HCMV感染与滋养细胞HLA-E蛋白表达的相关性

目的：研究HCMV感染与滋养细胞HLA-E蛋白表达的相关性。方法：体外分离、培养的滋养细胞系HTP-8,用HCMV AD169标准病毒株感染细胞。免疫荧光法检测HCMV感染后滋养细胞中HLA-E蛋白的表达。结果：病毒感染后10天内,细胞胞浆和胞膜均有HLA-E蛋白表达,感染后1-5天表达水平较低,6-10天表达强度显著增强。结论：滋养细胞HLA-E蛋白的表达与HCMV感染有相关性。     

人巨细胞病毒与胶质瘤相关性分子机制研究进展

人巨细胞病毒(human cytomegalovirus,HCMV)属于疱疹病毒β亚科,在人群中存在普遍感染.近年研究发现在人类胶质瘤等多种类型肿瘤组织中存在HCMV蛋白表达及相关基因组序列.随着对HCMV研究的深入,人们进一步探讨了HCMV及其相关蛋白在人胶质瘤中可能的致瘤分子机制.HCMV感染胶质瘤及其基因产物对PI3/AKT、SOX2、p-STAT3、NF-κB等多个信号通路及相关分子发挥重要作用,促进胶质瘤细胞的增殖、存活、侵袭、血管生成并抑制细胞凋亡.本文就HCMV与胶质瘤相关性分子机制研究进展进行综述.     

HCMV pp65蛋白在脑胶质瘤组织中的表达及临床意义

目的:探讨人巨细胞病毒（HCMV）pp65蛋白在脑胶质瘤组织中的表达及其与肿瘤级别和预后的相关性分析。方法:采用免疫组化方法检测HCMV pp65蛋白在不同级别脑胶质瘤组织中的表达情况,并通过统计学方法分析蛋白表达情况和患者预后的关系。结果:在89例脑胶质瘤组织中,60例（67.4%）在细胞核和（或）细胞质中出现HCMV pp65蛋白阳性表达,其中18例（56.3%）为Ⅱ级星形细胞瘤、20例（64.5%）为Ⅲ级星形细胞瘤以及22例（84.6%）为胶质母细胞瘤。在10例对照脑组织标本中没有发现HCMV pp65蛋白阳性表达。我们同时发现在不同级别脑胶质瘤组织中HCMV pp65蛋白表达差异性没有统计学意义,HCMV pp65蛋白表达水平与胶质瘤患者的无进展生存期（PFS）无相关性。结论:HCMV pp65蛋白在脑胶质瘤组织中高表达,为理解胶质瘤的形成和进展机制提供理论依据。     

基质金属蛋白酶-2在妊娠滋养细胞疾病中的表达及意义

目的:探讨基质金属蛋白酶-2(m atrix m etalloprote inase,MMP-2)在妊娠滋养细胞疾病中的表达。方法:应用免疫组化SP法检测正常早孕妇女胎盘绒毛10例、葡萄胎20例、侵蚀性葡萄胎32例及绒毛膜癌16例组织中MMP-2的表达情况。结果:MMP-2主要表达于合体滋养细胞、绒毛外滋养细胞。正常早孕绒毛组织、妊娠滋养细胞疾病组织中,MMP-2均有阳性表达,早孕绒毛与早孕期葡萄胎间有显著差异(P<0.05),与葡萄胎病理分型、胎次无相关性,早孕期葡萄胎MMP-2阳性表达率显著高于晚孕期葡萄胎(P<0.05)。正常绒毛组MMP-2阳性表达率与葡萄胎组、侵蚀性葡萄胎组和绒毛膜癌组相比有显著性差异(P<0.05)且有逐渐上升趋势,但其余各组间无统计学意义。MMP-2表达与侵蚀性葡萄胎、绒毛膜癌临床分期无相关性。侵蚀性葡萄胎组中未化疗者MMP-2阳性表达率显著高于化疗者(P<0.05);绒毛膜癌组中未化疗组MMP-2阳性表达率高于化疗组,但无统计学意义。结论:MMP-2与滋养细胞的浸润活性呈正相关,与孕周呈负相关,可能与正常滋养细胞浸润行为的时空阶段性和限制性有关。随着妊娠滋养细胞疾病恶性程度升高MMP-2逐渐呈强表达,化疗后其阳性表达明显下降,提示MMP-2可能作为临床早期诊断妊娠滋养细胞疾病、判断治疗疗效及预后的重要指标之一。     

