Identification and cloning of human astrocyte genes displaying elevated expression after infection with HIV-1 or exposure to HIV-1 envelope glycoprotein by rapid subtraction hybridization, RaSH
Identification and cloning of human astrocyte genes displaying elevated expression after infection with HIV-1 or exposure to HIV-1 envelope glycoprotein by rapid subtraction hybridization, RaSH
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DOI：
10.1038/sj.onc.1205445
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发表时间：
2002-05-16
期刊：
影响因子：
8
通讯作者：
Fisher, PB
中科院分区：
文献类型：
--
作者：
Su, ZZ;Kang, DC;Fisher, PB
神经变性和痴呆是艾滋病的常见并发症，由人类免疫缺陷病毒1型（HIV-1）感染中枢神经系统引起。脑中的HIV-1靶细胞包括小胶质细胞、浸润性巨噬细胞和星形胶质细胞，但很少有神经元。星形胶质细胞在维持突触微环境和神经元信号传递中起重要作用。为了研究与HIV-1感染星形胶质细胞相关的细胞基因表达的潜在变化，我们采用了一种高效灵敏的快速消减杂交方法，RaSH。从流产脑组织中分离原代人星形胶质细胞，并用HIV-1感染低传代细胞。为了鉴定在HIV-1感染后显示早期和晚期表达修饰的基因，并避免克隆在星形胶质细胞中显示正常细胞周期波动的基因，从6、12、24小时和3和7天未感染和感染的细胞中分离并合并RNA，并用于RaSH。从双链cDNA制备时间cDNA文库，所述双链cDNA被酶促消化成小片段，连接到衔接子，PCR扩增，并通过孵育测试和驱动PCR片段进行杂交。通过从HIV-1感染细胞制备的时间cDNA中减去来自未感染星形胶质细胞的时间cDNA，鉴定了在病毒感染细胞中显示升高表达的基因，称为星形胶质细胞升高基因（AEGs）。已知的和新的AEGs，目前的DNA数据库中没有报道，显示早期或晚期表达动力学后，HIV-1感染或治疗与重组HIV-1包膜糖蛋白（gp 120）。对于选定的AEGs，其蛋白质产物的表达也通过Western印迹法进行了测试，发现在HIV-1感染后显示出升高的表达。HIV-1感染或gp 120治疗后AEGs的调节模式类似，表明即使在没有生产性感染的情况下，星形胶质细胞的HIV-1暴露也可以诱导细胞基因表达的变化。
Neurodegeneration and dementia are common complications of AIDS caused by human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system. HIV-1 target cells in the brain include microglia, infiltrating macrophages and astrocytes, but rarely neurons. Astrocytes play an important role in the maintenance of the synaptic micro-environment and in neuronal signal transmission. To investigate potential changes in cellular gene expression associated with HIV-1 infection of astrocytes, we employed an efficient and sensitive rapid subtraction hybridization approach, RaSH. Primary human astrocytes were isolated from abortus brain tissue and low-passage cells were infected with HIV-1. To identify genes that display both early and late expression modifications after HIV-1 infection and to avoid cloning genes displaying normal cell cycle fluctuations in astrocytes, RNAs were isolated and pooled from 6, 12, 24 h and 3 and 7 day uninfected and infected cells and used for RaSH. Temporal cDNA libraries were prepared from double-stranded cDNAs that were enzymatically digested into small fragments, ligated to adapters, PCR amplified, and hybridized by incubation of tester and driver PCR fragments. By subtracting temporal cDNAs derived from uninfected astrocytes from temporal cDNAs made from HIV-1 infected cells, genes displaying elevated expression in virus infected cells, termed astrocyte elevated genes (AEGs), were identified. Both known and novel AEGs, not reported in current DNA databases, are described that display early or late expression kinetics following HIV-1 infection or treatment with recombinant HIV-1 envelope glycoprotein (gp120). For selected AEGs, expression of their protein products was also tested by Western blotting and found to display elevated expression following HIV-1 infection. The comparable pattern of regulation of the AEGs following HIV-1 infection or gp120 treatment suggest that HIV-1 exposure of astrocytes, even in the absence of productive infection, can induce changes in cellular gene expression.