喘可治抑制灭活HIV-1颗粒诱导人CD4+T细胞凋亡机制研究

Mechanism on the Inhibition and Inactivation of HIV-1 Granules and the Induction of Human CD4^+T Apoptosis by Chuankezhi

目的通过深入研究HIVp对CD4+T细胞凋亡的影响,以探讨HIV病中CD4+T细胞丢失的可能机制以及喘可治(CKZ)对CD4+T细胞凋亡的调控作用。方法 AT-2灭活HIV-1ⅢB型病毒颗粒,ELISA法检测p24抗原的含量;然后将HIVp加入到PBMCs中,使p24抗原终浓度为1 ng.mL-1;在相应的组中加入喘可治,使其终浓度为40μL.mL-1(v/v)。48 h后,运用荧光标记的An-nexin V染色技术结合流式细胞术评价CD4+T细胞的凋亡率;运用免疫荧光抗体染色技术结合流式细胞术检测CD4+T细胞CD95和DR5的表达;运用胞内免疫荧光抗体染色技术和分子探针染色技术结合流式细胞术检测CD4+T细胞Apo2.7和线粒体内膜膜电势(△ψm)的水平。结果与对照组相比,HIVp可以引起明显的CD4+T细胞凋亡(P<0.01),而喘可治能够有效抑制HIVp引发的CD4+T细胞凋亡(P<0.01)。结论 HIVp通过外源性凋亡途径和内源性凋亡途径删除CD4+T细胞,从而导致免疫缺陷;喘可治能够保护CD4+T细胞而有望开发成为艾滋病治疗的辅助药物。

Objective To explore the potential mechanism on CD4＋T cell loss in HIV and the regulation of CD4＋T apoptosis by chuankezhi（CKZ）through the deep research of the impacts of HIVp on CD4＋T apoptosis.Methods AT-2 was applied for the inactivation of HIV-1ⅢB viral granules.ELISA was used to detect the content of p24 antigen.HIVp was added into PBMCs so as to enable the terminal concentration of p24 antigen as 1ng·mL-1.CKZ was supplemented in corresponding groups so as to enable its terminal concentration as 40 μL·mL-1（v/v）.48 h later,the fluorescence labeling Annexin V staining technique combined with flow cytometry（FCM）was adopted to evaluate the apoptosis rate of CD4＋T cell.The immune fluorescent antibody staining technique combined with FCM was used to detect the expressions of CD95 and DR5 in CD4＋T cell.The intracellular immune fluorescent antibody staining technique and molecular probe dyeing technique as well as FCM were combined together to detect Apo2.7 in CD4＋7 cell and the level of mitochondrial membrane potential（△ψm）.Results Compared with control group,HIVp induced an apparent CD4＋T apoptosis（P0.01）.CKZ could effectively inhibit CD4＋T apoptosis induced by HIVp（P0.01）.Conclusion HIVp deletes CD4＋T cell via exogenous apoptosis and endogenous apoptosis and results in immune deficiency.CKZ can protect CD4＋T and is expected to be developed as an aided medicine for the treatment of AIDS.