基因芯片筛选尿道下裂雌激素敏感基因的研究

目的 寻找与尿道下裂发病密切相关的基因,探讨尿道下裂形成机制.方法 ①实验组为12例尿道下裂患儿,年龄6～12个月,平均8个月.尿道下裂中度5例,重度7例.组织标本取自尿道成形手术时切取的尿道板组织.②对照组为6例年龄匹配的男性患儿,留取包皮环切时的正常表皮组织.用Tri-Reagent分别提取总RNA,与含22 000个人类基因的寡核苷酸基因芯片杂交、洗脱、染色、扫描,基因强度变化行方差分析(ANOVA,P＜0.01)和Tukey分析,基因表达强度变化＞2倍作为有意义的基因,比较尿道下裂和正常组织之间基因表达差异.从上调表达的基因中选择雌激素敏感基因行RT-PCR,标本除上述患儿RNA外,再增加年龄配对的3例中度、1例重度尿道下裂和2例年龄配对的RNA作为对照,即对照、中度和重度尿道下裂患者RNA标本各8例,验证芯片结果.结果 尿道下裂组织与正常组织之间存在明显的基因表达差异,共筛选出表达强度变化＞2倍的基因94个,其中中度尿道下裂与正常比较,47个基因上调表达(P＜0.01);重度尿道下裂与正常比较,68个基因上调表达(P＜0.001);重度与中度比较,17个基因上调表达(P＞0.05).上调表达的基因中发现了4个雌激素敏感基因CYR61、结缔组织生长因子、ATF3和GADD45β,基因芯片和RT-PCR均证实其在尿道下裂组织中的表达明显高于正常对照.结论 异常表达的基因与尿道下裂的发生有关,尿道下裂组织中雌激素敏感基因上调表达参与了尿道下裂的发病机制.

Gene expression change study in hypospadias by microarray

Objective To detect the gene expression changes between urethra plates from hypospadias patients and foreskins from non-hypospadias patients by microarray and to investigate the underlying mechanisms of hypospadias.Methods Twelve hypospadias patients,aged 6-12 months (mean,8 months),were enrolled as the test group,including 5 moderate and 7 severe hypospadias patients.Six age-matched patients underwent circumcision were enrolled as controls.Samples from hypospaidas patient's urethra plates during hypospadias repair and samples from the foreskins during circumcision were obtained and processed into Tri-Reagent immediately for RNA extraction.Oligonucleotide expression microarrays were used to detect genes expression changes in tissues from patients with and without hypospadias.This microarray analysis incorporated 22 000 genes.The intensity of all genes present was analyzed by one-way ANOVA (P＜0.01) and Tukey's test.Four estrogen responsive genes,CYR61,CTGF,ATF3 and GADD45β,were tested by RT-PCR in 8 controls,8 moderate hypospadias and 8 severe hypospadias as well.Results Ninty-four genes were detected differentially expressed in hypospadias patients compared with phimosis patients.There were 47 genes upregulated in moderate hypospadias compared with controls(P＜0.01),68 genes up-regulated in severe hypospadias compared with controls(P＜0.001),17 genes up-regulated in severe hypospadias compared with moderate hypospadias(P＞0.05).These genes were involved in different cell functions such as growth regulation and signal transduction.CYR61,CTGF,ATF3 and GADD45β,known to be estrogen responsive or to interact with estrogen receptor were found up-regulated in microarray and the up-regulations were confirmed by RT-PCR.Conclusions The up-regulated genes contribute to the development of hypospadias.Up-regulation of estrogen responsive genes may play important roles in the development of hypospadias.