疱疹病毒HCMV和HHV-6感染与神经胶质瘤的相关性研究
发布时间：2018-09-05 16:34
【摘要】：胶质瘤是起源于神经胶质细胞、发病率最高的颅内肿瘤,占恶性脑肿瘤的80%以上。WHO根据胶质瘤的病理类型将其分为四个等级,其中I级和II级为低级别胶质瘤,III级和IV级为高级别胶质瘤。其中IV级多形性胶质母细胞瘤(Glioblastoma multiforme,GBM)是最具侵袭性的中枢神经系统原发肿瘤,约占原发恶性脑肿瘤的46.1%。患者的5年存活率仅为5.1%。目前主要通过放化疗以及手术切除等方法进行治疗,但效果并不理想。因此,寻找新的治疗手段是当前神经科学领域亟待解决的重要问题。神经胶质瘤的发病原因尚不明了,多种因素包括遗传因素、电离辐射和职业化学暴露等在胶质瘤的发生中可能具有重要作用。此外,已有研究表明多种病毒如乙型肝炎病毒(HBV)、丙型肝炎病毒(HCV)、人乳头瘤病毒(HPV)、EB病毒(EBV)等与原发性肝癌、宫颈癌以及鼻咽癌的发生密切相关。近年来,陆续有在胶质瘤组织中检出的病毒包括有人巨细胞病毒(HCMV)、EBV、人疱疹病毒6型(HHV-6)、猴空泡病毒40(SV40)和猿猴肉瘤病毒(WMSV)等的报道,但大都为西方的研究资料。由于遗传背景、生存环境的不同,且疾病的病因谱也不尽相同,积累我国胶质瘤与病毒感染的研究资料非常必要。前期我们关注了疱疹病毒与胶质瘤发生的关系,79例胶质瘤脑组织HHV抗原的初步检测结果显示:胶质瘤组织中单纯疱疹病毒1、2的阳性率与对照脑组织无明显差别,EBV阳性率较低,未检出水痘-带状疱疹病毒和HHV-8,但HCMV和HHV-6的检出率明显高于对照组,故本研究主要关注我国汉族人群中HCMV和HHV-6在胶质瘤组织中的检出率及其与疾病发生的可能关联。一.HCMV在胶质瘤发生中的可能作用HCMV属疱疹病毒科β亚科,是人疱疹病毒家族中基因组最大的病毒。基因组为双链DNA,长约180~240kb。HCMV基因组表达呈现一定的时序性,分即刻早期(IE)、早期(E)和晚期(L)基因,共编码227种病毒蛋白,其中结构蛋白pp65在感染早期和晚期均有丰富表达,易于检测。HCMV在人群中的感染率高达60~90%,而且一旦感染终生携带。HCMV感染具有潜伏-再激活的特点,可引起多种临床病症。2002年,Cobbs等首次发现胶质瘤组织中HCMV IE1抗原的表达率高达99%,提出HCMV感染与胶质瘤发生密切。在随后的十几年间,不同研究小组在胶质瘤组织中陆续检出HCMV抗原和核酸,包括有pp65、g B、pp28、pp71、p52/76k D、US28、UL55、UL123等,进一步支持Cobbs的实验结果和结论。然而,另外有研究在胶质瘤组织中并未检测到HCMV抗原或核酸,因而HCMV感染与胶质瘤的关系存在争议。目前认为出现以上不同研究结果的原因可能包括有不同的组织标本制备方法、不同的检测指标和方法、不同的研究人群等。以往我们在79例胶质瘤组织中发现HCMV pp65蛋白和DNA的表达阳性率分别为65.8%(52/79)和54.4%(43/79),且HCMV的阳性率和表达水平与胶质瘤的病理级别成正比。因此,结合以往的报道,我们认为HCMV在胶质瘤发生发展过程中发挥重要作用,但HCMV以何种机制参与胶质瘤的进展有待进一步阐明。已知血管生成是肿瘤生长和转移的必要条件,HCMV可通过与一些关键信号通路的相互作用参与血管生成的调节,进而参与胶质瘤的恶性表型。STAT3是信号转导及转录激活蛋白(signal transducer and activator of transcription,STAT)家族的重要成员。STAT3在人体的正常组织及细胞中有少许表达,被磷酸化后激活形成二聚体,是胚胎发育、分化,特别是神经干细胞和星形胶质细胞发育的重要调节因子。在肿瘤组织及细胞中STAT3异常高表达,可参与肿瘤细胞的恶性侵袭和转移。但HCMV是否通过激活STAT3信号通路参与胶质瘤的发生与发展,尚待进一步研究。胶质瘤是高度血管化的肿瘤,丰富的新生血管是胶质瘤生长、侵袭和转移的重要病理学基础,这一过程受多种血管生成因子调控,如血管内皮生成因子(VEGF)、血管生成素(Angiopoietin,Ang)以及endocan。其中,endocan是一个分子量为50k Da的硫酸皮肤素蛋白聚糖,主要由血管内皮细胞分泌,生理条件下,endocan在血液中的水平极低,其功能与细胞的粘附、炎症反应有关。部分炎症因子和VEGF可以刺激内皮细胞分泌endocan。已有研究发现,endocan在肿瘤组织和肿瘤病人血液中表达增加。在裸鼠荷瘤模型中,endocan异位表达可促进肿瘤的增长。在前期工作中我们发现,胶质瘤组织endocan高表达,其表达水平与胶质瘤病理级别呈正相关。由此派生如下问题:在肿瘤组织中高表达的HCMV和endocan是否存在相互作用进而促进胶质瘤进展?其中的分子机制是什么?为此,本研究利用HCMV AD169株,通过感染体外培养的胶质瘤细胞U87,探究HCMV感染对STAT3以及endocan表达的影响以及三者之间可能的调控关系。在此基础上,通过免疫组化的方法,检测了79例脑胶质瘤标本(肿瘤组织)及8例正常脑组织标本中p STAT3的表达水平及其与胶质瘤病理级别的相关性。本研究主要实验结果和结论如下:(1)HCMV感染上调U87细胞endocan和p STAT3的表达:利用HCMV感染U87细胞,感染后第1、2和4天收集细胞样本,qPCR检测endocan m RNA表达水平,发现endocan m RNA分别上升25%、270%和400%,与对照相比具有显著差别(p0.05和p0.01)。与此相一致,endocan蛋白水平的表达在感染后第1天变化不显著,第2天达峰值,为对照的1.6倍(p0.01),第4天仍维持在较高水平,为对照的1.3倍(p0.05)。提示HCMV感染可促进endocan的表达。同时,利用Western blot检测HCMV感染U87细胞中p STAT3的表达变化,结果显示,感染第1天p STAT3表达水平即升高至对照的1.7倍,差别显著(p0.01),且明显早于endocan表达上调的时间;感染后第2、4天p STAT3表达水平仍维持在较高的水平,和对照相比分别上调1.5倍和1.3倍。提示HCMV感染可促进p STAT3的表达。(2)STAT3下调和更昔洛韦处理可阻断HCMV介导的STAT3磷酸化和endocan表达增加利用RNAi技术建立U87-STAT3下调细胞株,命名为U87-STAT3-down,并实施HCMV感染实验,结果显示感染后第2天,细胞中endocan和p STAT3蛋白水平分别降低至对照组的80%和43%(p0.05),提示STAT3下调可能抑制HCMV感染引起endocan的上调表达,换言之HCMV可能通过STAT3调控endocan的表达。在HCMV感染U87细胞2h后,加入抗病毒药物更昔洛韦(GCV),抑制HCMV复制。于感染后1、2、4天收集细胞样本,检测STAT3和endocan的m RNA以及蛋白的表达。与HCMV感染组相比,GCV处理组的STAT3以及endocan的m RNA和蛋白均表达下调,提示抑制HCMV复制同样抑制了HCMV感染引起的U87细胞中endocan和STAT3的上调,进一步提示HCMV通过STAT3调控endocan的表达。(3)p STAT3蛋白在胶质瘤脑组织中的表达:本实验利用免疫组织化学法,对79例胶质瘤石蜡标本中的p STAT3表达情况进行了检测。结果显示,在胶质瘤标本中p STAT3的表达水平明显增加,而在对照脑组织中仅有微量表达。结合胶质瘤组织中HCMVpp65蛋白高表达,在体实验结果进一步支持“HCMV通过STAT3调控endocan的表达,进而参与胶质瘤进展”的推论。总之,本部分研究通过体外和体内实验证明HCMV感染能够通过激活STAT3上调胶质瘤细胞endocan表达,为深入揭示HCMV参与胶质瘤发生机制提供了资料,同时为临床上治疗胶质瘤提供了新思路。抗病毒治疗和多靶点的抗血管生成或许能成为胶质瘤的重要的辅助治疗手段。