Xing Hui Chun, Xu Xiao Yuan, Liu Zhen, Wang Qin Huan, Yu Min, Si Chong Wen
Department of Infectious Diseases, Peking University First Hospital, Beijing 100034, PR China.
Jpn J Infect Dis. 2004 Jun;57(3):91-6.
CXC-chemokine receptor (CXCR4) is one principle co-receptor for the entry of T cell line (T)-tropic HIV-1 virus into a cell. In order to find more efficacious therapeutic possibilities for people with an HIV-1 infection, we explored the inhibitory effects of antisense RNA on CXCR4 expression in MT4 cells. First, we used to RT-PCR to obtain DNA fragments from healthy adult peripheral blood mononuclear cells; these fragments targeted the initiation region of CXCR4 mRNA translation. We then constructed a recombinant retroviral vector, pLXSN-X4a (containing antisense RNA to CXCR4). After packaging by PA317 cells, the pseudovirion of the recombinant vector had formed and succeeded in transfecting MT4 cells (a kind of T-tropic HIV-1 susceptibility cell line). The PCR and RT-PCR results showed that the recombinant vector had integrated into the genome of MT4 cells and had been transcribed. The expression of CXCR4 on the surface of MT4 cells transfected with antisense RNA was reduced by 30%, compared with those cells transfected with blank vector or untransfected cells. No change in the DNA synthesis rates or in cell proliferation was found in any of the transfected cells. After a challenge with HIV-1 SF33, the cells transfected with antisense RNA vector (pLXSN-X4a) produced reduced p24 levels compared with the cells transfected with blank vector (pLXSN) or untransfected cells. These results indicated that these CXCR4-antisense expressing cells could resist T-tropic HIV-1 infection and could retain normal biological functions. These studies provide useful data for further experiments in this area.
CXC趋化因子受体（CXCR4）是T细胞嗜性HIV-1病毒进入细胞的一种主要共受体。为了找到针对HIV-1感染患者更有效的治疗方法，我们研究了反义RNA对MT4细胞中CXCR4表达的抑制作用。首先，我们用RT-PCR从健康成人外周血单个核细胞中获取DNA片段；这些片段靶向CXCR4 mRNA翻译的起始区域。然后我们构建了重组逆转录病毒载体pLXSN-X4a（含有针对CXCR4的反义RNA）。经PA317细胞包装后，重组载体的假病毒颗粒形成并成功转染MT4细胞（一种T细胞嗜性HIV-1易感细胞系）。PCR和RT-PCR结果显示，重组载体已整合到MT4细胞基因组中并已转录。与转染空载体的细胞或未转染的细胞相比，用反义RNA转染的MT4细胞表面CXCR4表达降低了30%。在任何转染细胞中均未发现DNA合成速率或细胞增殖有变化。用HIV-1 SF33攻击后，与转染空载体（pLXSN）的细胞或未转染的细胞相比，用反义RNA载体（pLXSN-X4a）转染的细胞产生的p24水平降低。这些结果表明，这些表达CXCR4反义的细胞能够抵抗T细胞嗜性HIV-1感染并能保持正常生物学功能。这些研究为该领域的进一步实验提供了有用的数据。
