Toll样受体3介导病毒促进垂体瘤增殖侵袭的机制研究

发布时间：2018-05-11 19:02

本文选题：垂体瘤 + 侵袭性　；参考：《第三军医大学》2016年博士论文

【摘要】：研究背景和目的:垂体瘤占颅内肿瘤约15%[1],其组织学为良性,但是有25-55%的垂体瘤朝周围结构浸润,比如鞍隔的骨性组织,筛窦和/或海绵窦等,这些垂体瘤称为侵袭性垂体瘤[2][3][4]。垂体瘤的侵袭性常与高Ki-67指数,并与严重的症状、更差的预后、高复发率、以及药物替代治疗的低反应性相关[5]。现有的研究已表明,染色体、基因(如:11p/q, p53),粘附分子(如:E-cadherin),金属基质蛋白(如:MMP2, MMP8),炎症因子(如:IL-6, IL-17)的异常变化与垂体瘤的进展、侵袭相关[5][6][7][8][9][10][11]。但评估、解释其侵袭性的特异性的组织学和分子生物学标准却依然缺乏。不仅如此,现有的研究多从垂体瘤自身基因、转录、转录后修饰等几个层面上的异常变化探索其侵袭性的来源,极少研究聚焦于外界环境如微生物感染等对垂体瘤发生发展的影响。15-20%的人类肿瘤都与促肿瘤病毒相关[12]。促肿瘤病毒促进肿瘤发生发展的一重要机制是慢性感染常导致氧化应激与炎症,受感染的细胞分泌炎症因子如IL-1β、IL-6及TNF-α,形成炎症环境(inflammatory milieu),炎症刺激、影响、激活NF-κB、STAT3及MAPKs等信号通路促进肿瘤的发展[13]。近年的研究发现,模式识别受体(PRRs)不仅表达在免疫细胞,还能在肿瘤细胞中有表达[14]。PRRs中最常见的是Toll样受体(TLR)家族,其家族的多个成员如TLR2[15],TLR3[16],TLR4[15][17]等均已证实在某些肿瘤中表达,且参与肿瘤的发生与发展,其中与病毒感染最为密切相关的是TLR3。各种来源双链RNA(dsRNA),坏死细胞来源或相关的RNA和人工合成的Poly(I:C)[18][19]均是TLR3的配体。病毒双向转录过程中形成的dsRNA可激活TLR3,这与病毒类型(DNA病毒、RNA病毒等)无关[20]。已有大量的研究表明,高危HPV16被发现与人类多种肿瘤发生相关,包括宫颈癌[21]、口咽癌[22]、肛门癌[23]以及头颈部癌[24]等。不仅如此,某些肿瘤中HPV16阳性的肿瘤细胞可更多朝周围结构侵袭,以及更远处的迁移[21]。HHV6是嗜B淋巴细胞病毒,与儿童淋巴瘤患者的预后密切相关[25]。而HSV1则与乳腺癌病人总体存活率相关,并且与吸烟、饮酒、HPV感染等因素综合决定头颈部癌的发生[26][27]。在垂体瘤中,尚未有报道其发生发展与某种病毒感染有关,因此,本研究拟检测侵袭性与非侵袭性垂体瘤中前述三个促肿瘤病毒(HPV16,HSV1以及HHV6B)的感染情况,探讨病毒感染与垂体瘤侵袭、增殖之间的关系。并且在体外垂体瘤细胞系中,探索病毒作用的分子机制,为临床上预估、治疗侵袭性垂体瘤提供新的思路。方法:1.利用免疫组织化学、巢式PCR等技术,研究HPV16、HSV1、HHV6B DNA、病毒蛋白在垂体瘤病人病理标本中的表达。2.利用qRT-PCR以及免疫组织化学的方法检测垂体瘤病人病理标本中TLR3 mRNA以及蛋白水平的表达。3.利用大鼠垂体瘤细胞系GH3细胞系在体外探究TLR3对细胞侵袭、增殖能力的影响以及分子机制探究。结果:1. HPV16和HHV6B在侵袭性垂体瘤标本中感染率更高60例垂体瘤标本中,HPV16在侵袭性垂体瘤阳性检测率为70%,非侵袭性垂体瘤阳性检测率为26.67%,侵袭性垂体瘤组中HPV 16的阳性检测率显著高于非侵袭性组(P0.01); HHV6B在侵袭性垂体瘤中的阳性检测率为55.33%,在非侵袭性垂体瘤中阳性检测率为23.33%,两者之间差异显著(P0.05); HSV1在侵袭性垂体瘤的检出率为50%,非侵袭性垂体瘤检出率为30%,差异无统计学意义(P0.05)。因此,HPV16、HHV6B感染与垂体瘤的侵袭性正相关,而HSV1的感染与垂体瘤侵袭性无关。2. TLR3的mRNA与蛋白在侵袭组中表达均显著高于非侵袭性PA组,且与HPV16、HHV6B感染率正相关。在mRNA水平上,我们检测了侵袭性垂体瘤组与非侵袭性垂体瘤组中与病毒感染相关的TLRs中的TLR3、4、7、8、9的表达,我们的结果发现,仅TLR3在侵袭性与非侵袭性组之间有差异(P0.01)。在侵袭组中,TLR3在mRNA水平上表达增加。紧接着,我们又用免疫组化的方法检测了 TLR3蛋白水平的表达。TLR3的蛋白在每一例垂体瘤患者标本中均检测出有表达,且TLR3表达在胞浆。通过对TLR3蛋白表达的定量统计,我们发现TLR3蛋白水平在侵袭性组显著高于非侵袭性组(P0.01)。通过统计学方法分析TLR3表达量与HPV16、HHV6B感染率之间的关系,我们发现病毒感染率与TLR3表达之间呈正相关。3. TLR3的激活可促进垂体瘤细胞系GH3的增殖我们在8个时间点上检测了 200 μg/ml Poly (I:C)对GH3细胞系的影响。生长曲线结果显示,在分析的时间点内,Poly (I:C)可以促进GH3细胞的增殖。4. TLR3的激活可影响GH3细胞系的凋亡与细胞周期Poly (I:C)处理组与对照组对比可以发现,处理组中更少的凋亡细胞,更多的细胞处于细胞周期的S期。抗凋亡的Bcl-2在Poly (I:C)处理组中表达增加,促凋亡剪切活化的Caspase 3在添加Poly (I:C)组中的表达更少。而促进凋亡的Bax蛋白在几个处理组之间差异无统计学意义。与此同时,我们检测了促进细胞从G1/G0期进入S期的蛋白CyclinD1,我们发现,CyclinD1的表达在Poly(I:C)处理组中增多。5. TLR3的激活可促进GH3细胞的侵袭在预先TLR3-siRNA预处理或未处理的Transwell上室中,加入Poly(I:C)。48小时后,侵袭的细胞被染色和计数。统计分析显示,Poly(I:C)处理组穿过上室的细胞更多。6. TLR3下游的NF-κB被激活我们运用免疫荧光的方法检测了 Poly (I:C)刺激后NF-κB p65亚基的亚细胞定位。不仅如此,我们还分别检测了 p65分别在细胞浆、核中的表达水平。在Poly (I:C)刺激24小时后,更多的p65转移到了细胞核内,同时p65蛋白的表达水平在核内也是高于胞浆。7. Poly (I:C)刺激GH3后,下游肿瘤侵袭相关蛋白与炎症因子的分泌增加。我们发现,GH3在Poly(I:C)刺激后MMP9表达的增加,与体外研究一致,我们发现在人侵袭性PA病理标本中MMP9的表达水平高于非侵袭性组。NF-κB的激活可导致某些炎症因子的分泌。Poly(I:C)刺激GH3细胞后,可导致下游NF-κB的激活。因此我们检测了 NF-κB转录调控的炎症因子IL-6, IL-1β与TNFα,三者在Poly (I:C)干扰组中分泌均上调。结论:1.HPV16, HHV6B的感染率与垂体瘤的侵袭、增殖正相关。2. TLR3在侵袭性垂体瘤中表达更高。3.在垂体瘤标本中,TLR3的表达与HPV16, HHV6B的感染率呈正相关。4.通过病毒类似物Poly (I:C)激活TLR3信号通路可促进大鼠垂体瘤细胞系GH3的增殖与侵袭。5. NF-κB的激活及其下游调控的炎症因子IL-6、IL-1β、TNF-α与MMP9亦参与促进GH3细胞系的增殖和侵袭。
