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Toll样受体4和B7-H6在胶质瘤发生发展及免疫逃逸中的作用和机制研究
中文摘要

胶质瘤起源于神经外胚层,是最常见的原发性中枢神经系统肿瘤,约占恶性脑肿瘤的80%[1,2]。根据世界卫生组织的分类,脑胶质瘤分为Ⅰ-Ⅳ四个级别。根据危险度分层,Ⅰ及Ⅱ级胶质瘤定义为低危性肿瘤,预后相对较好;Ⅲ及Ⅳ级胶质瘤定义为高危性恶性肿瘤,预后相对较差[3,4]。恶性胶质瘤呈浸润性生长,侵袭性强,恶性程度高,各年龄组的发病率和死亡率都很高[4]。胶质瘤患者早期无明显症状,随着肿瘤的生长,患者逐渐出现颅内压增高及脑组织受压迫的症状,非常容易被人们忽视,并延误最佳治疗时机。目前,临床上尚没有一种治疗方案能够有效地治疗恶性胶质瘤。胶质瘤的治疗仍以手术切除为主。但是,胶质瘤的肿瘤组织与正常脑组织之间没有明确的界限,手术经常难以完全切除肿瘤组织,而且,即使手术完全切除肿瘤组织,肿瘤也容易原位复发,最终导致患者死亡率较高[5]。另外,由于血脑屏障的存在及作用,阻挡了化疗药物进入肿瘤组织的部位,导致胶质瘤对化疗药物不敏感,各种预防和治疗的化疗药物疗效较差或毒副作用较大。而且,胶质瘤对放疗也表现为低敏感性。因此,胶质瘤患者预后极差,复发率高,平均生存期较短,约为1.5年,胶质母细胞瘤患者5年生存率小于3% [3,5]。根据2014年全国肿瘤中心的统计数据,我国脑胶质瘤位列10大致死肿瘤的第9位。因此,恶性胶质瘤的治疗仍是一个难点,亟待探索新的治疗靶点,研究新型治疗策略,以增强胶质瘤患者对治疗方案的敏感性,有效地杀灭肿瘤,从而改善患者的生存质量,并延长患者的生存时间。 第一部分脂多糖激活的Toll样受体4信号通路在胶质瘤CD133+肿瘤干细胞的发生发展及免疫逃逸中的作用及机制研究 目的: 胶质瘤恶性肿瘤组织内存在肿瘤干细胞(cancer stem cells,CSCs)。胶质瘤CSCs的肿瘤源性活性极强,并具有多种干细胞的特性,如多向分化、无限增殖、及自我更新能力等。目前,大部分研究将CD133作为胶质瘤CSCs的常用标记。文献报道,胶质瘤CSCs在胶质瘤的生长、入侵、血管形成、放化疗耐药及免疫逃逸过程中发挥重要作用。Toll-样受体4 (Toll-like receptor 4,TLR4)是目前研究最多的Toll样受体(Toll-like receptors,TLRs)。最近研究发现,多种肿瘤组织异常高表达TLR4,而且TLR4在肿瘤的发生发展过程中发挥着重要的作用。但是,胶质瘤CD133+CSCs是否表达TLR4及TLR4的具体功能如何?目前还不清楚。因此,该部分使用人胶质瘤细胞株及从患者肿瘤病理组织中分离得到的原代细胞,诱导生成胶质瘤CSCs,分离并获得胶质瘤CD133+CSCs,以胶质瘤CD133+CSCs作为研究对象。使用脂多糖(lipopolysaccharide,LPS)激活TLR4信号传导通路,并建立胶质瘤CD133+CSCs反应性细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL),研究TLR4信号传导通路在胶质瘤CD133+ CSCs发生发展及免疫逃逸过程中的作用及可能机制。 方法: 使用人胶质瘤细胞株SF295、U251及从4例胶质瘤患者肿瘤组织中分离得到的肿瘤原代细胞(pTl-4),诱导生成胶质瘤CSCs。通过流式细胞学方法,检测胶质瘤CSCs常见分子标记的表达水平。通过CD133磁珠分选方法,从胶质瘤CSCs中分离并获得6种胶质瘤CD133+CSCs。使用流式细胞学及Western Blot方法,检测胶质瘤CD133+CSCs中TLR4的表达水平;通过免疫组织化学染色方法(immunohistochemistry,IHC),检测胶质瘤患者肿瘤病理组织标本中TLR4的表达水平。使用CCK-8试剂盒(Cell Counting Kit-8,CCK-8)、实时定量聚合酶链反应(real time quantity polymerase chain reaction,qRT-PCR)、流式细胞学、酶联免疫吸附实验(enzyme linked immunosorbent assay,EUSA)及Western Blot方法,检测LPS刺激后,胶质瘤CD133+CSCs的增殖、分子标记表达、细胞因子分泌及相关信号传导通路分子表达情况。从健康供者外周血中分离获得的单个核细胞,使用磁珠分选方法,获得CD3+T细胞。胶质瘤CD133+CSCs经过辐照,与CD3+T细胞共培养,建立胶质瘤CD133+CSCs反应性CTL。通过乳酸脱氢酶(lactate dehydrogenase,LDH)释放实验,检测胶质瘤CD133+CSCs反应性CTL对其靶细胞的细胞毒性杀伤作用,并通过流式细胞学鉴定胶质瘤CD133+ CSCs反应性CTL的表型。 结果: 胶质瘤CSCs表达CD133、Nanog、SSEA-1、Msil及Nestin。胶质瘤CD133+ CSCs及患者组织标本表达TLR4蛋白。LPS促进胶质瘤CD133+CSCs的增殖并保护其免遭阿霉素诱导的凋亡。LPS在基因及蛋白质水平上调胶质瘤CD133+ CSCs标记CD133、Nanog及Nestin的表达。LPS促进胶质瘤CD133+CSCs分泌细胞因子单核细胞趋化蛋白-1(monocyte chemotactic protein-1,MCP-1)、巨噬炎性蛋白-lα(macrophage inflammatory protein-1α,MIP-lα)、肿瘤坏死因子-α (tumor necrosis factor-a,TNF-α)、白介素(interleukin,IL) -1β、IL-6及IL-10。 LPS激活TLR4信号传导通路,诱导胶质瘤CD133+CSC表达细胞周期蛋白酶4 (cyclin-dependent kinase,CDK)、CDK6、Cyclin E、B细胞淋巴瘤-2(B-cell lymphoma-2,bcl-2)、核因子-κB(nuclear factor-κB,NF-κB )、p-p38、p-Jun氨基末端激酶(Jun amino-terminal kinase,JNK)、p-细胞外信号相关激酶(extracellular signal related kinases,ERK)及p-蛋白激酶B(protein kinase B,Akt)。TLR4的表达抑制胶质瘤CD133+ CSCs反应性CTL的细胞毒性杀伤功能,而这种抑制作用在敲低TLR4表达的胶质瘤CD133+CSCs中不明显。而且,记忆性效应T细胞在TLR4信号传导通路介导的胶质瘤CD133+CSCs免疫逃逸过程中发挥重要的作用。 