中国HIV感染者VPR序列变异对细胞周期和致凋亡作用的影响 FREE

Effect of HIV1 Vpr on G2 arrest and apoptosis in Hela cell FREE

摘要:

目的研究带有不同变异位点的人免疫缺陷病毒1型（HIV1）vpr基因对感染细胞周期和凋亡的影响，以及其致细胞周期变化和致细胞凋亡的机制间的可能关系。方法以14个带有HIV1 vpr基因片段的pcDNA3.1（+）真核表达载体构建重组质粒，将其转染Hela细胞，并设立保守株vpr基因转染细胞、突变株vprFS基因转染细胞、空载体转染细胞和未转染细胞作为对照，经逆转录多聚酶链式反应（RTPCR）检测目的基因转染成功后，Pi染色，用流式细胞仪检测被转染细胞的细胞周期分布和细胞凋亡率。结果14个带有不同变异位点HIV1 vpr基因片段的Hela细胞经流式细胞仪检测,显示出不同的致细胞周期阻滞和致细胞凋亡的能力。发现转染保守片段HIV1 vpr的Hela细胞，其细胞周期出现G2期阻滞和细胞凋亡率明显升高，但转染vpr C末端截断的vprFS片段的细胞、空载体pcDNA3.1（+）转染细胞和未转染的Hela细胞无此现象。转染了HIV1 vpr基因序列相对应的Vpr蛋白中含有70V、85P、86G、94G突变的片段，较vpr保守片段致感染细胞G2期阻滞和凋亡的能力明显下降。初步发现vpr诱导G2期阻滞百分率越高，其所致凋亡率亦越高。结论HIV1vpr基因有明显的致感染细胞G2周期阻滞和致细胞凋亡的作用，但vpr C末端截断的vprFS片段无此功能。说明中国感染者HIV1 vpr基因表达蛋白的70V、85P、86G、94G位点突变能使其致感染细胞G2期阻滞和凋亡的能力下降。vpr诱导G2期阻滞的程度与其致凋亡水平可能相关，提示两者的发生机制可能有一定的关联。本研究为进一步探讨HIV1致病机制和探索可能的基因干预措施打下了基础。

Abstract:

ObjectiveTo explore the ability of  vpr gene in human immunodeficiency virus type 1 (HIV1 vpr) to induce cell G2 arrest and apoptosis, and the influence when it mutated, the relationship between vprinduced G2 arrest and apoptosis. MethodsFourteen mutated vpr fragments were selected from  patients with HIV. Both eukaryotic expression vector pcDNA3.1(+) and PCR products were purified, doublecut by HindⅢ and BamH Ⅰ, and the cut products were legated and  transformed into competent cells JM109. The 14 reconstructed plasmids were transfected into Hela cells.  Cells with pcDNA vprwt, pcDNA vprFs and pcDNA3.1  blank cells, and without pcDNA3.1 cell were established.  vpr mRNA expression was detected by RTPCR. The DNA content and percentage of apoptosis were monitored by flow cytometry. ResultsTransfected with 14 mutated HIV1 vpr fragments, cells displayed different G2 percentage and apoptosis ratio. HIV1 vpr induced cell cycle G2 arrest and apoptosis, whereas Vpr Fs with a Cterminal truncation, vector pcDNA3.1(+) and the blank cells can not. The  G2 percentage and apoptosis ratio reduced when transfected with Vpr expressing mutation of 70V, 85P, 86G, 94G compared with the Vpr wild type. The higher G2 percentage, the higher ratio of apoptosis was induced. ConclusionHIV1 vpr  can induce cell cycle G2 arrest and apoptosis, whereas Vpr Fs with a Cterminal truncation can not. The mutated sites of 70V, 85P, 86G, 94G may reduce the ability of Vpr to induce cell cycle G2 arrest and apoptosis. The extent of Vprinduced G2 arrest correlated with the levels of apoptosis, This study can  make a good foundation for further research on gene therapy.