B7-H4在胆管癌免疫逃逸中的作用和临床意义的初步研究

发布时间：2018-05-20 12:37

本文选题：胆管癌 + B7-H4　；参考：《第三军医大学》2015年博士论文

【摘要】：一、研究背景胆管癌作为一种常见的恶性肿瘤,发病率在我国消化系统肿瘤中排名第五,其发病率每年仍有增高的趋势。流行病学调查显示胆管癌患者发病年龄多为50-60岁,男性多于女性。胆管癌的发病率具有一定民族和区域分布差异,多发生亚洲,特别是东南亚以及中东国家,我国主要是华南以及东南沿海发病率较高。近些年,随着环境不断恶化以及临床诊断技术的发展进步,世界范围内胆管癌发病率的呈逐年上升趋势。尽管胆管癌的发病率仍低于消化系统其他恶性肿瘤,但是患者的死亡率却一直处于较高水平。胆管癌所处的特殊解剖部位以及高侵袭转移能力,是造成高死亡率的主要原因。目前针对胆管癌的治疗方法中,胆管癌根治性切除术是提高患者预后降低死亡率的主要手段。但是由于胆管癌起病隐匿、临床表现不典型以及缺乏早期诊断标志物,部分患者就诊时肿瘤已经进入晚期,有些甚至已经发生转移,因而失去了手术切除肿瘤的最佳时机。部分胆管癌患者即使接受根治性手术切除,肿瘤复发以及转移的风险仍然偏高。以上这些因素严重影响了胆管癌根治性切除术的远期疗效。近年来,针对胆管癌发生、发展的相关分子机制研究已经取得一定的进展,但是涉及到胆管癌的免疫逃逸以及侵袭转移的相关研究还不是很完善。所以我们把研究的重点放在了胆管癌的肿瘤微环境,特别是免疫逃逸以及侵袭转移的相关研究上。通过相关内容的研究,我们尝试发现新的分子以及相关通路作为诊断和治疗靶点,并且能够为创新临床诊断和治疗的方法提供理论依据。T细胞免疫作为肿瘤微环境中抗肿瘤免疫的重要组成部分,在肿瘤的发生、发展中起到十分关键的作用。其中T细胞的活化和增殖需两种信号途径。第一条信号途径是抗原递呈细胞通过呈递抗原肽并且与T细胞受体识别,传递活化信号。第二条信号途径是通过协同刺激信号分子活化T细胞。协同刺激分子决定了T细胞是被激活并且增殖还是转为抑制或者无应答状态。目前所知的协同刺激分子主要分为三类:肿瘤坏死因子家族、细胞因子家族以及B7家族。其中B7家族包括了B7-1、B7-2、B7-H1、B7-H2、B7-H3、B7-DC和B7-H4。B7家族作为重要的协同刺激分子,它们不仅能起到促进T细胞增殖活化以及产生细胞因子的作用,同时也能起到抑制、限制或者减弱T细胞反应的作用。B7-H4作为协同刺激分子B7家族中的成员,主要提供负性调节信号,通过抑制T细胞增殖、分泌相关细胞因子以及降低T细胞杀伤肿瘤细胞能力,从而促进肿瘤细胞逃过宿主免疫监视进而发生免疫逃逸现象;同时,B7-H4在部分肿瘤中特异性表达,还可以增强肿瘤细胞侵袭转移能力,促进肿瘤细胞恶性转化。既往文献报道,B7-H4在多种肿瘤中存在异常表达,并且与肿瘤临床病理指标以及病人预后密切相关。由于B7-H4在协助肿瘤细胞逃避机体免疫监视以及肿瘤发生发展中起到了十分重要的作用。根据既往研究成果以及前期研究结果,我们对B7-H4在胆管癌免疫逃逸中的作用和临床意义进行初步研究,并提出研究假设:B7-H4在胆管癌患者中特异性表达增高,并且通过抑制肿瘤微环境中的免疫淋巴细胞,降低对肿瘤细胞的杀伤能力,从而促进肿瘤细胞发生免疫逃逸。同时B7-H4还可促进胆管癌细胞发生恶性转化,增强癌细胞自身侵袭转移能力。二、研究方法为了验证上述假设,首先,我们通过免疫组化检测统计B7-H4在胆管癌病人中的表达情况,分析其表达水平与临床病理特征以及患者预后的关系,并分析肿瘤组织和非肿瘤组织中B7-H4的差异表达的情况;其次,我们通过免疫组化分别统计肿瘤实质和间质中CD4,CD8阳性T淋巴细胞浸润情况与B7-H4阳性表达之间的联系,分析B7-H4是否抑制T淋巴细胞在肿瘤微环境中浸润的情况;再次,我们在体外试验中干扰B7-H4在肿瘤细胞中的表达,分析B7-H4表达水平的改变对细胞毒性淋巴细胞的杀伤作用和相关细胞因子分泌情况的影响。最后,通过下调B7-H4在胆管癌细胞中表达水平,观察胆管癌细胞侵袭和迁移能力的变化情况。本项研究目的在于探讨B7-H4在胆管癌中的表达情况以及相关临床意义,以及它如何促进胆管癌细胞在肿瘤微环境中发生免疫逃逸以及恶性转化,尤其是增强癌细胞侵袭迁移能力的可能机制,开拓胆管癌发生肿瘤免疫逃逸以及侵袭转移的研究思路,为发现新的胆管癌预后指标以及新的免疫治疗靶点提供科学研究依据。1.收集从2005年至2012年第三军医大学西南医院肝胆外科研究所进行根治性切除并经病理诊断明确为胆管癌的病例。上述收集的病例均未进行放疗或者化疗。同时收集19例慢性胆管炎、8例胆管腺瘤标本和18例胆管癌淋巴结转移组织标本。收集的临床资料包括病人性别、年龄、肿瘤位置、大小、分化程度、分期分级、淋巴结转移以及远处转移的情况。采用免疫组化的方法检测B7-H4在各种组织中的表达情况,并对比肿瘤组织和非肿瘤组织中B7-H4的表达差异;同时分析B7-H4与患者临床病理特征的相关性。2.收集110例胆管癌患者肿瘤组织,进行免疫组化染色,检测统计CD4、CD8阳性T淋巴细胞分别在肿瘤实质以及肿瘤间质组织中的浸润表达情况,并结合对应患者的B7-H4表达情况,分析B7-H4与CD4、CD8阳性T淋巴细胞表达情况的相关性。3.收集110例胆管癌患者临床随访资料(包括患者术后总存活时间和无瘤生存时间)。总生存时间的计算方式是从病人手术时间到病人死亡时间或者最后一次联系随访时间;无瘤生存时间的计算方式是从病人手术时间到发现肿瘤复发的时间。肿瘤的复发主要依靠影像学以及病理诊断为主。单因素生存分析采用KM法(KM method),多因素分析采用Cox多因素逐步回归(向前,极大似然法)。分析B7-H4在肿瘤组织中的表达与患者预后之间的关系。4.选择胆管癌细胞株QBC939和RBE作为研究对象,利用慢病毒作为载体,构建sh RNA-B7-H4序列,下调两株细胞中B7-H4的表达水平,然后将其与细胞毒性T淋巴细胞共培养,观察细胞毒性T淋巴细胞对胆管癌细胞杀伤作用的变化情况。同时我们还检测共培养后培养基上清液相关细胞因子分泌水平的变化情况。5.选择胆管癌细胞株QBC939和RBE作为研究对象,利用慢病毒作为载体,构建sh RNA-B7-H4序列,下调两株细胞中B7-H4的表达水平,然后通过观察两株胆管癌细胞的损伤修复实验、Transwell侵袭实验,观察B7-H4表达对胆管癌细胞侵袭和迁移能力的影响。