结论: 胶质瘤CD133+CSCs表达TLR4,LPS激活的TLR4信号传导通路促进胶质瘤CD133+CSCS的增殖、化疗耐药、分子标记表达及细胞因子分泌,并保护其免遭胶质瘤CD133+CSCs反应性CTL的细胞毒性杀伤作用。因此,TLR4信号传导通路是胶质瘤CD133+CSCs发生发展及免疫逃逸的重要因素,阻断TLR4信号传导通路可能为胶质瘤患者提供治疗新策略。 第二部分:B7-H6在胶质瘤发生发展中的作用及机制研究 目的: 近年来,尽管治疗策略方面的研究有了很大的进展,胶质瘤仍然是一种不可治愈的疾病,患者5年生存率较低。靶向一些调控肿瘤发生发展及免疫应答反应等分子方面的研究,可能为胶质瘤的治疗提供新的理论依据及策略。据报道,与正常组织不同,肿瘤组织及细胞特异性表达B7-H6。而且,B7-H6在肿瘤的发生发展及免疫应答反应过程中发挥重要的作用。但是,B7-H6在胶质瘤方面的研究较少。因此,本部分以胶质瘤肿瘤细胞株及患者原代细胞为研究对象,采用分子生物学及免疫学的研究方法,研究胶质瘤B7-H6的表达水平;研究细胞因子及炎症介质刺激后胶质瘤细胞B7-H6表达水平的变化,探索胶质瘤B7-H6表达的具体调控机制。研究胶质瘤患者肿瘤病理组织标本中B7-H6的表达水平。阐述B7-H6在胶质瘤发生发展过程中的作用及可能机制。旨在为胶质瘤的有效治疗方案的建立提供新的理论依据。 方法: 通过PCR方法,检测人胶质瘤肿瘤细胞株及患者原代肿瘤细胞B7-H6的表达水平。通过IHC方法,检测胶质瘤患者肿瘤病理组织标本中B7-H6的表达水平。使用LPS刺激人胶质瘤肿瘤细胞,通过qRT-PCR及Western Blot方法,检测不同时间的LPS作用后,人胶质瘤肿瘤细胞株B7-H6表达水平的变化。通过小干扰RNA (small interfering RNA,siRNA)方法敲低胶质瘤细胞B7-H6的表达水平。通过CCK-8、克隆形成实验及细胞计数等方法,检测人胶质瘤肿瘤细胞的增殖能力。通过划痕愈合实验、transwell迁移和侵袭实验,检测人胶质瘤肿瘤细胞的转移和侵袭能力。通过Western Blot方法,检测人胶质瘤肿瘤细胞相关蛋白分子的表达水平,分析B7-H6导致的胶质瘤发生发展过程中涉及到的信号传导通路及相关蛋白分子。 结果: PCR方法检测显示,人胶质瘤肿瘤细胞株及从患者肿瘤病理组织中分离得到的原代细胞表达B7-H6。IHC方法检测显示,胶质瘤患者肿瘤病理组织标本表达B7-H6。使用LPS,在不同的时间点刺激人胶质瘤肿瘤细胞株后,胶质瘤细胞B7-H6的mRNA和蛋白水平表达增加。使用siRNA方法,敲低人胶质瘤肿瘤细胞株及患者原代细胞中B7-H6的表达水平。敲低肿瘤细胞B7-H6的表达水平后,人胶质瘤肿瘤细胞株及患者原代细胞的增殖、迁移及侵袭能力降低,其可能的机制是通过降低vimentin、N-cadherin、基质金属蛋白酶(matrix metalloproteinase, MMP) 2、MMP9及survivin的表达水平,并增加E-cadherin和bcl-2相关x蛋白(bcl-2 associated x Protein,Bax)的表达水平来介导。 结论: 人胶质瘤肿瘤细胞株、患者原代细胞及肿瘤病理组织标本中均可检测到B7-H6的表达。LPS诱导人胶质瘤肿瘤细胞株B7-H6的表达。B7-H6在人胶质瘤的发生发展过程中发挥着重要的作用,通过调控相关蛋白分子的表达促进肿瘤的增殖、侵袭及转移的发生。因此,该研究将为靶向B7-H6的胶质瘤治疗方案的研究提供新的理论依据和靶点。 关键词:胶质瘤;肿瘤干细胞;TLR4; B7-H6;免疫逃逸

英文摘要

Glioma occurs in neuroderm and is the most common primary tumor of the central nervous system, accounting for about 80% of malignant brain tumors [1,2]. According to the classification of WHO, glioma is divided into grade Ⅰ-Ⅳ. Grade Ⅰ and grade Ⅱ gliomas are low-risk tumors with relatively good prognosis. Ⅲ and Ⅳ grade gliomas are high-risk malignant tumors with poor prognosis [3,4], Malignant glioma is highly invasive and highly malignant, with high morbidity and mortality in all age groups [4], The symptoms of glioma patients are not obvious at early stage. As the tumor grows, the symptoms of intracranial hypertension and compression of brain tissue gradually appear, which are easily ignored and delay the best treatment time. So far, there is no effective clinical treatment for malignant glioma. Surgical resection is the main treatment strategy at present. However, there is no definite boundary between malignant glioma and normal brain tissue, and it is difficult to remove the tumor tissues completely. Moreover, even if the tumor is completely removed, the recurrence easily occurs and the mortality is high. In addition, the presence and function of the blood-brain barrier blocks the entry of chemotherapeutic agents into the tumor tissue, causing glioma to be insensitive to chemotherapeutic agents, and the efficacy of