二.HHV-6与胶质瘤的相关性研究HHV-6是疱疹病毒家族的成员,属于玫瑰疹病毒属,1986年首次分离于艾滋病(AIDS)晚期患者。HHV-6与HCMV同属于β疱疹病毒亚科,两病毒具有相似的生物学特性包括包膜糖蛋白以及核衣壳结构等。病毒基因的转录和翻译与HCMV相似,分为三个阶段:即刻早期基因、早期基因和晚期基因。成熟的病毒颗粒采用杀死宿主细胞的方式进行释放。HHV6对星形胶质细胞具有亲嗜性,感染早期会引发特异性的炎症反应,病毒通过调节多种前炎性因子和趋化因子的活性,逃避免疫系统的攻击。HHV-6与胶质瘤的关系的研究刚刚起步。近年来,陆续有研究小组报道在胶质瘤组织中检出HHV-6病毒抗原和核酸。例如,Crawford JR小组通过PCR和ISH的方法检测到88例小儿胶质瘤,发现HHV-6 U57基因的阳性表达。与HCMV相似,也有与上述相反的研究结论,例如Chan PK小组应用巢式PCR技术检测98例石蜡包埋胶质瘤组织中HHV-6的DNA,结果显示仅有8例HHV-6阳性。可见HHV-6与胶质瘤的关系尚需深入研究。为进一步明确HHV-6与胶质瘤的关系,本部分实验通过免疫组化的方法检测GBM中HHV-6的表达情况。主要研究结果如下:(1)病人一般情况:从首都医科大学附属三博脑科医院收集70例胶质母细胞瘤(GBM)标本和6例对照脑组织(取自脑外伤手术病人)。其中GBM患者男女比例接近1:1,平均年龄52.3岁(6-73岁)。对照脑组织取自脑外伤病人,无糖尿病、高血压等心脑血管疾病和感染性疾病。(2)HHV-6抗原在脑组织的分布:利用免疫细胞化学方法检测脑组织(石蜡切片)中HHV-6抗原的表达。结果显示,在GBM组织中HHV-6的阳性率为36%(25/70),在25例阳性组织中,呈弱阳性率为88%(22/25)、阳性率4%(1/25)、强阳性率8%(2/25),而对照组(外伤)脑组织中未检出HHV-6的表达。HHV-6在胞质和细胞核中均有表达。本研究仅仅检测了HHV-6在高级别胶质瘤组织中的表达,其阳性率远远高于对照组,提示HHV-6可能与胶质瘤的发生存在一定的关联。尚需扩大样本量、结合临床随访进行进一步研究HHV-6表达率以及阳性强度与临床指征、病人预后的相关性。病毒感染与胶质瘤的关系尚存争议。本研究首先通过实验证明HCMV感染通过激活STAT3上调胶质瘤细胞endocan表达,为深入阐明HCMV参与胶质瘤进展的机制提供新的线索,抗HCMV和endcoan治疗可能会成为胶质瘤治疗的重要辅助手段。再次,我们发现中国汉族胶质母细胞瘤患者中HHV-6抗原表达明显升高,HHV-6感染可能与胶质瘤的发生存在一定的关联,但具体机制有待于扩大样本量、结合临床进一步阐明。
[Abstract]:Glioblastoma is the most common intracranial tumor originating from glial cells, accounting for more than 80% of malignant brain tumors. According to the pathological types of gliomas, the WHO classifies them into four grades: grade I and II are low grade gliomas, and grade III and IV are high grade gliomas. GBM is the most aggressive primary tumor of the central nervous system, accounting for 46.1% of the primary malignant brain tumors. The 5-year survival rate of the patients is only 5.1%. At present, it is mainly treated by radiotherapy, chemotherapy and surgical resection, but the effect is not satisfactory. Therefore, it is an important problem to find new treatment methods in the field of neuroscience. The etiology of gliomas is unknown. Many factors, including genetic factors, ionizing radiation and occupational chemical exposure, may play an important role in the development of gliomas. Hepatocellular carcinoma, cervical carcinoma and nasopharyngeal carcinoma are closely related. In recent years, there have been reports of human cytomegalovirus (HCMV), EBV, human herpesvirus 6 (HHV-6), simian vacuole virus 40 (SV40) and simian sarcoma virus (WMSV) in glioma tissues, but most of them are western research data. It is necessary to accumulate the data on the relationship between herpes virus and the occurrence of gliomas in China. Preliminary results of HHV antigen detection in 79 gliomas showed that the positive rate of herpes simplex virus 1,2 in gliomas was higher than that in controls. There was no significant difference in brain tissue, EBV positive rate was low, no varicella-zoster virus and HHV-8 were detected, but the detection rate of HCMV and HHV-6 was significantly higher than that of the control group. Therefore, this study focused on the detection rate of HCMV and HHV-6 in glioma tissues and its possible correlation with the occurrence of glioma. 