石家庄肾病医院搜集，中华儿童肾病网摘录2pU中华儿童肾病网

　　【摘要】 目的 研究和探索与肾脏移植相关的差异表达基因。方法 选择肾移植患者30例。分术前当日和术后4周采新鲜外周血液10ml，血标本按术前（对照）和术后归类分组。用Ficoll技术，获取外周血单个核细胞。标本均置于液氮罐保存。采用一步法提取总RNA，同组RNA标本同量混合，用荧光染料Cy3-dUTP标记对照组总RNA，用Cy5-dUTP标记术后组总RNA。逆转录标记cDNA探针。采用高通量基因表达谱芯片（16，920点基因）进行芯片杂交，并用ScanArray4000扫描仪扫描芯片，得到Cy3/Cy5图像文件，通过划格确定杂交点范围，过滤背景噪声，提取基因表达的荧光信号强度值。ArrayPro Analyzer4.0分析Cy3和Cy5两种荧光信号的强度和比值。结果 术后组和对照组存在差异表达的基因，其中重复2次差异表达的基因有433条（2.56%）。显示上调基因26条，其中已知基因11条，未知基因15条;下调基因407条，其中已知基因252条，未知基因155条。结论 运用高通量、大规模基因表达谱芯片分析和研究患者基因表达谱系，能够有效筛选有关移植免疫方面的相关基因。2pU中华儿童肾病网
　2pU中华儿童肾病网
　　关键词 肾脏 移植 基因芯片 基因表达谱      2pU中华儿童肾病网

　　Guo Yifeng，Zheng Junhua，Tan Jianming，et al. 2pU中华儿童肾病网

　　Department of Urology，Changzheng Hospital，Second Military Medical University，Shanghai200003.2pU中华儿童肾病网

　　【Abstract】 Objective To explore and discover the relative genes with transplant immunity.Methods By virtue of gene microarray to analyze the differential expression of the genes in the PBMCs of the patients having acceptˉed renal transplantation.The30patients having accepted renal transplantation（4weeks after operation）were collectˉed fresh blood according to preoperation（as control group）and postoperation4weeks（as postoperation group）.By Fiˉcoll method，PBMCs was collected and preserved in liquid nitrogen.Total RNA were extracted by one-step techˉnique and purified.Samples of per group were merged into the whole by the same quantity.The total RNA of postopˉeration group were labeled with Cy5-dUTP respectively，and total RNA of control group were labeled with Cy3-dUTP，then by reverse transcript way to label the cDNA probe.High throughout gene chip（16，920）was hybrided and scanned by ScanArray4000.Cy3/Cy5image files were copied.The area of point was determined with grids，the noise of background was filtered and fluorescent signal value of gene-expressing was obtained.ArrayPro Analyzer4.0computed and analyzed the fluorescent signal intensity and ratio of Cy3and Cy5.Results There are differential exˉpression433genes（2.56%），including up-regulated26（known11and unknown15）and down-regulated407（known252and unknown155）.Conclusion cDNA microarray for analysis of gene expression proflies is a powerful method to identify associated genes with transplant immunity.2pU中华儿童肾病网

　　Key words kidney transplantation gene microarray Gene expression profiles   2pU中华儿童肾病网

　　器官移植，包括肾脏移植已成为治疗终末期器官功能衰竭唯一有效的根治方法。目前在移植界仍然面临许多难题，难题之一在于如何解决肾移植术后机体产生排斥和抗排斥之间的平衡。免疫抑制剂用量过大，则造成抑制过度，全身免疫力下降，诱发各种感染 ［1，2］  ，甚至肿瘤的发生 ［3］  。免疫抑制剂用量过小则易出现抑制力不足，急性排斥反应的发生。了解肾移植术后免疫反应状态是必要的。能否采用高通量、大规模的基因芯片去筛选和发现肾脏移植差异 表达基因，从基因水平去检测免疫反应状态，找出它们的内在联系。为临床提供理论依据和参考。2pU中华儿童肾病网

　　1 材料和方法2pU中华儿童肾病网

　　1.1 材料2pU中华儿童肾病网

　　1.1.1 试剂 2pU中华儿童肾病网

　　Ficoll-Hypaque分离液（比重1.077±0.001，上海试剂二厂出品）。Cy3-dCTP，Cy5-dCTP（美国Amerˉsham Phamacia Biotech Co.出品），Superscript II（美国Gibco BRL Co.出品）。2pU中华儿童肾病网

　　1.1.2 芯片 2pU中华儿童肾病网

　　16，920点基因表达谱芯片由上海联合基因科技有限公司提供。ScanArray4000扫描仪购自美国General Scanning公司。2pU中华儿童肾病网
  2pU中华儿童肾病网
　　1.1.3 主要仪器 2pU中华儿童肾病网

　　ScanArray4000扫描仪，美国General Scanning Co.出品。电泳仪，美国Phamacia Biotech Co.出品。恒温杂交箱，美国Robbins Scientific Corporation出品。