三、研究结果1.在110例患者的胆管癌组织中,54例呈B7-H4染色阳性,阳性率为49%;8例胆管腺瘤组织中,全部呈B7-H4染色阴性;19例慢性胆管炎组织中,4例呈B7-H4染色阳性,阳性率为21.1%。本部分结果提示:B7-H4在肿瘤组织中表达的阳性率明显高于非肿瘤组织(p=0.023),B7-H4在胆管癌组织中表达异常增高。通过分析B7-H4表达情况和患者临床病理特征的相关性发现:B7-H4的表达与肿瘤分期,分级和淋巴转移有关;而与性别、年龄、部位,远处转移无关。本部分结果提示:B7-H4的异常表达可能与肿瘤恶性程度以及侵袭转移相关。2.在110例患者的胆管癌肿瘤间质组织中,CD4阳性T淋巴细胞轻度浸润97例,重度浸润13例;CD8阳性T淋巴细胞轻度浸润56例,重度浸润54例。在110例患者的胆管癌肿瘤实质组织中,CD4阳性T淋巴细胞轻度浸润88例,重度浸润22例;CD8阳性T淋巴细胞轻度浸润76例,重度浸润34例。胆管癌组织中B7-H4的表达水平与CD8阳性T淋巴细胞在肿瘤间质细胞浸润程度有关。本部分结果提示:B7-H4分子作为一个负性免疫调控分子,可能通过降低CD8阳性T淋巴细胞在肿瘤间质细胞浸润的程度,从而在促进胆管癌细胞发生免疫逃逸中起到重要作用。3.通过对110胆管癌患者生存数据的统计。Kaplan—Meier模型分析提示:B7-H4阳性组患者生存时间明显低于B7-H4阴性组患者(p=0.015)。我们按照相同方法发现:B7-H4阳性组患者复发时间明显低于B7-H4阴性组患者(p=0.045)。为了排除相关混杂因素的干扰,我们将患者年龄、性别、肿瘤部位、分化程度以及B7-H4表达情况带入Cox模型中进行术后存活时间以及复发时间的多因素相关性分析。结果显示:B7-H4阳性表达是判定患者术后存活时间和复发时间的一个相对独立危险因素(HR=1.786,95%CI=1.110-2.872,p=0.017;HR=2.062,95%CI=1.160-3.665,p=0.014)。本部分结果提示:B7-H4阳性表达与胆管癌患者预后较差显著相关而且可以作为判断患者预后的独立分子标志物。4.我们通过Western-blot方法检测,发现:两株胆管癌细胞株QBC939和RBE均有B7-H4蛋白的表达。我们采用慢病毒转染sh RNA-B7-H4进入细胞株,成功下调B7-H4表达水平;在效靶比为(20:1或者10:1)时,特异性CD8+CTL对B7-H4表达水平下调的胆管癌细胞杀伤能力明显高于对照组的杀伤能力。淋巴细胞和B7-H4表达水平下调的胆管癌细胞株混合培养后,TGF-β,IL-6细胞因子分泌水平明显低于对照组。本部分结果提示B7-H4有可通过能增强细胞因子TGF-β,IL-6的分泌水平,诱导CD8阳性T分化为不具有细胞毒性Th17,从而降低CD8+CTL对肿瘤细胞的杀伤能力。5.采用慢病毒转染sh RNA-B7-H4进入细胞株,成功下调B7-H4表达水平;通过观察损伤修复实验、Transwell侵袭实验发现下调胆管癌细胞中B7-H4蛋白表达水平能明显抑其癌细胞侵袭和迁移能力。本部分结果提示:B7-H4明显促进了胆管癌的侵袭和转移。四、研究结论胆管癌中存在B7-H4异常表达,其高表达提示患者预后不良。分析原因可能是B7-H4有可能通过促进肿瘤侵袭迁移,或者通过增强TGF-β,IL-6分泌水平,降低细胞毒性T淋巴细胞对肿瘤细胞的杀伤作用,从而促进胆管癌细胞发生免疫逃逸,进而影响患者预后。
[Abstract]:The incidence of cholangiocarcinoma, a common malignant tumor, is fifth in the digestive system tumor in China. The incidence of cholangiocarcinoma is still increasing every year. The epidemiological survey shows that the incidence of cholangiocarcinoma is more than 50-60 years old, and the incidence of cholangiocarcinoma has a certain ethnic and regional distribution. In recent years, the incidence of cholangiocarcinoma in the world is increasing year by year. Although the incidence of cholangiocarcinoma is still lower than other evil of the digestive system, the incidence of cholangiocarcinoma in the world is increasing year by year, especially in Southern China and the southeast coastal countries. The death rate of the patients is at a high level. The special anatomical location and the high invasion and metastasis of bile duct cancer are the main causes of high mortality. Carcinoma of tube occult, atypical clinical manifestations and lack of early diagnostic markers. Some patients have advanced tumors at the time of treatment and some even have metastasize. Therefore, the best time to remove the tumor is lost. Some patients with bile duct cancer are still at risk of recurrence and metastasis even