Differential gene expression during HIV-1 infection analyzed by suppression subtractive hybridization.
Differential gene expression during HIV-1 infection analyzed by suppression subtractive hybridization.
复制标题
通过抑制消减杂交分析 HIV-1 感染期间的差异基因表达。
DOI：
10.1097/00002030-200403050-00002
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Finkel,TerriH
中科院分区：
文献类型：
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作者：
Yin,Jiyi;Chen,MariaF;Finkel,TerriH
目的：探讨HIV-1感染和细胞凋亡对细胞和病毒基因表达的影响。方法：采用流式细胞术分析HIV-1 iiib感染的CEM-SS T细胞的感染和凋亡情况。对不同感染时间点的细胞进行抑制消减杂交（Suppression subtractive hybridization， SSH），构建消减互补DNA文库。差异筛选和Northern blots证实了差异基因的表达，并对这些基因进行了测序并与数据库进行了比较。结果：T细胞在HIV-1 IIIB感染早期（感染后5-7天）发生凋亡。令人惊讶的是，细胞在第9天开始恢复，到第18天，几乎所有被感染的细胞都能存活，即使它们保持相同的感染水平。通过SSH，鉴定了HIV-1 IIIB感染后第7天和第18天的差异基因表达谱。SSH得到两个缺失的cDNA文库；差异筛选表明，864个菌落中有200个在各自的时间点高度表达。DNA序列分析确定了特定的凋亡相关基因、HIV-1病毒基因和其他感兴趣的候选基因。Northern blot分析证实，其中一些基因主要在“凋亡”或“非凋亡”时间点表达。结论：已知的和新的细胞基因产物已经被确定与细胞凋亡直接（或负）相关，并可能调节HIV-1感染中的细胞死亡。这些结果为差异表达基因的功能研究提供了一个框架，并可能为治疗hiv -1感染者提供新的治疗方法。
Objective:Characterization of the effects of HIV-1 infection and apoptosis on cellular and viral gene expression.Methods:Flow cytometry was used to analyze infection and apoptosis concurrently in HIV-1 IIIB-infected CEM-SS T cells. Suppression subtractive hybridization (SSH) was applied to cells from different time points of infection to construct subtracted complementary DNA (cDNA) libraries. Differential screening and Northern blots confirmed differential gene expression and these genes were sequenced and compared with database.Results:T cells undergo apoptosis at early stages of HIV-1 IIIB infection (days 5–7 post-infection). Surprisingly, cells begin to recover after day 9 and by day 18 almost all infected cells are viable, even though they maintain the same level of infection. By SSH, differential gene expression profiles between day 7 and day 18 after HIV-1 IIIB infection were characterized. SSH yielded two subtracted cDNA libraries; differential screening of the subtracted cDNA libraries suggested that 200 out of 864 colonies were highly expressed at their respective time points. DNA sequence analysis identified specific apoptosis-related genes, HIV-1 viral genes, and other candidate genes of interest. Northern blot analysis confirmed that some of these genes were expressed predominantly at the ‘apoptotic’or ‘non-apoptotic’time points.Conclusions:Known and novel cellular gene products have been identified that are directly (or inversely) correlated with apoptosis and may regulate cell death in HIV-1 infection. These results provide a framework for functional studies on the differentially expressed genes and may suggest novel therapeutic approaches for treatment of HIV-1-infected individuals.