HLA-E基因表达沉默对人乳腺癌细胞生物学行为的影响

目的：探讨HLA-E基因表达对人乳腺癌细胞增殖、凋亡、侵袭、迁移等生物学行为的影响,分析靶向HLA-E基因的小分子RNA干扰(siRNA)技术应用于乳腺癌治疗的可行性。方法：设计并合成靶向HLA-E基因的特异性siRNA序列,转染至人乳腺癌细胞MDA-MB-231,同时设立空白对照组、阴性对照组及脂质体组。实时荧光定量PCR(qPCR)和Western blot法分别检测各组乳腺癌细胞HLA-E mRNA及蛋白的表达;CCK-8法和流式细胞术检测各组乳腺癌细胞增殖率和凋亡率;Transwell实验检测各组乳腺癌细胞侵袭和迁移能力。结果：乳腺癌细胞转染HLA-E siRNA后,HLA-E mRNA及蛋白表达水平明显降低。与阴性对照组相比,HLA-E siRNA转染组乳腺癌细胞增殖率降低(P<0.01);凋亡率增加(P<0.01);侵袭迁移能力降低(P<0.01)。结论：靶向人乳腺癌细胞HLA-E基因的siRNA能有效抑制其基因表达,进而抑制细胞增殖,诱导细胞凋亡,降低乳腺癌细胞侵袭和迁移能力。     

MMP-9/TIMP-1在葡萄胎中表达及与临床病理参数相关性

[目的]研究基质金属蛋白酶（MMP-9）及其组织抑制剂（TIMP-1）在葡萄胎滋养细胞中的表达及其与临床病理参数的相关性.[方法]应用链霉菌抗生物素蛋白-过氧化物酶免疫组织化学方法检测15例部分性葡萄胎、55例完全性葡萄胎滋养细胞中MMP-9、TIMP-1表达.[结果]葡萄胎患者MMP-9的表达与年龄和清宫次数有相关性（分别为rs=0.262,P=0.028;rs=0.390,P=0.001）.TIMP-1的表达与卵巢黄素化囊肿、清宫前血β-HCG值、血β-HCG转阴时间、妊娠呕吐、合并妊娠高血压综合征及清宫次数有相关性.[结论]MMP-9与葡萄胎患者的年龄和清宫次数有关,HCG可能影响葡萄胎TIMP-1的表达,MMPs/TIMPs比例的改变也可能是滋养细胞疾病发生妊娠高血压综合征的原因之一.     

Hp感染与胃癌组织中Bcl-2、Bax表达的相关性研究

目的 揭示Bcl-2,Bax在胃癌组织的表达与Hp感染的相关性.方法 采用快速尿素酶试验及组织学改良Giemsa染色法联合检测130例胃癌组织与70例慢性胃炎组织中Hp感染情况,免疫组织化学PV9000法,检测130例胃癌组织与70例慢性胃炎组织中Bcl-2,Bax蛋白的阳性表达情况.结果 Hp(+)组Bcl-2蛋白的阳性表达率明显高于Hp(-)组(P<0.01);胃癌组织中Hp感染与Bcl-2蛋白表达之间存在正相关关系(P<0.01,r=0.288);Hp(+)组Bax蛋白的阳性表达率明显低于Hp(-)组(P<0.01);胃癌组织中Hp感染与Bax蛋白表达之间存在负相关关系(P<0.01,r=-0.536).结论 Hp感染与Bcl-2蛋白表达存在正相关性,与Bax蛋白表达存在负相关性,提示Hp感染与细胞凋亡,二者可能共同参与胃癌的发生发展过程.     

PUMA基因对胰腺癌细胞BxPC-3的促凋亡作用及其可能机制

目的:研究P53正向凋亡调节因子基因(P53 up-regulate modulator of apoptosis,PUMA)对胰腺癌细胞株BxPC-3凋亡的影响及其可能的作用机制.方法:以100 MOI的携PUMA基因重组腺病毒(Ad-PUMA)感染BxPC-3细胞0～96 h,流式细胞术检测BxPC-3细胞凋亡率,Western blotting检测BxPC-3细胞中PUMA、Bcl-2、Bax、Cytochrome C和Caspase-3蛋白的表达,Western blotting检测BxPC-3细胞中细胞质和线粒体内Bax的表达及Bax寡聚体.结果:随着Ad-PUMA感染时间的延长,BxPC-3细胞凋亡率逐渐增加,48 h时最高.Ad-PUMA感染促进BxPC-3细胞中PUMA、Cytochrome C和Caspase-3蛋白的表达,抑制BxPC-3细胞中Bcl-2蛋白的表达.Ad-PUMA感染后BxPC-3细胞的凋亡率与BxPC-3细胞中PUMA蛋白的表达具有明显的相关性.AdPUMA感染不影响BxPC-3细胞中Bax蛋白的总表达量,但细胞质中的Bax几乎完全消失,而线粒体中的Bax表达明显增加;AdPUMA感染诱导BxPC-3细胞中Bax蛋白的寡聚化.结论:PUMA基因通过线粒体途径促进胰腺癌细胞凋亡.     