1. HCMV in the occurrence of glioma may be. HCMV is the largest genome in the human herpesvirus family, belonging to the beta subfamily of the Herpesviridae family. The genome is a double-stranded DNA, about 180-240 kb in length. The HCMV genome expresses in a certain time sequence. It can be divided into immediate early (IE), early (E) and late (L) genes, encoding 227 viral proteins, including the structural protein pp65 in the early and late stages of infection. HCMV infection rate in the population is as high as 60-90%, and once the infection carries. HCMV infection has a latent-reactivation characteristics, can cause a variety of clinical symptoms. In 2002, Cobbs et al. first found in glioma tissue HCMV IE1 antigen expression rate as high as 99%. HCMV infection and glioma closely related. Over the next decade, different research groups have detected HCMV antigens and nucleic acids in glioma tissues, including pp65, g B, pp28, pp71, p52/76kD, US28, UL55, UL123 and so on, further supporting Cobbs'experimental results and conclusions. At present, it is believed that the causes of the above different results may include different preparation methods of tissue specimens, different detection indicators and methods, and different research groups. In the past, we found that the positive rates of HCMV pp65 protein and DNA expression in 79 glioma tissues were 65.8% (52/79) and 54.4% (43/79) respectively. Therefore, combined with previous reports, we believe that HCMV plays an important role in the occurrence and development of glioma, but the mechanism of HCMV involved in the progression of glioma remains to be further clarified. Angiogenesis is known to be a necessary condition for tumor growth and metastasis, HCM. STAT3 is an important member of the signal transducer and activator of transcription (STAT) family. STAT3 is slightly expressed in normal tissues and cells and is phosphorus-bound. Activation after acidification to form dimers is an important regulatory factor in embryonic development and differentiation, especially in the development of neural stem cells and astrocytes. Abnormally high expression of STAT3 in tumor tissues and cells may participate in malignant invasion and metastasis of tumor cells. Gliomas are highly vascularized tumors. Abundant neovascularization is an important pathological basis for the growth, invasion and metastasis of gliomas. This process is regulated by a variety of angiogenic factors, such as vascular endothelial growth factor (VEGF), angiopoietin (Ang) and endocan. Among them, endocan is a molecular weight. 50 kDa dermatin sulfate proteoglycan is mainly secreted by vascular endothelial cells. Under physiological conditions, endocan is very low in the blood. Its function is related to cell adhesion and inflammatory reaction. Some inflammatory factors and VEGF can stimulate endocan secretion by endothelial cells. It has been found that endocan is in tumor tissue and blood of tumor patients. In a nude mice tumor-bearing model, ectopic expression of endocan promotes tumor growth. In our previous work, we found that the expression of endocan in glioma tissue was high, and the expression level was positively correlated with the pathological grade of glioma. In this study, we investigated the effect of HCMV infection on the expression of STAT3 and endocan and the possible relationship between them by infecting glioma cell U87 in vitro with HCMV AD169 strain. The main results and conclusions of this study are as follows: (1) HCMV infection up-regulates the expression of endocan and pSTAT3 in U87 cells: U87 cells were infected with HCMV, and cell samples were collected on the 1st, 2nd and 4th day after infection, and endocan was detected by qPCR. The expression level of endocan m RNA increased by 25%, 270% and 