if they are treated with radical hand resection. These factors have seriously affected the long-term effect of radical resection of cholangiocarcinoma. In recent years, some progress has been made in the study of the molecular mechanism of the development of cholangiocarcinoma. However, the related research on the immune escape and invasion and metastasis of cholangiocarcinoma is not very perfect. So we focus on the research. In the study of tumor microenvironment, especially immune escape and invasion and metastasis, we try to find new molecules and related pathways as targets for diagnosis and treatment, and to provide a theoretical basis for innovative clinical diagnosis and treatment methods based on.T cell immunization as tumor microring. The important part of anti-tumor immunity in the environment plays a key role in the occurrence and development of the tumor. The activation and proliferation of T cells require two signal pathways. The first signal pathway is that the antigen presenting cells pass the antigen peptide and recognize the T cell receptor and transmit the activation signal. The second signal pathway is through synergy. Stimulator molecules activate T cells. Synergistic stimulators determine whether T cells are activated, proliferated or converted to inhibitory or non responsive states. The known synergistic stimuli are divided into three categories: the tumor necrosis factor family, the cytokine family and the B7 family. Among them, the B7 family includes B7-1, B7-2, B7-H1, B7-H2, B7-H3, B7-DC, and the B7 family. As an important synergistic stimulator, the B7-H4.B7 family can not only promote the proliferation and activation of T cells and produce cytokine, but also inhibit, restrict or weaken the role of T cell response,.B7-H4 as a member of the B7 family of synergistic stimulators. The main purpose is to provide negative regulatory signals, by inhibiting T cells to increase. Colonization, secreting related cytokines and reducing the ability of T cells to kill tumor cells, thus promoting tumor cells escaping from host immune surveillance and immune escape; meanwhile, the specific expression of B7-H4 in some tumors can also enhance the invasion and metastasis of tumor cells and promote malignant transformation of tumor cells. Previously reported, B7-H4 Abnormal expression exists in various tumors and is closely related to tumor clinicopathological indicators and patient prognosis. B7-H4 plays an important role in assisting tumor cells to escape immune surveillance and tumor development. According to previous research results and previous research results, we immune to B7-H4 in cholangiocarcinoma. A preliminary study of the role and clinical