| Background:vpr is one of seven HIV virus additional regulatory genes. vpr protein produced by vpr gene plays an important role in the process of HIV infect host cells and in the pathogenesis of AIDS. It is involved in nuclear translocation of proviral integration complex, transcription activation of the virus, induces cell cycle G2/M phase arrest and promotes apoptosis of infected cells. NKG2D is a major activating receptor of NK cell, transducte activation signal after binding ligand ULBPs and induced NK cytotoxicity. This paper would research the impact of HIV-1Vpr to the expression on ULBPs, confirme vpr is one of viral factors activated NKG2D receptor and guided NK cytotoxic effect。Objective:Vitro cell experiment observe the expression of NKG2D ligands ULBP1and ULBP2increase on the vpr positive jurkat cell surface, confirmed the conclusions that the expression of ULBP1and ULBP2on the surface of CD4+T cells increased significantly in hiv-infected patients peripheral blood, which observed by our laboratory; cytotoxicity test discovery the lethality to the vpr positive jurkat cell decreased after blocking NKG2D on NK cell surface; put forward the molecular mechanisms that HIV-1Vpr possiblely through upregulation of NKG2D ligands, activating NK cytotoxic effect of CD4+T lymphocytes.This study will help to clarify the pathogenesis of HIV-1and provide an effective molecular target for the treatment of AIDS. Methods:The full length sequences of vpr gene was acquired from the plasmid BSXCYPYVPRXCTHY o To determine the primary structure of vpr, the DNA sequences of plasmids were analyzed by automatic DNA sequencing. Vpr was transfected into jurkat cell by Liposome, after culturing three days, mRNA expression of vpr of transfected cells was detected by RT-PCR。Flow cytometry detected the expression of double positive expression of ULBP1and ULBP2on jurkat cell surface (the group after vpr transfected, PCD transfected, blank group)。Peripheral blood mononuclear cells (PBMCs) were obtained after Ficoll-Hypaque density gradient centrifugation of whole blood. Take the NK cells and the ones after blocking NKG2D with NKG2D antibody from healthy people and HIV-1infected untreated patients (HIV group) as effector cells, Vpr negative jurkat cells as target cells,vitro cytotoxicity experiments compare the cytotoxic effect.Results:The sequencing result of the vpr equaled the known vpr sequence in the primary plasmid;VPr and PCD empty vector was transfected into jurkat cells by Liposome, RT-PCR detected the expression of target genes vpr. Flow cytometry elevated that the expression of ULBP1/ULBP2double positive increased on the vpr positive jurkat cells surface. NK cell cytotoxicity experiments showed that the cytotoxic effect of NK cell after blocking NKG2D to the vpr positive jurkat cell decreased.Conclusion:1. The expression of ULBP1/ULBP2on the surface of vpr positive jurkat cells increased than the blank group and PCD group, indicating that HIV-1Vpr can upregulate the expression of NKG2D ligands on the surface of jurkat cells, confired the conclusions that our Laboratory studies had made out through vitro experiments:the expression of ULBP1and ULBP2on the surface of CD4+Tcells increased in hiv-infected patients peripheral blood,and excludes the impact of other gene products, further demonstrates the key role of vpr in it.2.The lethality of NK cells to the vpr positive jurkat cells after blocking the NKG2D declined,reveals that after vpr infection,the NK cytotoxicity enhanced through NK cell activating receptor NKG2D Handing NKG2D ligands ULBP-1and ULBP-2, signal transduction after vpr infected。Compared with NK cells from normal people, The Cell lethality of NK cells from HIV-infected persons decrease, suggesting that the cytotoxicity of NK cells may be impaired after HIV infected。... |
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