400% respectively, which was significantly different from that of the control (p0.05 and p0.01). Similarly, the expression of endocan protein did not change significantly on the first day after infection, reached a peak value on the second day, which was 1.6 times as high as that of the control (p0.01), and remained at a high level on the fourth day, 1.3 times as high as that of the control (p0.05). At the same time, Western blot was used to detect the expression of P STAT3 in U87 cells infected with HCMV. The results showed that the expression of P STAT3 in U87 cells infected with HCMV increased to 1.7 times of the control on the first day of infection, and the difference was significant (p0.01), and the expression of P STAT3 was significantly earlier than that of endocan. At a higher level, the expression of P STAT3 was up-regulated by 1.5 and 1.3 times, respectively, compared with the control group. (2) STAT3 down-regulation and ganciclovir treatment could block HCMV-mediated STAT3 phosphorylation and endocan expression. U87-STAT3 down-regulated cell line was established by RNAi technique, named U87-STAT3-down, and HCMV infection experiment was carried out. The results showed that the levels of endocan and P STAT3 decreased to 80% and 43% respectively (p0.05) on the 2nd day after infection, suggesting that STAT3 down-regulation may inhibit the up-regulation of endocan expression induced by HCMV infection. In other words, HCMV may regulate the expression of endocan through STAT3. HCMV replication was made. Cell samples were collected 1,2,4 days after infection to detect the expression of STAT3 and endocan m RNA and protein. Compared with HCMV infection group, the expression of STAT3 and endocan m RNA and protein in GCV treatment group were down-regulated, suggesting that inhibiting HCMV replication also inhibited the up-regulation of endocan and STAT3 in U87 cells induced by HCMV infection. The results showed that HCMV regulated the expression of endocan by STAT3. (3) The expression of P STAT3 protein in brain tissues of glioma: The expression of P STAT3 in paraffin-embedded samples of 79 glioma specimens was detected by immunohistochemistry. The results showed that the expression of P STAT3 in glioma specimens was significantly increased, but only in control brain tissues. Combining with the high expression of HCMVpp65 protein in glioma tissues, in vivo experimental results further support the hypothesis that HCMV regulates endocan expression through STAT3 and then participates in glioma progression. It provides information for further revealing the mechanism of HCMV involvement in gliomas and provides new ideas for clinical treatment of gliomas. Antiviral therapy and multi-target anti-angiogenesis may be important adjuvant therapy for gliomas. 2. Correlation between HHV-6 and gliomas. HHV-6 is a member of herpes virus family and belongs to Mei. Rosea virus, first isolated from advanced AIDS patients in 1986. HHV-6 and HCMV belong to the same subfamily of beta-herpesvirus. The two viruses have similar biological characteristics, including envelope glycoprotein and nucleocapsid structure. Because mature virus particles are released by killing host cells, HHV6 is a cytophilic agent to astrocytes and can induce specific inflammation in the early stage of infection. The virus escapes the attack of the epidemic system by regulating the activity of various pro-inflammatory and