应用抑制消减杂交克隆支气管哮喘病人嗜酸细胞差异表达基因

目的 探讨嗜酸细胞在支气管哮喘发病中的分子机制。方法 提取1名哮喘发作病人嗜酸细胞总RNA为实验子,治疗后作为对照的嗜酸细胞的RNA为驱动子。检测子、驱动子的总RNA由SMART cDNA合成方法合成双链cDNA,进而由抑制消减杂交方法获得消减杂交产物。消减杂交产物克隆到T载体建立消减文库,对部分克隆进行杂交筛选及序列比对。结果 消减杂交文库挑取400个克隆进行PCR扩增,阳性率约为80%。部分阳性克隆经2轮杂交筛选及序列对比后获得6个差异片段,涉及与编码炎症介质、细胞信号传导、能量代谢和细胞凋亡相关的基因。结论 抑制消减杂交技术有效地克隆了支气管哮喘发作期和缓解期嗜酸细胞差异表达的基因,为阐明嗜酸细胞在支气管哮喘发病中的分子机制奠定了良好的基础,并为临床防治支气管哮喘、寻找新的药物和方法提供了依据。

Cloning of differentially expressed genes of eosinophils from asthmatic patients by suppression subtractive hybridization

Objective To explore the molecular mechanism of eosinophils for its role in bronchial asthma. Methods The total RNA extracted from the eosinophils of patients during the onset of asthma was used as the tester and the total RNA obtained after treatment served as the driver. cDNA suppression subtractive hybridization (SSH) was performed using the protocols described in the Clontech SMART PCR cDNA Sythesis Kit and PCR-Select cDNA Subtraction Kit. The subtracted cDNA was then inserted into T vector to generate subtracted cDNA library. Clones of the subtracted cDNA library were screened by hybridization and the insert sequence of the positive clones was compared with the sequence in the GenBank. Results A total of 400 clones selected from the subtracted cDNA library were amplified by PCR and about 85% of these clones contained inserts. Six differential cDNA fragments were acquired after two differential screening. These genes were involved in the regulation of proinflammatory response, signal transduction, energy metabolism and cell apoptosis. Conclusion Differentially expressed genes of the eosinophils during the onset and the remission stage of bronchial asthma can be effectively cloned by SSH, which provides a solid foundation for clarifying the molecular mechanism of eosinophils in asthma and a theoretical base for clinical treatment and prevention of asthma.