基因芯片筛选非综合征型唇腭裂差异表达基因的初步研究

A pilot study of differential genes in nonsyndromic cleft lip with or without cleft palate（NSCL/P） by expression microarray

摘要:

目的：利用全基因组表达谱芯片技术，分析非综合征型唇腭裂（nonsyndromic cleft lip with or without palate，NSCL/P）患儿与正常儿童基因表达的差异，筛选出非综合征型唇腭相关基因。方法：采用离心柱法分别提取正常儿童和NSCL/P患儿腭部组织各3例的总RNA，经纯化后逆转录为cDNA，利用荧光染料（Cy3）标记aaUTP，转录合成标记的cRNA，经纯化及质检后与Agilent 4 × 44 k人类全基因组表达谱芯片杂交，扫描荧光信号图像，对芯片原始数据进行归一化处理，利用倍数差异和t检验筛选差异表达基因，采用上海博豪生物在线分析系统进行基因的功能注释和关联分析，明确差异基因的生物学功能，选取特定的差异表达基因，应用实时荧光PCR技术验证芯片结果的可靠性。结果：筛选出差异基因共254个，其中表达上调的有151个，表达下调的有103个。选取5条差异表达基因进行实时荧光定量PCR验证，ADH1C（t=5.132，P=0.000）、RDH10（t=2.960，P=0.039）、HIST1H2BD（t=4.446，P=0.001）3条基因表达下调，KAT2B（t=-4.945，P=0.000）、FOSB（t=-3.666，P=0.005）2条基因表达上调，差异有统计学意义，且与芯片结果表达趋势一致。结论：NSCL/P患儿基因表达与正常儿童存在明显差异，分析这些差异基因，有助于阐明NSCL/P的发病机制，为疾病的预防提供理论依据。

Abstract:

Objective：To analyze the deferentially expressed genes between nonsyndromic cleft lip with or without cleft palate（NSCL/P） patients and healthy control using gene expression microarray technology and to screen NSCL/P related genes. Methods：Total RNA was extracted from 3 NSCL/P patients and 3 controls by centrifugal column method. The RNA was purified and qualitatively qual－ified，then reverse transcribed into cDNA. The cDNA was transcribed into cRNA；fluorescence dye Cy3 was used to label the cRNA. The labeled cRNA was used to hybrid with the Agilent 4 × 44 k human genome-wide expression of the gene chip；the fluorescence signal was scanned by the computer and the original data were normalized. The differential expression genes was screened by t-test and fold difference.The gene functional annotation and correlation analysis were carried out by the online analysis system of Shanghai Bohao biology to clarify the biological functions of the different genes. The specific differential expression genes were selected and the reliability of the chip results was verified by real-time PCR. Results：Totally 254 differentially expressed genes were screened out，among which 151 had up-regulated expression and 103 had down-regulated expression. Five differential expression genes were selected for real-time PCR. ADH1C（t=5.132，P=0.000），RDH10（t=2.960，P=0.039），HIST1H2BD（t=4.446，P=0.001） had down-regu－lated expression，KAT2B（t=-4.945，P=0.000），FOSB（t=-3.666，P=0.005） had up-regulated expression. The results showed that three genes had down-regulated expression and two genes had up-regulated expression，which was consistent with the result of chip ex－pression. Conclusion：There are significant differences in gene expression between normal children and children with NSCL/P. Analyzing these diferentially expressed genes may help clarify the pathogenesis of NSCL/P and provide a theoretical basis for disease prevention.