chemokines. In recent years, a number of research groups have reported detection of HHV-6 virus antigens and nucleic acids in gliomas. For example, the Crawford JR team detected 88 cases of childhood gliomas by PCR and ISH, and found positive expression of HHV-6 U57 gene. Similar to HCMV, there are also contrary research findings, such as the Chan PK team using nested PCR technology. In order to further clarify the relationship between HHV-6 and glioma, immunohistochemical method was used to detect the expression of HHV-6 in GBM. The main results were as follows: (1) Patients in general Situation: Seventy specimens of glioblastoma (GBM) and six control brain tissues were collected from Sanbo Brain Hospital Affiliated to Capital Medical University. The male-female ratio of GBM patients was close to 1:1, with an average age of 52.3 years (6-73 years). The control brain tissues were taken from brain trauma patients without diabetes, hypertension and other cardiovascular and cerebrovascular diseases and infections. Disease. (2) Distribution of HHV-6 antigen in brain tissues: Immunocytochemical method was used to detect the expression of HHV-6 antigen in brain tissues (paraffin section). The results showed that the positive rate of HHV-6 in GBM tissues was 36% (25/70). In 25 positive tissues, the weak positive rate was 88% (22/25), the positive rate was 4% (1/25), and the strong positive rate was 8% (2/25) compared with the control group (trauma). HHV-6 expression was not detected in brain tissues. HHV-6 was expressed in both cytoplasm and nucleus. The positive rate of HHV-6 expression in high grade glioma tissues was much higher than that in control group, suggesting that HHV-6 may be associated with the occurrence of glioma. The relationship between HCMV infection and glioma remains controversial. This study first demonstrated that HCMV infection up-regulates endocan expression in glioma cells by activating STAT3, providing new clues to further elucidate the mechanism of HCMV involvement in glioma progression and anti-HCMV and E. Ndcoan therapy may be an important adjuvant therapy for glioma. thirdly, we found HHV-6 in Chinese Han glioblastoma patients
【学位授予单位】：首都医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41
本文编号：2224831
[Abstract]:Glioblastoma is the most common intracranial tumor originating from glial cells, accounting for more than 80% of malignant brain tumors. According to the pathological types of gliomas, the WHO classifies them into four grades: grade I and II are low grade gliomas, and grade III and IV are high grade gliomas. GBM is the most aggressive primary tumor of the central nervous system, accounting for 46.1% of the primary malignant brain tumors. The 5-year survival rate of the patients is only 5.1%. At present, it is mainly treated by radiotherapy, chemotherapy and surgical resection, but the effect is not satisfactory. Therefore, it is an important problem to find new treatment methods in the field of neuroscience. The etiology of gliomas is unknown. Many factors, including genetic factors, ionizing radiation and occupational chemical exposure, may play an important role in the development of gliomas. Hepatocellular carcinoma, cervical carcinoma and nasopharyngeal carcinoma are closely related. In recent years, there have been reports of human cytomegalovirus (HCMV), EBV, human herpesvirus 6 (HHV-6), simian vacuole virus 40 (SV40) and simian