| 摘要: |
| 目的:筛选辐射诱导的支气管上皮细胞恶性转化模型中的差异表达基因。方法:应用抑制差减杂交方法（SSH）构建辐射诱导的细胞转化模型差异表达基因的cDNA文库。对差减文库进行PCR筛选，对得到的差异片段进行测序及BLAST分析；对部分筛选出来的差异表达基因使用荧光定量PCR方法检测并确认其变化；将新的序列表达标签（EST）登录到GenBank上。结果:在80个进行测序的克隆中，得到确定序列的共73个。在转化细胞中表达下降的序列41条，BLAST比较分析结果：得到已知序列6条；未知基因的EST序列20条；空载体序列7条；8条为重复测定序列。在转化细胞中表达上升的序列32条，BLAST分析：已知序列14条；未知基因的EST序列9条；重复测定的序列9条。对其中的部分基因改变进行荧光定量PCR检测，结果表明辐射转化组中MY06、HACE1、ZNF143、HNRPH1的表达量明显增加（与对照组相比，转化组中的mRNA分别增加了3.49、29.38、12.99、5.00倍）；而PCBP2、RPL15、TCERG1的表达量下降（与对照组相比，转化组中的mRNA分别减少了1.89、48.77、11.95倍）。将得到的29个未知序列登录到GenBank，序列ID:EB643220~EB643248。结论:利用抑制差减杂交方法成功建立了恶性转化细胞模型差异表达cDNA文库，包含大量功能未知的新基因；ZNF143表达增加,其与细胞的增殖与分裂有关，TCERG1作为转录辅助激活因子在mRNA转录和后期的修饰过程中起到重要的作用，PCBP2是一个Polyc连接蛋白，具有蛋白翻译调节功能，这些基因在辐射致癌中尚无研究报道。 |
| 关键词: 抑制差减杂交 细胞恶性转化模型 辐射 差异表达基因 cDNA文库 |
| DOI：10.3724/SP.J.1008.2008.00756 |
| 投稿时间：2007-07-11修订日期：2008-06-23 |
| 基金项目:国家自然科学基金（30370359）. |
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| Suppression subtraction hybridization in screening of differentially expressed genes in radiation-induced malignant transformation cellular model |
| GAO Fu,CAI Jian-ming,CUI Jian-guo,LI Bai-long*,MIN Rui,HUANG Yue-cheng,NI Jin,SUN Ding,JIANG Hao |
| (Department of Radiation Medicine,Faculty of Navy Medicine,Second Military Medical University,Shanghai 200433,China) |
| Abstract: |
| Objective:To screen for the differentially expressed genes during irradiation-induced malignant transformation of human bronchus epithelium cells (BEAS-2B).Methods: Suppression subtraction hybridization (SSH) was used to construct a subtracted cDNA library of differentially expressed genes during irradiation-induced malignant transformation of BEAS-2B cells.Then the subtracted library was screened by PCR and the differential fragments were sequenced and analyzed with BLAST.Fluorescent real-time quantitative PCR was used to investigate some of the differentially expressed genes.The new EST was registered in GenBank.Results: Then 40 clones were chosen to be sequenced from the library of increased expression and decreased expression respectively according to the length of insertion element.Totally 73 sequences were obtained from the 80 sequenced clones.Forty-one sequences were decreased in the transformed cells; BLAST analysis indicated that there were 6 known sequences,20 unknown sequences,7 void sequences and 8 repeated sequences.Thirty-two sequences were increased in the transformed cells; Blast analysis indicated that there were 14 known sequences,9 unknown sequences,and 9 repeated sequences.Fluorescent quantitative PCR revealed that,compared with control group,the expression of MY06,HACE1,ZNF143,and HNRPH1 were significantly increased in the radiation transforming group,with their mRNAs increased by 3.49,29.38,12.99 and 5.00 folds,respectively.Compared with control group,the expression of PCBP2,RPL15,and TCERG1 in the radiation transforming group was significantly decreased,with their mRNAs decreased by 1.89,48.77 and 11.95 folds,respectively.The 29 unknown sequences were registered in the GenBank (ID: EB643220-EB643248).Conclusion: The cDNA library has been successfully established for malignant transformation cellular model by suppression subtractive hybridization;the library includes a number of unknown genes.The increased gene ZNF143 is associated with cell proliferation and cell division.TCERG1,as an assistant transcription activation factor,plays an important role in the mRNA transcription and later modification.PCBP2,a Polyc connection protein,plays a modulating role in protein translation.These genes have not been reported in the radiation carcinogenicity. |
| Key words: suppression subtractive hybridization malignant transformations cellular model irradiation differential expression genes cDNA library |
