人单核细胞株巨细胞病毒编码miR-US25-1-3p过表达后蛋白质组学变化分析

摘要:目的分析人 单核细胞株(THP-1)过表达人巨细胞病毒( HCMV)编码miR-US25-1-3p后蛋白质表达谱变化及价值。方法采用脂质体将 hemv-miR-US25-1-3p模拟物转染THP-1细胞构建hemv-miR-US25-1-3p过表达细胞株，RT-qPCR验证转染 效果;采用串联质谱标签标记联合液相色谱-串联质谱对hemv-miR-US25-1-3p 过表达后THP-1细胞蛋白质组学进行定量分析,生物信息学分析差异表达蛋白质生理病理功能;western blot验证重要差异蛋白表达变化。结果与对照相比， THP-1 细 胞过表达hcmv-miR-US25-1-3p后65种蛋白质呈现差异表达,其中17种上调,48种下调。GO、COG和KEGG注释及功能预测结果显示,差异表达蛋白主要参与炎症反应信号通路、代谢过程、细胞及遗传信息功能,与肿瘤、病毒性心肌炎、非酒精性脂肪 肝、原发性免疫缺陷病、类风湿关节炎、多种病原体感染、阿尔茨海默病、亨廷顿病等多种疾病相关。48种下调蛋白质中,文献调研和生物信息学分析发现，肿瘤坏死因子相关凋亡诱导配体(TRIL)和受激活调节正常T细胞表达和分泌因子(RANTES) 参与抗病毒及炎症反应,上述蛋白质下调表达经western blot 验证。结论HCMV 编码的hcmv-miR-US25-1-3p 能够影响宿主蛋白表达谱,并可能通过抑制TRAIL和RANTES表达,促进病毒潜伏、免于宿主细胞杀伤。     

活动性人巨细胞病毒感染的病毒基因表达特点

目的:探讨活动性人巨细胞病毒(HCMV)感染的病毒基因表达特点.方法:建立持续稳定感染(A组)及活动感染(B组)两种细胞感染状态,FQ-PCR和RT-PCR法监测HCMV DNA复制和MCP mRNA转录,免疫组化法联合计算机病理图文分析系统检测pUL44表达水平,光镜观察细胞病变、电镜检查细胞超微结构改变.结果:B组HCMV DNA负荷量(5.61～9.04 lgGE/106HEL)明显高于A组(4.32～5.91 lgGE/106HEL,P<0.05或P<0.01),pUL44也持续升高表达(area:339.6～1 431.9μm2,IA:56.4～319.8,P<0.01),MCP mRNA水平逐渐升高.A组pUL44少量、随机表达(area:219.2～296.7μm2,IA:31.0～58.3),未检到MCP mRNA,亦未发现细胞病变,B组出现进行性加重的细胞病变和细胞超微结构破坏.结论:HCMV DNA负荷量持续升高及MCP mRNA转录是活动性HCMV感染的重要标志.     

白颖苔草提取物抗人巨细胞病毒作用机制的实验研究

目的初步探讨白颖苔草提取物抗人巨细胞病毒（human cytomegaloviras,HCMV）的作用机制。方法通过不同时间点干预HCMV—HEL感染细胞模型,确定提取物是在病毒入侵细胞前或后发挥抗病毒作用;应用RT—PCR检测即刻早期基因UL122、早期基因UI朋、晚期基因UL86mRNA的转录表达,探讨提取物发挥抗病毒作用的时段;通过观察HCMV—UL32荧光病毒在HEL增殖过程中的荧光信号变化,确定提取物是在HCMVUL32基因特录前或者转录后发挥作用。结果无论将提取物在病毒入侵细胞前、后进行干预HCMV—HEL感染模型,均可抑制细胞病变的发生;RT-PCB．结果提示干预模型的HCMVUL122、UL44基因的结果为阳性,而晚期基因UL86的转录表达为阴性;提取物可有效抑制荧光病毒的荧光信号的表达。结论白颖苔草提取物抗HCMV的作用机制主要是抑制中晚期的基因转录,而对病毒的入侵过程和即刘早期、早期基因的转录表达无明显影响。     

