慢性阻塞性肺疾病肺气虚证和肺阴虚证患者T淋巴细胞相关基因表达对比研究.doc

慢性阻塞性肺疾病肺气虚证和肺阴虚证患者T淋巴细胞相关基因表达对比研究
作者:李泽庚,童佳兵,彭波,张念志,郑汉城,季红燕
【摘要】目的利用基因芯片研究慢性阻塞性肺疾病(COPD)肺气虚证和肺阴虚证患者T淋巴细胞基因表达的差异。方法采集肺气虚证、肺阴虚证患者和健康人的外周血作为实验血样和对照血样。用Ficoll技术分离外周血淋巴细胞,用分选式流式细胞仪分选并纯化T淋巴细胞,并采用一步法提取总RNA,逆转录合成双链cDNA后,体外转录合成生物素标记的cRNA,片断化后,采用人类全基因表达谱芯片进行芯片杂交,扫描后筛选出差异表达基因。结果肺气虚证患者/健康人外周血T淋巴细胞相关差异表达基因45条,其中上调41条,下调4条;肺阴虚证患者外周血T淋巴细胞相关差异表达基因32条,其中上调19条,下调13条;肺气虚证组/健康人组和肺阴虚证组/健康人组均高表达的差异基因5条。结论基因芯片技术能有效地研究基因的表达谱,筛查出COPD肺气虚证、肺阴虚证患者T淋巴细胞相关差异基因。
【关键词】慢性阻塞性肺疾病;肺气虚证;肺阴虚证;T淋巴细胞;基因表达;基因芯片
Abstract:Objective To study the associated gene expression in thymus-dependent lymopholyte of the COPD patients with pulmonary qi deficiency syndrome (PQDS) and pulmonary yin deficiency syndrome (PYDS) by gene chips. Methods Collect peripheral blood of patients with PQDS and PYDS (as experimental samples) and of nomal man (as control samples). By Ficoll method, peripheral blood lymphocyte was collected, then thymus-dependent lymphocyte were extracted and purified by flow cytometry. Total RNA were extracted by one-step technique and purified. Then, synthesis double strand cDNA template from total RNA, transcription of cRNA probe with biotin labeling, subsequently, cRNA sample were fragmented. The gene chip (Human Genome U133 Plus Array, 38500 genes) was hybridized and scanned. Then fluorescent signal value of gene expressing was obtained, and differential expression genes were sifted. Results There were 45 genes expressed differently among PQDS and normal man, including up-regulated 41 and down-regulated 4;32 genes expressed differently between PYDS and normal man, including up-regulated 19 and down-regulated 13;5 up-regulated genes expressed differently between PQDS and PYDS with normal man. Conclusion Gene chip can be applied to study gene expression profiles effectively and to screen PQDS and PYDS associated genes.

Key words:chronic obstructive pulmonary disease;pulmonary qi deficiency syndrome;pulmonary yin deficiency syndrome;thymus-dependent lymphocyte;gene expression;gene chi