不同变异位点的HIV-1 vpr重组真核表达载体对转染细胞凋亡作用的观察

Effects of different mutated sites in vpr gene of HIV on apoptosis of host cells ： experiment with HeLa cells

目的观察不同的人免疫缺陷病毒1型（HIV-1）的vpr基因变异株致宿主细胞凋亡作用的区别，及其可能的机制。方法14个带有不同变异位点的中国感染者HIV-1 vpr基因片断，其PCR产物纯化后经HindⅢ和BamHⅠ双酶切，以pcDNA3．1（＋）真核表达质粒连接转化实验，将重组质粒用脂质体瞬时转染至HeLa细胞，G418筛选后收获细胞。RT-PCR检测目的基因mRNA水平的表达，荧光染色在显微镜下观察Hoeschst凋亡细胞并计算凋亡率，DNA琼脂糖电泳观察各转染细胞凋亡条带，膜联蛋白V-FITC流式细胞检测各株细胞凋亡率，并比较各株细胞的半胱酸蛋白水解酶3活性差别。结果14个vpr基因片段转染HeLa细胞后，发现有第70、85、86或94位变异的vpr转染细胞Hoeschst染色凋亡率和半胱酸蛋白水解酶3活性检测（分别为0．1225，0．1675，0．1975，0．1575和11．356，8．021，14．6875，10．521）较无这些变异的保守序列vpr转染细胞（0．355和182．4875）为低，DNA琼脂糖电泳法和膜联蛋白细胞凋亡检测也提示同样的规律；第1～7号重组质粒（均为vpr AE亚型）的致细胞凋亡能力也明显小于第8～14号重组质粒（vpr B，AB，C和C／BC亚型）。结论首次观察到有第70、85、86或94位变异的HIV-1 vpr序列，其致HeLa细胞凋亡的能力低于无这些位点变异的保守序列，AE亚型诱导宿主细胞凋亡能力低于其他亚型，其机制之一与半胱酸蛋白水解酶3途径激活降低有关。

Objective To investigate the effects of different mutated sites in the vpr gene of HIV on the apoptosis of host ceils, and the possible mechanism thereof. Methods Fourteen HIV-1 vpr fragments were obtained from HIV-infected persons. Eukaryotic expression vector pcDNA3. 1 （ ＋ ） plasmid was extracted, the PCR purified product was double-cut by Hind Ⅲ and BamH, and the cut products were legated to vectors, thus establishing the JM109 competent cells. Sequencing was used to confirm the reconstruction of pcDNA-vpr eukaryotic expression vectors that were then transfected into HeLa cells. Blank vectors were transfected as control group. Cells were harvested after ：24 hours and underwent Hoechst 33258 staining and observed under fluorescence microscope. Annexin-FITC-PI staining and flow cytometry were used to observe the percentage of apoptosis. The caspase-3 activity was detected by enzyme labeling instrument. Results The apoptotic rates shown by Hoechst and annexin--FITC-PI staining methods, and caspace-3 activity levels of the HeLa cells transfected with the gene fragments with mutated sites 70, 85, 86, and 94 ells were all lower than the cells trasnsfected with the gene fragments without these mutated sites. The apoptosis causing ability levels of the No 1 -7 recombinant plasmids （all of the Vpr AE subtype） were all lower than those of the No 8 - 14 plasmids （of Vpr B, AB, C, and C/BC subtypes）. Conclusion The apoptosis causing ability of the HIV with the vpr sequence with mutated sites 70, 85, 86, 94 is significantly lower than those without these sites. AE subtype induces lower apoptotic behavior in the hoist cells, and decreased activation of the caspase-3 pathway may be one of the mechanisms.