various preventive and therapeutic drugs is not satisfactory or toxic and side effects are great. Malignant glioma is also less sensitive to radiotherapy. Therefore, the prognosis of glioma patients is poor and the recurrence rate is high [5], The mean survival time of glioma patients is 1.5 years. The 5 years survival rate for glioblastoma is less than 3% [3,5], According to the 2014 National Cancer Center, glioma is the ninth most lethal tumor in China. Therefore, the treatment of malignant glioma remains challenging. It is urgent to explore new target and therapy strategy to improve the sensitivity to therapies, kill tumors effectively, and finally improve the life quality and survival time of glioma patients. Part 1 The function and mechanism of LPS activated toll like receptor 4 signal pathway in the tumorgenesis, progression and immune evasion of glioma CD133+ cancer stem cells Objective: Cancer stem cells (CSCs) is found in the malignant tumor tissue of glioma. Glioma CSCs exhibit high tumorigenic activity and has many characteristics of stem cells, such as multilineage differentiation, unlimited proliferation and self-renewal. Most studies now use CD 133 as a common marker for glioma CSCs. It is reported that glioma CSCs plays an important role in glioma growth, invasion, angiogenesis, resistance to chemotherapy and radiotherapy, and immune escape. Toll-like receptor 4 (Toll-like receptor 4, TLR4) is the most studied Toll like receptors (Toll-like receptors, TLRs) at present. Recent studies have found that many tumor tissues abnormally express high levels of TLR4, and TLR4 plays an important role in the development and progression of tumors. However, whether glioma CD 133+ CSCs expresses TLR4? And what is the specific functions of TLR4? It's not clear yet. Therefore, this part uses human glioma cell lines and primary cells isolated from patients' tumor pathology tissue to induce glioma CSCs, and obtains glioma CD 133+ CSCs and uses them as the research object. Lipopolysaccharide (lipopolysaccharide, LPS) was used to activate TLR4 signaling pathway and the CD133+ glioma CSCs reactive CTL was established in order to study the roles and possible mechanisms of TLR4 signal transduction pathway in development and immune escape of glioma CD133+ CSCs. Methods: Human glioma cell lines SF295, U251 and four primary tumor cells, isolated from human glioma samples (pT1-4), were used to induce and generate glioma CSCs. The expression levels of molecular markers of glioma CSCs were identified via flow cytometry. Six kind of glioma CD133+ CSCs were separated and purified by CD133 magnetic-activated cell separation purification system. The expression of TLR4 in glioma CD 133+ CSCs and patient tissues were detected via flow cytometry, Western Blot and immunohistochemical staining (IHC). After LPS stimulation, the