抗人巨细胞病毒免疫球蛋白G在脑胶质瘤的表达及与预后的相关性

目的:探讨抗人巨细胞病毒免疫球蛋白G（HCMV IgG）水平在脑胶质瘤的表达及与预后的相关性。方法:选择2013年1月至2017年8月在我院就诊并接受手术切除的脑胶质瘤患者作为研究对象，入组后测定患者抗HCMV IgG水平，观察抗HCMV IgG表达及水平与脑胶质瘤患者特征与预后的相关性。结果:患者抗HCMV IgG阳性表达率为76.17％。抗HCMV IgG与年龄、性别、肿瘤部位、肿瘤大小、病程、KPS评分等均无相关性（P＞0.05），与肿瘤分级存在正相关（P＜0.05）。肿瘤分级、手术方式、替莫唑胺疗程和抗HCMV IgG表达是影响患者1年死亡率的独立预后因素（P均＜0.05）。对IgG阳性患者进一步分析显示，抗HCMV IgG＜10 U/ml患者生存期低于10～29 U/ml和≥30 U/ml患者（P＜0.05），抗HCMV IgG≥30 U/ml患者生存期高于10～29 U/ml患者但差异无统计学意义（P＞0.05）。结论:抗HCMV IgG表达与胶质瘤恶性程度呈正相关，与生存期呈负相关，抗HCMV IgG低表达患者具有更差的预后，应引起临床关注。     

非经典HLA I类分子（HLA—G和HLA—E）在人肿瘤细胞系的表达及IFN—γ的调节作用

目的：探讨非经典HLA I类分子在人肿瘤细胞系的表达及IFN-γ的调节作用。方法：用RT-PCR法检测12种肿瘤细胞系和人脑胶质瘤组织及妊娠妇女滋养层组织标本HLA-G和HLA-E mRNA的表达。结果：人脑胶质瘤组织及妊娠妇女滋养层组织均有HLA-G和HLA-E mRNA的表达；12株肿瘤细胞中仅人T细胞淋巴瘤Karpas存在HLA-G3的mRNA表达，经IFN-γ处理后，Karpas出现HLA-G1/G5和HLA-G2/G4的表达，宫颈癌Hela细胞、黑色素瘤M21细胞和膀胱癌T24细胞出现HLA-G3 mRNA的表达；12株肿瘤细胞中有9株表达HLA-E mRNA。结论：肿瘤存在HLA-G和HLA-E mRNA的表达，IFN-γ可促进HLA-G mRNA的表达。肿瘤细胞HLA-G和HLA-E的表达可能是肿瘤逃避免疫系统监视的机制之一。     

Human cytomegalovirus induces cellular tyrosine kinase signaling and promotes glioma cell invasiveness

Given our previous findings that human cytomegalovirus (HCMV) nucleic acids and proteins are expressed in human malignant glioma in vivo, we investigated cellular signaling events associated with HCMV infection of human glioma and astroglial cells. HCMV infection caused rapid activation of the phosphatidylinositol-3 kinase (PI-3K) effector AKT kinase in human astro-glial and fibroblast cells, and induced tyrosine phosphorylation of phospholipase Cγ (PLCγ). Co-immunoprecipitation experiments revealed association of the p85 regulatory subunit of PI-3K with a high-molecular weight protein phosphorylated on tyrosine, following short-term exposure to HCMV. In contrast to a previous report, we were unable to confirm the identity of this high-molecular weight protein as being the epidermal growth factor receptor (EGFR). Stimulation of glioma and fibroblast cell lines over-expressing EGFR with HCMV (whole virus) or soluble glycoprotein B did not induce tyrosine phosphorylation of the receptor, as did the genuine ligand, EGF. Furthermore, we found that expression levels of the human ErbB1-4 receptors were not rate-limiting for HCMV infection. Dispensability of EGFR function during early HCMV infection was substantiated by demonstration of viral immediate early gene expression in cells lacking the EGFR gene, indicating that HCMV may promote oncogenic signaling pathways independently of EGFR activation. Among non-receptor cellular kinases, HCMV infection induced phosphorylation of focal adhesion kinase (FAK) Tyr397, which is indispensable for integrin-mediated cell migration and invasion. HCMV-induced FAK activation was paralleled by increased extracellular matrix-dependent migration of human malignant glioma but not normal astro-glial cells, suggesting that HCMV can selectively augment glioma cell invasiveness. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Modulation of oncogenic phenotype in human glioma cells by cytomegalovirus IE1-mediated mitogenicity