[Abstract]:Background and purpose: pituitary adenomas account for about 15%[1] of intracranial tumors, and their histology is benign, but 25-55% pituitary tumors are infiltrated around the surrounding structures, such as the osseous tissue of the sellar septum, ethmoid sinus and / or cavernous sinus, which are known as invasive pituitary tumor [2][3][4]. pituitary tumor invasiveness and high Ki-67 index, and with severe symptoms, more Poor prognosis, high recurrence rate, and low responsiveness related [5]. studies in drug replacement therapy have shown that chromosomes, genes (such as 11p/q, p53), adhesion molecules (such as: E-cadherin), metal matrix proteins (such as MMP2, MMP8), abnormal changes in inflammatory factors (such as IL-6, IL-17), and the progression of pituitary adenomas, and the invasion of [5][6][7][8][9][10][11]. However, the assessment of its invasive specific histology and molecular biological standards is still lacking. Not only that, the existing studies have explored the invasiveness of the pituitary tumor at several levels, such as its own genes, transcriptional, posttranscriptional modifications, and so on, and rarely study the pituitary tumor focusing on the external environment, such as microbiological infection. Human tumors that affect the development of.15-20% are an important mechanism for the tumor - related [12]. - promoting oncology promoting the development of tumors. Chronic infections often lead to oxidative stress and inflammation, and the infected cells secrete inflammatory factors such as IL-1 beta, IL-6 and TNF- a, and form the inflammatory environment (inflammatory milieu), inflammatory stimuli, and effects. The activation of NF- kappa B, STAT3 and MAPKs signaling pathway promotes tumor development in recent years. It has been found that the pattern recognition receptor (PRRs) is not only expressed in immune cells, but also the most common [14].PRRs in the tumor cells is the Toll like receptor (TLR) family, and a number of members of the family such as TLR2[15], TLR3[16], TLR4[15][17] and so on have been confirmed. It is expressed in some tumors and participates in the occurrence and development of the tumor. The most closely related to the virus infection is TLR3. RNA (dsRNA). The source of the necrotic cells or the associated RNA and the synthesized Poly (I:C) [18][19] are the ligands of TLR3. The dsRNA activable TLR3 formed during the bi-directional transformation of the virus is the type of the virus. A large number of studies (DNA virus, RNA virus, etc.) not related to [20]. have shown that high risk HPV16 has been found to be associated with a variety of human tumors, including cervical cancer [21], oropharyngeal cancer [22], anal cancer [23], and head and neck cancer [24] and so on. Not only so, the HPV16 positive tumor cells in some tumors can be more invasive to the surrounding structures, as well as more distant migration. [21].HHV6 is a B lymphocytic virus, which is closely related to the prognosis of patients with children's lymphoma, while HSV1 is associated with the overall survival of patients with breast cancer, and it is associated with smoking, drinking, HPV infection and other factors that determine the occurrence of [26][27]. in the pituitary tumor, which has not been reported to be associated