proliferation, expression of molecular markers, cytokine secretion and expression of related molecules in signal pathways are investigated via Cell Counting Kit-8 (CCK-8), real time quantity polymerase chain reaction (qRT-PCR), flow cytometry, enzyme linked immunosorbent assay (ELISA) and Western Blot. Mononuclear cells were isolated from peripheral blood of healthy donors, and CD3+ T cells were obtained by magnetic bead sorting. Glioma CD133+ CSCs was irradiated and cultured with CD3+T cells to establish glioma CD 133+ CSCs reactive cytotoxic T lymphocyte (CTL). lactate dehydrogenase (LDH) cytotoxicity test was used to detect the cytotoxic lysis of glioma CD 133+ CSCs reactive CTL to different target cells, and its phenotype was identified by flow cytometry. Results: Glioma CSCs expressed CD133, Nanog, SSEA-1, Msil and Nestin. TLR4 protein was expressed in gliomas CD133+ CSCs and patient tissues. LPS promoted the proliferation of glioma CD133+ CSCs and protects them against doxorubicin induced apoptosis. LPS up-regulated the expression of CD133, Nanog and Nestin at the gene and protein levels in glioma CD133+CSCs. LPS promoted the secretion of cytokines including monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1 α (MIP-lα), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and IL-10. LPS activated TLR4 signal pathway and induced the expression of cyclin-dependent kinase (CDK)4, CDK6, Cyclin E, B-cell lymphoma-2 (bcl-2), nuclear factor κB (NF-κB), p-p38, p-Jun amino-terminal kinase (JNK), pextracellular signal related kinases (ERK) and p- protein kinase B (Akt) in glioma CD133+CSCs. The capacity of glioma CD133+ CSC-reactive cytotoxic T lymphocyte to selectively kill glioma CD 133+ CSCs was reduced by LPS, and this effect was not apparent after TLR4 knockdown in glioma CD 133+ CSCs. Memory effect T cells play roles in the immune evasion of glioma CD133+CSCs mediated by TLR4 signaling pathway. Conclusion: TLR4 is expressed in glioma CD 133+ CSCs expression TLR4. LPS acitivated TLR4 signaling pathway, promoted the proliferation, drug resistance, molecular marker expression and cytokine secretion of glioma CD133+ CSCs, and protected glioma CD133+ CSCs from glioma CD133+ CSCs reactive CTL induced cytolysis. Therefore, TLR4 signaling pathway is an important factor in the development and immune escape of glioma CD133+ CSCs, and blocking TLR4 signaling pathway may provide a new therapeutic strategy for glioma patients. Part 2 The role and mechanism of B7-H6 in tumorigenesis and progression of glioma Objective: Although great progress has been made in treatment regimens, gliomas are still incurable and the 5-year survival remains poor. Studies focusing on molecules that regulate tumorigenesis or immunity may provide potential therapeutic strategies for patients with glioma. It is reported that, different from the normal