Recent evidence indicates that human cytomegalovirus (HCMV) infection occurs in a high percentage of human malignant gliomas in vivo, as the HCMV immediate early-1 (IE1) protein is detected in >90% of these tumors. The HCMV IE1 protein is essential for viral infection and has potent trans-activating and oncomodulatory properties. To investigate a potential role of HCMV in glioma biology, we stably expressed the HCMV IE1 gene product in immortalized and malignant human glial cells. Here we show that stable IE1 expression can differentially affect the growth of human glioblastoma cells, resulting in either growth proliferation or arrest. IE1 expression led to dysregulation of phosphatidylinositol 3-kinase/AKT activity, Rb phosphorylation, and expression of the p53 family of proteins. In U87 and U118 glioblastoma cells, IE1 induced cellular proliferation paralleled by reduction in steady-state expression level of Rb and p53 family proteins (including p53, p63, or p73) and simultaneous induction of the phosphatidylinositol 3-kinase/AKT signaling pathway. In contrast, IE1 expression in LN229 and U251 glioblastoma cells and immortalized human astrocytes was associated with increased expression of p53 family proteins, accompanied by growth arrest or lack of enhanced proliferation. Moreover, IE1 promoted cell cycle entry and DNA synthesis of human glioma cells on both stable expression in tumor-derived cell lines as well as transient expression in primary glioblastoma cells. These findings indicate that HCMV IE1 can significantly affect important oncogenic signaling pathways in glioblastoma cells.     

Effect of HCMV IE1 Protein on Cytokines Secretion and Apoptosis of Macrophages

Objective： To investigate the effect of human cytomegalovirus （HCMV） IE1 protein on the secretory activity and apoptosis of macrophages. Methods： The eukaryotic expression vector pEGFP-C1/IE1 was used to transfect THP-1-macrophages. 48 h after transfection, the expression and localization of GFP or GFP-IE1 was observed under fluorescent microscope. The levels of IL-1β and TNF-α in the culture media were examined by ELISA, and the mRNA expression of them was analyzed by RT-PCR. Cell undergoing apoptosis were determined by flow cytometry using the propidium iodide （PI） staining method. The data were analyzed by SPSSI3.0. Results： As observed under fluorescent microscope, the expressions of GFP-IEI and GFP by plasmid pEGFP-C1/IE1 or pEGFP-C1 in THP-1-macrophages could be found in nuclei or whole cells. Conclusion： As demonstrated by RT-PCR and ELISA, mRNA and protein expressions of IL-1β and TNF-α and promotes apoptosis in THP-1-macrophages.     

Absence of human cytomegalovirus infection in childhood brain tumors

Human cytomegalovirus (HCMV) is a common human pathogen which induces different clinical manifestations related to the age and the immune conditions of the host. HCMV infection seems to be involved in the pathogenesis of adult glioblastomas. The aim of our study was to detect the presence of HCMV in high grade gliomas and other pediatric brain tumors. This hypothesis might have important therapeutic implications, offering a new target for adjuvant therapies. Among 106 pediatric patients affected by CNS tumors we selected 27 patients with a positive HCMV serology. The serological analysis revealed 7 patients with positive HCMV IGG (≥14 U/mL), whom had also a high HCMV IgG avidity, suggesting a more than 6 months-dated infection. Furthermore, HCMV IGM were positive (≥22 U/mL) in 20 patients. Molecular and immunohistochemical analyses were performed in all the 27 samples. Despite a positive HCMV serology, confirmed by ELISA, no viral DNA was shown at the PCR analysis in the patients’ neoplastic cells. At immunohistochemistry, no expression of HCMV antigens was observed in tumoral cells. Our results are in agreement with recent results in adults which did not evidence the presence of HCMV genome in glioblastoma lesions. We did not find any correlation between HCMV infection and pediatric CNS tumors.