with a certain viral infection. This study is intended to detect the infection of three HPV16, HSV1, and HHV6B in invasive and non invasive pituitary adenomas and to explore the relationship between viral infection and the invasion and proliferation of pituitary adenomas. In vitro, the molecular mechanism of the action of the virus is explored to provide a clinical prediction for the treatment of invasive pituitary tumors. New ideas. Methods: 1. using immunohistochemistry, nested PCR and other techniques, the expression of HPV16, HSV1, HHV6B DNA, the expression of viral protein in the pathological specimens of pituitary tumor patients,.2. using qRT-PCR and immunohistochemical method to detect the expression of TLR3 mRNA and protein level in the pathological specimens of pituitary tumor patients,.3. using rat pituitary tumor cells. The GH3 cell line was used to explore in vitro the effect of TLR3 on cell invasion, proliferation and molecular mechanisms. Results: 1. HPV16 and HHV6B had higher infection rates in invasive pituitary tumor specimens, the positive detection rate of HPV16 in invasive pituitary tumor was 70%, the positive detection rate of non invasive pituitary tumor was 26.67%, invasive hypophysis. The positive detection rate of HPV 16 in the tumor group was significantly higher than that in the non invasive group (P0.01), the positive detection rate of HHV6B in invasive pituitary tumor was 55.33%, the positive detection rate in non invasive pituitary tumor was 23.33%, the difference was significant (P0.05), the detection rate of HSV1 in invasive pituitary tumor was 50%, and the detection rate of non-invasive pituitary tumor was 30%, the difference was 30%. There was no statistical significance (P0.05). Therefore, HPV16, HHV6B infection was positively related to the invasiveness of pituitary adenomas, while HSV1 infection and pituitary tumor invasiveness were not related to the.2. TLR3 mRNA and protein expression in the invasive group significantly higher than that in the non invasive PA group, and was positively related to the HPV16, HHV6B infection rate. In mRNA level, we detected invasive pituitary tumor groups and in the group of invasive pituitary adenomas. The expression of TLR3,4,7,8,9 in TLRs associated with virus infection in the non invasive pituitary tumor group, our results showed that only TLR3 was different between the invasive and non invasive groups (P0.01). In the invasive group, the expression of TLR3 was increased at the level of mRNA. Then, we also detected the expression of.TLR3 in the TLR3 protein level by immunohistochemical method. The protein was detected in every patient with pituitary adenoma, and TLR3 was expressed in the cytoplasm. By quantitative analysis of the expression of TLR3 protein, we found that the level of TLR3 protein was significantly higher in the invasive group than in the non invasive group (P0.01). The relationship between the expression of TLR3 and the rate of HPV16 and HHV6B infection was analyzed by statistical method, and we found that the relationship between the expression of TLR3 and the rate of HHV6B infection was analyzed. The positive correlation between the virus infection rate and the expression