sarcoma virus (WMSV) in glioma tissues, but most of them are western research data. It is necessary to accumulate the data on the relationship between herpes virus and the occurrence of gliomas in China. Preliminary results of HHV antigen detection in 79 gliomas showed that the positive rate of herpes simplex virus 1,2 in gliomas was higher than that in controls. There was no significant difference in brain tissue, EBV positive rate was low, no varicella-zoster virus and HHV-8 were detected, but the detection rate of HCMV and HHV-6 was significantly higher than that of the control group. Therefore, this study focused on the detection rate of HCMV and HHV-6 in glioma tissues and its possible correlation with the occurrence of glioma. 1. HCMV in the occurrence of glioma may be. HCMV is the largest genome in the human herpesvirus family, belonging to the beta subfamily of the Herpesviridae family. The genome is a double-stranded DNA, about 180-240 kb in length. The HCMV genome expresses in a certain time sequence. It can be divided into immediate early (IE), early (E) and late (L) genes, encoding 227 viral proteins, including the structural protein pp65 in the early and late stages of infection. HCMV infection rate in the population is as high as 60-90%, and once the infection carries. HCMV infection has a latent-reactivation characteristics, can cause a variety of clinical symptoms. In 2002, Cobbs et al. first found in glioma tissue HCMV IE1 antigen expression rate as high as 99%. HCMV infection and glioma closely related. Over the next decade, different research groups have detected HCMV antigens and nucleic acids in glioma tissues, including pp65, g B, pp28, pp71, p52/76kD, US28, UL55, UL123 and so on, further supporting Cobbs'experimental results and conclusions. At present, it is believed that the causes of the above different results may include different preparation methods of tissue specimens, different detection indicators and methods, and different research groups. In the past, we found that the positive rates of HCMV pp65 protein and DNA expression in 79 glioma tissues were 65.8% (52/79) and 54.4% (43/79) respectively. Therefore, combined with previous reports, we believe that HCMV plays an important role in the occurrence and development of glioma, but the mechanism of HCMV involved in the progression of glioma remains to be further clarified. Angiogenesis is known to be a necessary condition for tumor growth and metastasis, HCM. STAT3 is an important member of the signal transducer and activator of transcription (STAT) family. STAT3 is slightly expressed in normal tissues and cells and is phosphorus-bound. Activation after acidification to form dimers is an important regulatory factor in embryonic development and differentiation, especially in the development of neural stem cells and astrocytes. Abnormally high expression of STAT3 in tumor tissues and cells may participate in malignant invasion and metastasis of tumor cells. Gliomas are highly vascularized tumors. Abundant neovascularization is an important pathological basis for the growth, invasion and metastasis of gliomas. This process is regulated by a variety of angiogenic factors, such as vascular endothelial growth factor (VEGF), angiopoietin (Ang) and endocan. Among them, endocan is a molecular weight. 