organization, B7-H6 is selectively expressed in tumor cells and plays vital roles in tumorigenesis and tumor immunity. However, B7-H6 has been less studied in gliomas. Therefore, we investigate glioma cell lines and primary cells obtained from glioma patient tissues via molecular biological and immunological methods. We explored the expression level of B7-H6 in gliomas; and the expression of B7-H6 in glioma cells after stimulated with cytokines and inflammatory mediators, trying to elucidate the specific regulatory mechanisms of B7-H6 expression in gliomas. We analyzed the role and possible mechanism of B7-H6 in tumorigenesis and progression of glioma. We aimed to provide a new target for the effective treatment of glioma and a new approach for the design of new biological therapy strategies. Methods: The expression levels of B7-H6 in glioma cell lines, primary cells and tissue samples were detected by PCR and IHC. The expression levels of B7-H6 in glioma cells were detected by qRT-PCR and Western Blot after LPS stimulation at different time points. The expression level of B7-H6 in glioma cells was knocked down by small interfering RNA (siRNA). The proliferation of glioma cells was detected via CCK-8, clone formation assay and cell counting. The migration and invasion capacity of glioma cells were detected via transwell migration and invasion assay. The expression levels of related proteins in human glioma tumor cells were detected by Western Blot, and the signal transduction pathway and related protein molecules involved in the development and progression of glioma caused by B7-H6 were analyzed. Results: The PCR results demonstrated that B7-H6 was expressed in glioma cell lines and primary cells isolated from human glioma tissues. IHC detection showed that B7-H6 was expressed in glioma patient tissues. After stimulated with LPS at different time pointss, the expression of B7-H6 increased at mRNA and protein levels. siRNA was used to knockdown B7-H6 expression levels in glioma cells. After B7-H6 knockdown, the proliferation, migration and invasion capacity of glioma cells were inhibited, possibly via decreasing the expression of vimentin, N-cadherin, matrix metalloproteinase (MMP) 2, MMP9 and survivin, and increasing the expression of E-cadherin and bcl-2 associated x protein (Bax). Conclusion: B7-H6 was expressed in glioma cell lines, primary cells and tissues of patients. LPS upregulated the expression levels of B7-H6 in glioma cells. B7-H6 played important roles in the tumorigenesis and progression of glioma. B7-H6 promote tumor proliferation, migration and invasion via regulating the expression of related proteins. Therefore, targeting B7-H6 may provide a novel therapeutic strategy for glioma patients. Key words: glioma; cancer stem cells; TLR4; B7-H6; immune evasion

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