先天性人巨细胞病毒肝炎小鼠模型的建立

目的探讨建立人巨细胞病毒（HCMV）先天性感染致新生鼠肝炎模型的可行性。方法将HCMV-AD169接种至10周龄Balb/c雌雄小鼠腹腔后,随机选择配对。待雌鼠分娩后取出新生鼠肝脏,进行病毒分离、病理学检测及原位分子杂交检测。结果病理学研究结果证实,HCMV感染的新生小鼠肝组织中见点、灶状坏死,并可见巨核细胞。坏死区炎细胞浸润。核内可见偏于一端的包涵体,使细胞呈＂猫头鹰眼＂样,汇管区可见炎细胞浸润。局部肝脏包膜增厚。原位杂交结果显示,病毒核酸存在于受感染肝细胞内。病毒分离结果证实在新生鼠肝组织上清液中可分离出HCMV。结论 HCMV能侵袭Balb/c小鼠,并通过胎盘感染其子代,引起肝炎。这种模拟人类先天性巨细胞病毒肝炎的小鼠模型的建立为该病动物实验研究提供了可能。     

甘草酸对感染人巨细胞病毒的人胚肺成纤维细胞凋亡和炎性反应的影响

目的 探讨甘草酸对感染人巨细胞病毒(HCMV)的人胚肺成纤维细胞MRC-5增殖、凋亡和炎性反应的影响。方法 体外培养MRC-5细胞,并将其分为对照组、病毒组、更昔洛韦组(50μg/mL)、甘草酸低剂量组(0.25 mg/mL)、甘草酸中剂量组(0.50 mg/mL)、甘草酸高剂量组(1.00 mg/mL)。接种HCMV不同时间后,分别使用CCK8试剂盒检测细胞增殖活性,使用流式细胞仪检测细胞凋亡情况,采用蛋白质印迹法(Western blot)检测细胞中活化含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved-caspase3)等蛋白表达情况,采用酶联免疫吸附试验(ELISA)检测细胞培养上清液中肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)水平。结果 CCK8实验结果显示,经HCMV感染48、72 h时,病毒组增殖活性均低于对照组,更昔洛韦组、甘草酸中剂量组、甘草酸高剂量组增殖活性均高于病毒组,差异有统计学意义(P<0.05);甘草酸中剂量组与甘草酸高剂量组增殖活性比较,差异无统计学意义(P>0.05),故选择甘草酸中剂量组(0.50 mg/mL)进行后续实验,并...     

免疫低下状态群体感染人巨细胞病毒的研究进展

人巨细胞病毒(HCMV)是疱疹病毒科中最大的病毒,结构复杂,其感染在人群中非常普遍,近年来,因免疫低下状态群体尤其是移植群体中的HCMV潜伏感染和激活感染而越来越受到临床重视。本文就HCMV生物学特性,HCMV的感染与免疫,HCMV的致病机制,HCMV感染情况及HCMV相关研究的问题进行阐述。     

人巨细胞病毒抑制人巨核系祖细胞增殖的体外研究

目的 探讨人巨细胞病毒（HCMV）对人巨核系祖细胞增殖的影响。方法 收集20份脐血标本,采用巨核系祖细胞体外半固体培养技术,观察HCMV AD169株对巨核细胞集落形成单位（CFU－MK）生长的影响,分别用原位聚合酶链反应（IS－PCR）和RT－PCR检测集落细胞中的HCMV DNA与抑刻早期抗原（IEA）mRNA。结果 HCMV AD169株在体外能明显抑制CFU－MK生长,抑制程度与病毒感染滴度呈剂量依赖关系,经IS－PCR检测发现病毒感染组CFU－MK细胞中有HCMV DNA存在;RT－PCR检测发现病毒感染组CFU－MK细胞中有IEAmRNA的表达。结论 HCMV AD169株可直接感染巨核系祖细胞,并抑制其增殖与分化;HCMV对巨核系祖细胞的直接抑制作用可能是该病毒感染后引起血小板减少的原因之一。     

