人巨细胞病毒对巨核细胞生长的影响及其反义寡核苷酸的抗病毒活性  

Effect of HCMV on the Development of Human Megakaryocytes and Antiviral Effect of Antisense Oligonucleotides to HCMV

作　　者：姚军霞[1] 刘莉[2] 刘仲萍[1] 黄士昂[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院干细胞中心,武汉430022 [2]华中科技大学同济医学院附属协和医院肿瘤中心,武汉430022

出　　处：《华中科技大学学报（医学版）》2007年第3期322-325,共4页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家杰出青年科学基金资助项目(No.30225038)

摘　　要：目的探讨人巨细胞病毒(HCMV)对人巨核系祖细胞的抑制作用及其机制,初步研究HCMV反义寡核苷酸(ASON)的抗病毒感染作用。方法采用体外悬浮培养巨核系细胞,观察HCMV AD169株对巨核系祖细胞生长的影响。用流式细胞术检测HCMV感染巨核系细胞上HCMV pp65抗原的表达。用HCMV感染经ASON预处理的巨核细胞,计数存活巨核细胞数,用FACS检测巨核细胞上HCMV pp65蛋白表达,并用MTT法测定ASON的细胞毒性。结果HCMV AD169株在体外能明显抑制巨核系祖细胞的增殖,悬浮培养第12天,与对照组相比,HCMV感染组的巨核细胞数减少了67.86%(P<0.05)。HCMV感染的巨核系细胞有HCMV pp65抗原的表达,其表达率为(24.5±3.1)%。0.08μmol/L的UL36反义寡核苷酸(UL36Anti)能对抗HCMV的作用,使巨核细胞数保持在未被感染时的水平,与HCMV感染组巨核细胞数比较有显著差异(P<0.05)。UL36Anti组巨核细胞上几乎无HCMV pp65蛋白的表达。MTT结果表明UL36Anti显著影响细胞生长的浓度为90μmol/L。结论HCMV感染引起的血小板减少与其抑制巨核系祖细胞增殖有关。特异的ASON有一定的抗HCMV活性,有可能作为一种新的手段用于HCMV感染引起的血小板减少症的防治。Objective To investigate the inhibitory effect and the mechanism of human cytomegalovirus （HCMV） on human megakaryocyte precursors and study the antiviral effect of antisense phosphorothioate deoxyoligonucleotide （ASON） against HCMV. Methods Megakaryocytes of 18 cord blood samples in a liquid culture system and pretreated CD34＋ cells with ASON against HCMV named UL36Anti were infected by HCMV AD169 strain. The number of megakaryocytes was counted. FACS was used to detect the expression of HCMV pp65 protein in megakaryocytes. UL36Anti＇s cytotoxicity was evaluated by the MTT assay. Results HCMV AD169 suppressed the formation of megakaryocytes in vitro significantly. Compared with control group, the number of megakaryocytes infected by HCMV AD169 was decreased by 67.86% （P〈0.05）. FACS detected 2-color fluorescent staining with antibodies directed against CD41a and HCMV pp65 antigen on megakaryocytes infected by HCMV, and the expression rate was （24.5±3.1）M. Compared with the infected group by HCMV, UL36Anti treatment at 0.08μmol/L could recover the number of megakaryocytes significantly （P%0.05）. In the infected megakaryocytes pretreated with UL36Anti of 0.08μmol/L, HCMV pp65 was not detected. MTT assay revealed that the concentration for UL36Anti significantly inhibiting the cell growth was 90μmol/L. Conclusion HCMV AD169 strain inhibited the proliferation and differentiation of megakaryocytes and their precursors by directly infecting them. The specific ASON has sure anti-HCMV activity. As a new means, it may be used to prevent and treat thrombocytopenic purpura caused by HCMV infection.

关 键 词：巨细胞病毒 巨核系祖细胞 反义寡核苷酸 

分 类 号：R373.4[医药卫生—病原生物学]