significance of B7-H4 has been carried out, and the hypothesis is proposed that the specific expression in the patients with cholangiocarcinoma is increased, and the tumor cells can be reduced by inhibiting the immune lymphocytes in the tumor microenvironment and reducing the killing ability of the tumor cells, and the immune escape of the tumor cells is promoted. At the same time, B7-H4 can also promote the development of cholangiocarcinoma cells. Two, in order to verify the hypothesis, first of all, we detected the expression of B7-H4 in the patients with cholangiocarcinoma by immunohistochemistry, and analyzed the relationship between the expression level and the clinicopathological features as well as the prognosis of the patients, and analyzed the B7-H4 in the tumor and non tumor tissues. Secondly, we analyzed the relationship between CD4, CD8 positive T lymphocyte infiltration and B7-H4 positive expression in tumor substance and interstitial tissue by immunohistochemistry, and analyzed whether B7-H4 inhibited the infiltration of T lymphocytes in the tumor microenvironment; again, we interfered with B7-H4 in the tumor cells in vitro. The effects of B7-H4 expression level on cytotoxic lymphocyte killing and cytokine secretion were analyzed. Finally, the changes in the invasion and migration of bile duct cancer cells were observed by down regulation of the expression level of B7-H4 in cholangiocarcinoma cells. The purpose of this study was to explore the table of B7-H4 in cholangiocarcinoma. As well as the relevant clinical significance, and how it promotes the immune escape and malignant transformation of bile duct cancer cells in the tumor microenvironment, especially the possible mechanism of enhancing the invasion and migration of cancer cells, exploring the research ideas of the immune escape and invasion and metastasis of the bile duct cancer, in order to find a new prognostic indicator of bile duct cancer. And the new immunotherapy targets provide scientific research based on.1. collection from 2005 to 2012 at the Department of hepatobiliary surgery, Southwest Hospital, Third Military Medical University, which had undergone radical resection and pathologically diagnosed as cholangiocarcinoma. All cases collected were not treated with radiotherapy or chemotherapy. 