人巨细胞病毒特异性T淋巴细胞检测的研究进展

人巨细胞病毒 (HCMV)是疱疹病毒的一种 ,在不同人群中的感染率为 5 0 %～ 90 %。初次感染后 ,在宿主免疫的控制下潜伏于骨髓等细胞中。当免疫功能受损时 (移植、获得性免疫缺陷综合征、肿瘤等 ) ,潜伏的病毒易被激活 ,严重者可引起致死性肺炎、肝炎[1] 。大量研究表明 ,特异性细胞免疫对HCMV感染的控制和疾病的恢复起重要作用 ,尤其是CD8+ T对控制HCMV感染更为重要[1,2 ] 。Cwynarski等[3 ] 研究发现 ,HCMV潜伏感染期 ,如特异性CD8+ T在免疫正常者中为 0 4～ 0 8× 10 7/L ;而肾移植受者为≥ 1× 10 7/L时 ,均可阻止病毒的激活…     

携带增强绿色荧光蛋白的慢病毒载体感染人脂肪源性干细胞的有效性

背景:细胞移植前,将目的基因转染干细胞可增强细胞治疗效果.但要实现目的基因的高效转移并适度表达,载体系统的选择是关键.目的:将携带绿色荧光蛋白的慢病毒载体感染人脂肪源性干细胞,检测病毒感染效率及感染后的细胞活力.方法:利用脂质体转染法获取慢病毒原液.设定不同感染复数(1,5,10,15,20),将带有增强绿色荧光蛋白的慢病毒载体感染人脂肪源性干细胞.荧光显微镜下观察病毒转染后人脂肪源性干细胞内增强绿色荧光蛋白的表达,流式细胞仪测定病毒感染效率.茜素红染色及油红O染色鉴定病毒转染后人脂肪源性干细胞的多向分化潜能.MTT法检测转染后人脂肪源性干细胞的增殖情况.结果与结论:随病毒感染时间及感染复数值的增加,人脂肪源性干细胞内绿色荧光表达明显增强,感染后第3天,感染复数为20时人脂肪源性干细胞内可见明显绿色荧光表达,病毒感染效率为60%:第5～7天,病毒感染效率可达90%～95%.转染后人脂肪源性干细胞经成骨及成脂诱导液培养后,茜素红染色及油红O染色阳性,表明病毒感染未影响人脂肪源性干细胞的多向分化能力.病毒感染后,人脂肪源性干细胞的增殖能力未受明显影响.说明携带增强绿色荧光蛋白的慢病毒载体可以有效感染人脂肪源性干细胞.     

人巨细胞病毒感染状态与免疫

人巨细胞病毒(humancytomegalovirus,HCMV)是疱疹病毒科β属病毒,基因为双股线性DNA,具有种属特异性[1]。发达国家人群HCMV的感染率为40%～60%,而发展中国家几乎达到100%,且多在婴幼儿时期发生[2]。由于与人类免疫系统共同进化,HCMV侵入人体后形成多种独立机制逃避免疫系统的追剿,宿主细胞不能清除这部分病毒,使得其在体内潜伏或呈低度增殖,并与宿主保持相对平衡状态,可长期或终身存在于宿主体内[13]。在机体免疫状态改变后,这种平衡状态常常被打破,病毒占优势,发生HCMV原发感染或潜伏—激活。由于该病毒感染的普遍性、致病的复杂性…     

Detection of human cytomegalovirus in medulloblastomas reveals a potential therapeutic target

