肾移植术后受者外周血淋巴细胞的早反应基因表达谱变化

Changes of early response gene expression profile of peripheral lymphocytes in human renal allograft recipients

背景:同种异体免疫应答过程中有许多基因参与淋巴细胞的活化及其调控。目的:研究肾移植后受者外周血淋巴细胞差异表达基因,尤其是早反应基因。设计、时间及地点:基因水平的对照观察实验,于2003-09/2004-02在解放军第二军医大学长征医院泌尿外科及器官移植中心以及上海博星基因芯片公司完成。对象:选取无明显急性排斥反应的同种异体肾移植受者16例。方法:应用包含4096个人cDNA克隆的微矩阵芯片,分析肾移植受者移植后24h外周血淋巴细胞的基因表达谱。每位受者于移植前当日和术后24h分别抽取外周血10mL,分离外周血淋巴细胞。16例受试者同一时间点的淋巴细胞混合。按一步法抽提两个时间点细胞总RNA并纯化mRNA,分别反转录合成、标记cDNA探针,与包含4096个人cDNA克隆的微矩阵芯片杂交,结果通过生物信息学方法分析。主要观察指标:差异表达的基因。结果:肾移植后24h与移植前相比,肾移植受者外周血淋巴细胞差异表达的基因共有216个,其中下调基因103个,上调基因113个。这些基因主要涉及细胞信号转导、细胞凋亡等多个种类。结论:肾移植后24h,基因芯片技术可有效地筛选出受者外周血淋巴细胞早反应基因的差异表达。

BACKGROUND： There are various genes in activation and regulation of lymphocytes in the allogeneic immune response. OBJECTIVE： To explore differentially expressed genes of peripheral lymphocytes in recipients after kidney transplantation, especially those of early response genes. DESIGN, TIME AND SETTING： The observational experiment based on genes was performed at the Urology and Organ Transplantation Center of Changzheng Hospital Affiliated to the Second Military Medical University of Chinese PLA and Shanghai Biostar Gene Chip Co., Ltd. from September 2003 to February 2004. PARTICIPANTS： 16 patients underwent kidney transplantation without evidence of acute rejection were enrolled in the study. METHODS： Early gene expression profile of peripheral lymphocytes in human renal allograft recipients 24 hours later were investigated by means high-density cDNA microarray containing 4 096 human genes. Before and 24 hours after renal transplantation, 10 mL peripheral blood sample were taken from every patients. The lymphocytes were isolated and mixed in two groups respectively. Total RNA of lymphocytes was isolated by one-step method and mRNA was purified, reversely transcribed, labeled and hybridized to gene chip containing 4 096 target genes. The results were analyzed by bioinformatics. MAIN OUTCOME MEASURES： Differentially expressed genes. RESULTS： Comparison analysis revealed that expression of 116 genes were up-regulated and 103 genes down-regulated in lymphocytes 24 hours after transplantation. These genes were associated with signal transduction, cell apoptosis and cell proliferation and others. CONCLUSION： High-density cDNA microarray technology is effective in screening early response differentially expressed genes of peripheral lymphocytes 24 hours after trasplantation.