered by Bel-2 showed that Bcl-2 gene plays a role in developing of asthma. While the amount of Bcl-2 related proteinin serum seems to have no relation with asthma.