Medulloblastomas are the most common malignant brain tumors in children. They express high levels of COX-2 and produce PGE2, which stimulates tumor cell proliferation. Human cytomegalovirus (HCMV) is prevalent in the human population and encodes proteins that provide immune evasion strategies and promote oncogenic transformation and oncomodulation. In particular, HCMV induces COX-2 expression; STAT3 phosphorylation; production of PGE2, vascular endothelial growth factor, and IL-6; and tumor formation in vivo. Here, we show that a large proportion of primary medulloblastomas and medulloblastoma cell lines are infected with HCMV and that COX-2 expression, along with PGE2 levels, in tumors is directly modulated by the virus. Our analysis indicated that both HCMV immediate-early proteins and late proteins are expressed in the majority of primary medulloblastomas. Remarkably, all of the human medulloblastoma cell lines that we analyzed contained HCMV DNA and RNA and expressed HCMV proteins at various levels in vitro. When engrafted into immunocompromised mice, human medulloblastoma cells induced expression of HCMV proteins. HCMV and COX-2 expression correlated in primary tumors, cell lines, and medulloblastoma xenografts. The antiviral drug valganciclovir and the specific COX-2 inhibitor celecoxib prevented HCMV replication in vitro and inhibited PGE2 production and reduced medulloblastoma tumor cell growth both in vitro and in vivo. Ganciclovir did not affect the growth of HCMV-negative tumor cell lines. These findings imply an important role for HCMV in medulloblastoma and suggest HCMV as a novel therapeutic target for this tumor.     

Lobucavir is phosphorylated in human cytomegalovirus-infected and -uninfected cells and inhibits the viral DNA polymerase.

Lobucavir (LBV) is a deoxyguanine nucleoside analog with broad-spectrum antiviral activity. LBV was previously shown to inhibit herpes simplex virus (HSV) DNA polymerase after phosphorylation by the HSV thymidine kinase. Here we determined the mechanism of action of LBV against human cytomegalovirus (HCMV). LBV inhibited HCMV DNA synthesis to a degree comparable to that of ganciclovir (GCV), a drug known to target the viral DNA polymerase. The expression of late proteins and RNA, dependent on viral DNA synthesis, was also inhibited by LBV. Immediate-early and early HCMV gene expression was unaffected, suggesting that LBV acts temporally coincident with HCMV DNA synthesis and not through cytotoxicity. In vitro, the triphosphate of LBV was a potent inhibitor of HCMV DNA polymerase with a Ki of 5 nM. LBV was phosphorylated to its triphosphate form intracellularly in both infected and uninfected cells, with phosphorylated metabolite levels two- to threefold higher in infected cells. GCV-resistant HCMV isolates, with deficient GCV phosphorylation due to mutations in the UL97 protein kinase, remained sensitive to LBV. Overall, these results suggest that LBV-triphosphate halts HCMV DNA replication by inhibiting the viral DNA polymerase and that LBV phosphorylation can occur in the absence of viral factors including the UL97 protein kinase. Furthermore, LBV may be effective in the treatment of GCV-resistant HCMV.     

Cytomegalovirus replication in primary and passaged human placental cells

Human cytomegalovirus (HCMV) was found to replicate in passaged fibroblastic human first trimester and term placental cells. The time-course of viral DNA replication as well as virus production in these human placental fibroblasts was similar to that in human embryo fibroblast cultures. In contrast, HCMV did not replicate in primary placental epithelioid cells. Continued passage (5 or more) of primary placental epithelioid cells was necessary to convert these cells to a state of permissiveness. The permissive cells were, however, fibroblasts. HCMV DNA replication in passaged placental fibroblastic cells was not affected by treatment with insulin or human chorionic gonadotropin. Furthermore, no replication of HCMV DNA occurred in choriocarcinoma cells, the epithelioid cells derived from cancer of the placenta. These results suggest that epithelial placental trophoblasts, either normal or transformed, were nonpermissive for HCMV. The permissiveness of HCMV infection to secondary placental cells which was observed might be due to the strong selection of fibroblastic cells in vitro.     