50 kDa dermatin sulfate proteoglycan is mainly secreted by vascular endothelial cells. Under physiological conditions, endocan is very low in the blood. Its function is related to cell adhesion and inflammatory reaction. Some inflammatory factors and VEGF can stimulate endocan secretion by endothelial cells. It has been found that endocan is in tumor tissue and blood of tumor patients. In a nude mice tumor-bearing model, ectopic expression of endocan promotes tumor growth. In our previous work, we found that the expression of endocan in glioma tissue was high, and the expression level was positively correlated with the pathological grade of glioma. In this study, we investigated the effect of HCMV infection on the expression of STAT3 and endocan and the possible relationship between them by infecting glioma cell U87 in vitro with HCMV AD169 strain. The main results and conclusions of this study are as follows: (1) HCMV infection up-regulates the expression of endocan and pSTAT3 in U87 cells: U87 cells were infected with HCMV, and cell samples were collected on the 1st, 2nd and 4th day after infection, and endocan was detected by qPCR. The expression level of endocan m RNA increased by 25%, 270% and 400% respectively, which was significantly different from that of the control (p0.05 and p0.01). Similarly, the expression of endocan protein did not change significantly on the first day after infection, reached a peak value on the second day, which was 1.6 times as high as that of the control (p0.01), and remained at a high level on the fourth day, 1.3 times as high as that of the control (p0.05). At the same time, Western blot was used to detect the expression of P STAT3 in U87 cells infected with HCMV. The results showed that the expression of P STAT3 in U87 cells infected with HCMV increased to 1.7 times of the control on the first day of infection, and the difference was significant (p0.01), and the expression of P STAT3 was significantly earlier than that of endocan. At a higher level, the expression of P STAT3 was up-regulated by 1.5 and 1.3 times, respectively, compared with the control group. (2) STAT3 down-regulation and ganciclovir treatment could block HCMV-mediated STAT3 phosphorylation and endocan expression. U87-STAT3 down-regulated cell line was established by RNAi technique, named U87-STAT3-down, and HCMV infection experiment was carried out. The results showed that the levels of endocan and P STAT3 decreased to 80% and 43% respectively (p0.05) on the 2nd day after infection, suggesting that STAT3 down-regulation may inhibit the up-regulation of endocan expression induced by HCMV infection. In other words, HCMV may regulate the expression of endocan through STAT3. HCMV replication was made. Cell samples were collected 1,2,4 days after infection to detect the expression of STAT3 and endocan m RNA and protein. Compared with HCMV infection group, the expression of STAT3 and endocan m RNA and protein in GCV treatment group were down-regulated, suggesting that inhibiting HCMV replication also inhibited the up-regulation of endocan and STAT3 in U87 cells induced by HCMV infection. The results showed that HCMV regulated the expression of endocan by STAT3. (3) The expression of P STAT3 protein in brain tissues of glioma: The expression of P STAT3 in paraffin-embedded samples of 79 glioma specimens was detected by immunohistochemistry. The results showed that the expression of P STAT3 in glioma specimens was significantly increased, but only in control brain tissues. Combining with the high expression of HCMVpp65 protein in glioma tissues, in vivo experimental results further support the hypothesis that HCMV regulates endocan expression through STAT3 and then participates in glioma progression. It provides information for further revealing the mechanism of HCMV involvement in gliomas and provides new ideas for clinical treatment of gliomas. Antiviral therapy and multi-target anti-angiogenesis may be important adjuvant therapy for gliomas. 