建立HHV-6体外感染U251细胞模型并对其生物学功能及机制的研究

发布时间：2018-05-22 14:53

本文选题：人类疱疹病毒6型 + 神经胶质瘤　；参考：《南京医科大学》2010年硕士论文

【摘要】： 人类疱疹病毒6型(Human herpesvirus 6,HHV-6)是一类嗜人淋巴细胞的双链DNA病毒,于1986年由美国癌症中心的Salahuddin等首先从淋巴增生性疾病患者外周血单个核细胞中分离到,对其进行抗原性和酶切图谱分析表明,它有别于其它疱疹病毒,是一种具有疱疹病毒科形态特征和嗜淋巴细胞特性的新型疱疹病毒,故命名为人类疱疹病毒6型,属于疱疹病毒β亚科。HHV-6可分为A和B两个亚型。HHV-6感染具有广泛的嗜细胞性,主要侵犯CD4+T细胞、单核巨噬细胞及神经胶质细胞等。1988年日本学者Yamanishi确认了HHV-6是婴幼儿急疹(ES)的病原体,后来有报道神经胶质瘤、脑炎、慢性疲劳综合症、器官移植后感染、多发性硬化症等多种疾病有关,但对其致病机制尚不清楚。神经胶质瘤(Glioma)是人类最常见的原发性脑肿瘤,到目前为止对其病因不清楚。近年来病毒在神经胶质瘤发生发展中的作用引起了多学科的广泛关注。有多篇报道在神经胶质瘤患者瘤组织中能检测到HHV-6 DNA的存在,但未能拿到病毒。人神经胶质瘤U251细胞是一种星形胶质瘤细胞株,具有星形胶质细胞部分特性,因此若能够建立HHV-6体外感染U251细胞模型,对研究HHV-6与神经胶质瘤的关系有指导意义。本论文分三个部分,(一)HHV-6GS株在CBMCs中的培养扩增及病毒效价的滴定。通过PCR检测到HHV-6的DNA,间接免疫荧光检测到HHV-6抗原。病毒的滴度最终确定约为5×103TCID50/ml。(二)HHV-6感染神经胶质瘤U251细胞建立体外感染模型。结果发现HHV-6能感染U251细胞引起细胞病变,促进细胞增殖,使细胞周期发生改变及细胞因子IL-6分泌增加;成功建立了体外感染的模型。(三)对有丝分裂原活化蛋白激酶(mitogen-activated protein kinase , MAPK)信号通路的分支JNK和P38通路进行研究。结果发现在HHV-6的作用下磷酸化JNK和P38蛋白表达增加,病毒感染可能通过活化JNK和P38信号通路,引起肿瘤细胞的分化。在成功建立HHV-6感染U251细胞体外模型的基础上,对HHV-6感染神经胶质瘤U251细胞的功能及机制作了初步研究,结果说明HHV-6与神经胶质瘤的发生发展存在一定的联系,为进一步研究神经胶质瘤的病毒病因和发病机理提供理论和实验依据。
[Abstract]:Human herpesvirus 6 (HHV-6) is a human lymphocytic double-stranded DNA virus. It was first isolated from peripheral blood mononuclear cells (PBMC) of patients with lymphoproliferative diseases by Salahuddin et al of the American Cancer Center in 1986. The results of antigenicity and restriction endonuclease analysis showed that it was a new type of herpesvirus, which was different from other herpesviruses and had morphologic and lymphophilic characteristics of herpesviridae, so it was named human herpesvirus type 6. The infection of herpesvirus 尾 subfamily. HHV-6 can be divided into A and B subtypes. HHV-6 infection has a wide range of cytophilia, mainly invading CD4 T cells. In 1988, the Japanese scholar Yamanishi confirmed that HHV-6 was the pathogen of infantile rash ES.After that, glioma, encephalitis, chronic fatigue syndrome, infection after organ transplantation were reported. Multiple sclerosis and other diseases are related, but its pathogenesis is not clear. Glioma (Glioma) is the most common primary brain tumor in humans. In recent years, the role of virus in the development of glioma has attracted wide attention. The presence of HHV-6 DNA was detected in glioma tissues, but the virus was not obtained. Human glioma U251 cell line is a kind of astroglioma cell line, which has some astrocytic properties. Therefore, if HHV-6 can be established to infect U251 cells in vitro, it will be helpful to study the relationship between HHV-6 and glioma. This paper is divided into three parts: culture and amplification of HHV-6GS strain in CBMCs and titration of virus titer. HHV-6 was detected by PCR and HHV-6 antigen was detected by indirect immunofluorescence. The titer of the virus was determined to be about 5 脳 103 TCID 50 / ml. (HHV-6 infected glioma U251 cells) in vitro infection model was established. The results showed that HHV-6 could induce cytopathic changes, promote cell proliferation, change cell cycle and increase the secretion of cytokine IL-6 in U251 cells, and the model of infection in vitro was successfully established. (3) the branching JNK and P38 pathways of mitogen-activated protein kinase, MAPK) signaling pathway were studied. The results showed that the expression of phosphorylated JNK and P38 protein increased under the action of HHV-6. Virus infection may induce the differentiation of tumor cells by activating JNK and P38 signaling pathway. On the basis of successfully establishing HHV-6 infected U251 cell model in vitro, the function and mechanism of HHV-6 infected glioma U251 cells were studied. The results showed that there was a certain relationship between HHV-6 and the occurrence and development of glioma. To provide theoretical and experimental basis for further study of viral etiology and pathogenesis of glioma.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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