of TLR3.3. TLR3 activates the proliferation of the pituitary tumor cell line GH3. We detected the effect of 200 mu g/ml Poly (I:C) on the GH3 cell line at 8 time points. The growth curve shows that in the time point of analysis, Poly (I:C) can promote the activation of GH3 cell proliferation.4.. The apoptosis of the GH3 cell line and the cell cycle Poly (I:C) treatment group and the control group were found to be less apoptotic cells in the treatment group and more cells in the S phase of the cell cycle. The expression of anti apoptotic Bcl-2 in the Poly (I:C) treatment group increased, and the expression of Caspase 3 promoting apoptotic shear activation was less in the addition of Poly (I:C) group. There was no statistical difference between the apoptotic Bax protein in several treatment groups. At the same time, we detected the protein CyclinD1 that promoted the cell to enter S phase from the G1/G0 phase. We found that the expression of CyclinD1 in the Poly (I:C) treatment group was increased by the activation of.5. TLR3, which could promote the invasion of GH3 cells in pre TLR3-siRNA preconditioning or untreated Tra. In the upper chamber of nswell, the cells that were attacked were stained and counted after the addition of Poly (I:C).48 hours. Statistical analysis showed that the NF- kappa B of the Poly (I:C) treatment group passing through the cells of the upper chamber more downstream of.6. TLR3 was activated and we detected the subcellular localization of the Poly (I:C) stimulation by the immunofluorescence method. The expression level of p65 in the cytoplasm and nucleus was detected. After 24 hours of Poly (I:C) stimulation, more p65 was transferred into the nucleus, and the expression level of p65 protein was also higher than that of.7. Poly (I:C) stimulated GH3 in the nucleus, and the secretion of the downstream tumor invasion related proteins and inflammatory factors increased. We found that GH3 was in Poly (I:C). The increase of MMP9 expression after excitation is consistent with the in vitro study. We found that the expression level of MMP9 in human invasive PA pathological specimens is higher than the activation of.NF- kappa B in the non invasive group, which leads to the secretion of.Poly (I:C) stimulated GH3 cells by some inflammatory factors, which can lead to the activation of the downstream NF- kappa B. Therefore, we detected the inflammatory factors of NF- kappa B transcriptional regulation. IL-6, IL-1 beta and TNF alpha, three in the Poly (I:C) interference group all up regulation. Conclusion: 1.HPV16, the infection rate of HHV6B and the invasion of pituitary tumor, the proliferation of.2. TLR3 in invasive pituitary tumor is higher.3. in the pituitary tumor specimens, TLR3 expression is positively correlated with the infection rate of HPV16, TLR3 signaling pathway can promote the proliferation of rat pituitary tumor cell line GH3 and the activation of.5. NF- kappa B and its downstream regulation of the inflammatory factor IL-6. IL-1 beta, TNF- A and MMP9 also participate in promoting the proliferation and invasion of the GH3 cell line.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R736.4

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