2. Correlation between HHV-6 and gliomas. HHV-6 is a member of herpes virus family and belongs to Mei. Rosea virus, first isolated from advanced AIDS patients in 1986. HHV-6 and HCMV belong to the same subfamily of beta-herpesvirus. The two viruses have similar biological characteristics, including envelope glycoprotein and nucleocapsid structure. Because mature virus particles are released by killing host cells, HHV6 is a cytophilic agent to astrocytes and can induce specific inflammation in the early stage of infection. The virus escapes the attack of the epidemic system by regulating the activity of various pro-inflammatory and chemokines. In recent years, a number of research groups have reported detection of HHV-6 virus antigens and nucleic acids in gliomas. For example, the Crawford JR team detected 88 cases of childhood gliomas by PCR and ISH, and found positive expression of HHV-6 U57 gene. Similar to HCMV, there are also contrary research findings, such as the Chan PK team using nested PCR technology. In order to further clarify the relationship between HHV-6 and glioma, immunohistochemical method was used to detect the expression of HHV-6 in GBM. The main results were as follows: (1) Patients in general Situation: Seventy specimens of glioblastoma (GBM) and six control brain tissues were collected from Sanbo Brain Hospital Affiliated to Capital Medical University. The male-female ratio of GBM patients was close to 1:1, with an average age of 52.3 years (6-73 years). The control brain tissues were taken from brain trauma patients without diabetes, hypertension and other cardiovascular and cerebrovascular diseases and infections. Disease. (2) Distribution of HHV-6 antigen in brain tissues: Immunocytochemical method was used to detect the expression of HHV-6 antigen in brain tissues (paraffin section). The results showed that the positive rate of HHV-6 in GBM tissues was 36% (25/70). In 25 positive tissues, the weak positive rate was 88% (22/25), the positive rate was 4% (1/25), and the strong positive rate was 8% (2/25) compared with the control group (trauma). HHV-6 expression was not detected in brain tissues. HHV-6 was expressed in both cytoplasm and nucleus. The positive rate of HHV-6 expression in high grade glioma tissues was much higher than that in control group, suggesting that HHV-6 may be associated with the occurrence of glioma. The relationship between HCMV infection and glioma remains controversial. This study first demonstrated that HCMV infection up-regulates endocan expression in glioma cells by activating STAT3, providing new clues to further elucidate the mechanism of HCMV involvement in glioma progression and anti-HCMV and E. Ndcoan therapy may be an important adjuvant therapy for glioma. thirdly, we found HHV-6 in Chinese Han glioblastoma patients
【学位授予单位】：首都医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41
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