In vitro generation of human cytomegalovirus pp65 antigenemia, viremia, and leukoDNAemia.

Immunocompromised patients with disseminated human cytomegalovirus (HCMV) infection have circulating PMN carrying HCMV pp65 (antigenemia), infectious virus (viremia), and viral DNA (leukoDNAemia). Because HCMV does not fully replicate in PMN, it is generally hypothesized that virions and viral materials are taken up by phagocytosis from fully permissive HCMV-infected endothelial cells. However, no experimental evidence has ever been provided for these PMN-endothelium interactions. PMN from 11 donors were cocultured with endothelial cells infected with an endothelium-adapted HCMV strain and with human fibroblasts infected with low-passaged clinical and laboratory-adapted HCMV strains. pp65-positive PMN were detected after coculture with both HCMV-infected endothelial and fibroblast cells, provided that wild and not laboratory-adapted strains were used. In addition, cocultured PMN carried infectious virus as demonstrated by virus isolation and presence of complete virus particles by electron microscopy. Moreover, high levels of viral DNA were consistently detected by quantitative PCR in cocultured PMN. Thus, we have generated in vitro the three most important viral parameters detected in patients with disseminated HCMV infection (antigenemia, viremia, and leukoDNAemia). The failure of laboratory-adapted HCMV strain to induce this phenomenon demonstrates that important modifications have occurred in attenuated viral strains affecting basic biological functions.     

Broadly targeted human cytomegalovirus-specific CD4+ and CD8+ T cells dominate the memory compartments of exposed subjects

Human cytomegalovirus (HCMV) infections of immunocompetent hosts are characterized by a dynamic, life-long interaction in which host immune responses, particularly of T cells, restrain viral replication and prevent disease but do not eliminate the virus or preclude transmission. Because HCMV is among the largest and most complex of known viruses, the T cell resources committed to maintaining this balance have never been characterized completely. Here, using cytokine flow cytometry and 13,687 overlapping 15mer peptides comprising 213 HCMV open reading frames (ORFs), we found that 151 HCMV ORFs were immunogenic for CD4(+) and/or CD8(+) T cells, and that ORF immunogenicity was influenced only modestly by ORF expression kinetics and function. We further documented that total HCMV-specific T cell responses in seropositive subjects were enormous, comprising on average approximately 10% of both the CD4(+) and CD8(+) memory compartments in blood, whereas cross-reactive recognition of HCMV proteins in seronegative individuals was limited to CD8(+) T cells and was rare. These data provide the first glimpse of the total human T cell response to a complex infectious agent and will provide insight into the rules governing immunodominance and cross-reactivity in complex viral infections of humans.     

Circulating endothelial giant cells permissive for human cytomegalovirus (HCMV) are detected in disseminated HCMV infections with organ involvement.

Giant cells fully permissive for human cytomegalovirus (HCMV) were found to circulate, at a variable proportion, in peripheral blood of 21 out of 25 immunocompromised patients with disseminated HCMV infection. Circulating endothelial giant cells (EGC) were identified by a specific monoclonal antibody of endothelial origin and shown to express immediate-early, early, and late viral proteins. Immunostaining patterns of different viral proteins were comparable to those detected in vitro in cultured human umbilical vein endothelial cells. EGC counts > 10 were associated with high levels (> 100) of HCMV viremia and antigenemia, as well as with an overt clinical syndrome in transplanted patients, and to an untreated long lasting organ localization in AIDS patients. On the other hand, EGC counts were < 10 during disseminated HCMV infections of both transplant recipients with no apparent organ syndrome and AIDS patients with recent organ involvement. In tissue sections from AIDS patients, infected endothelial cells were found to progressively enlarge till detaching from the small vessel wall and entering blood stream. HCMV-infected EGC represent a new systemic parameter suitable for the diagnosis of HCMV organ involvement and for the study of the pathogenesis of disseminated infections.