摘要
目的研究非梗阻性无精症(NOA)睾丸基因表达谱变化,为研究其发病机制奠定基础。方法知情同意下获取正常青年人和NOA患者的睾丸活检标本各3例,采用Q IA GEN RNA提取试剂盒提取总RNA,分别等量混合后制备探针。利用ClonTech公司生产的包含8000个已知基因的人类cDNA基因芯片[Atlas Plastic Human 8K Mi2croarray(Cat.#790521)]筛选差异表达基因。按基因功能分类方法对睾丸组织中差异表达的基因进行归类分析。从基因芯片结果中选择我们感兴趣的基因用RT-PCR、T-A克隆测序方法验证。结果筛选到显著差异表达基因4117个(上调基因1564个,下调基因2553个)。在表达上调的基因中代谢相关基因(11.4%),与基因和蛋白表达相关的基因(34.8%),信号通路相关基因(28.3%),细胞分化相关基因(16.5%),细胞结构和运动相关基因(4.2%),细胞或内环境稳态相关基因(24.9%)。在表达下调的基因中代谢相关基因(13.6%),与基因和蛋白表达相关的基因(31.5%),信号通路相关基因(36.7%),细胞分化相关基因(13.5%),细胞结构和运动相关基因(3.6%),细胞或内环境稳态相关基因(20.1%)。RT-PCR及T-A克隆测序进一步证实TGF-β信号通路中TGFβRⅡ和Smad2基因在NOA患者睾丸组织显著上调。结论NOA患者睾丸组织中基因表达谱发生了明显变化,NOA与多种基因表达变化有关,其中TGF-β信号通路显著上调可能对进一步研究NOA的机制具有重要意义。
Objective To understand the mechanism of non-obstructive azoospermia (NOA), we studied the testis gene profile of the disease. Methods Under the approvement by the institutional review board of Peking University, three NOA patients with were recruited into this study. Three age-matched volunteer donors with healthy record according to their consent provided their normal testicular biopsies as control. Total RNA was extracted from these testis biopsies using QIAGEN RNAeasy kit. Total RNA from three NOA patients was pooled together. Likewise, the control was done. Differentiations of gene expression were studied by Clon-Tech cDNA microarray method. The differential expression gene in NOA were classified by Venter's classify system and the selected candidate genes were identified by RT-P.CR analysis and sequence analysis. Results In the microarray result, there were 4117 genes with significantly differential expression. Among of them 1564 genes were up-regulated and 2553 genes were down-regulated. Up-regulated genes included relating metabolism (11.4%), regulating gene or protein expression (34.8%), cell signaling (28.3%), cell differentiations (16.5%), cell structure or motility (4.2%) and homeostasis (24.9%). Down-regulated genes included regulating metabolism (13.6%), regulating gene or protein expression (31.5%), cell signaling (36.7%), cell differentiations (13.5%), cell structure or motility (3.6%) and homeostasis (20.1%). The microarray result, that TGF- β receptor TGFβR Ⅱ and Smad2 were significantly increased in NOA testes compared with that of the normal testes which was confirmed by RT - PCR analysis with sequence analysis by T-A cloning. Conclusion The gene expression profile in NOA was changed significantly compared with the control; NOA male may be related with multi-gene changes, especially the increased level of TGF- β signaling, which may be important for further study on NOA.
出处
《中国男科学杂志》
CAS
CSCD
2008年第5期1-4,共4页
Chinese Journal of Andrology
基金
国家自然科学基金211工程(No.219)
国家自然科学基金(No.30772285)
关键词
少精子症
寡核苷酸序列分析
基因表达谱
oligospermia
oligonucleotide array sequence analysis
gene expression profiling