19 cases of chronic cholangitis and 8 cases of bile duct adenoma were collected at the same time. Specimens and 18 specimens of lymph node metastases of cholangiocarcinoma were collected. The clinical data included patients' sex, age, tumor location, size, differentiation, stage classification, lymph node metastasis, and distant metastasis. Immunohistochemical method was used to detect the expression of B7-H4 in various tissues and to compare tumor tissue and non tumor tissue. The difference in expression of B7-H4, and the correlation between B7-H4 and the patient's clinicopathological features.2. collection of 110 cases of cholangiocarcinoma tumor tissue, immunohistochemical staining, the detection of CD4, CD8 positive T lymphocytes in tumor substance and tumor interstitial tissue infiltration table, and the corresponding B7-H4 expression in the corresponding patients, The correlation of the expression of B7-H4 and CD4, CD8 positive T lymphocytes was analyzed in 110 cases of cholangiocarcinoma patients (including total survival time and non tumor survival time after operation). The total survival time was calculated from the patient's operation time to the patient's death time or the last contact time of the patients. The method of calculation is from the operation time of the patient to the time to discover the recurrence of the tumor. The recurrence of the tumor is mainly based on the imaging and pathological diagnosis. The single factor survival analysis uses the KM (KM method) method. The multifactor analysis adopts the stepwise regression of Cox multiple factors (forward, maximum likelihood). The expression of B7-H4 in the tumor tissue and the prognosis of the patient are analyzed. The relationship between.4. and QBC939 and RBE was selected as the research object. Using the lentivirus as the carrier, the SH RNA-B7-H4 sequence was constructed and the expression level of B7-H4 in two cells was downregulated. Then the cytotoxic T lymphocyte was co cultured to observe the changes of cytotoxic T lymphoblastic cells' killing effect on the cholangiocarcinoma cells. We also detected the changes in the level of cytokine secretion related to the supernatant of the culture medium after co culture..5. selected bile duct cancer cell lines QBC939 and RBE as the research object, using the lentivirus as the carrier, constructing the SH RNA-B7-H4 sequence, down the expression level of B7-H4 in two cells, and then observing the damage repair of the two bile duct cancer cells. The effect of B7-H4 expression on the invasion and migration of cholangiocarcinoma cells was observed by Transwell. Three. Results 1. in 110 cases of cholangiocarcinoma, 54 cases were positive with B7-H4 staining, the positive rate was 49%; 8 cases of bile duct adenoma were all negative in B7-H4 staining; 19 cases of chronic cholangitis, 4 cases were B7-H4 dyed Yang The positive rate of 21.1%. showed that the positive rate of B7-H4 expression in tumor tissues was significantly higher than that of non tumor tissues (p=0.023), and the expression of B7-H4 in cholangiocarcinoma tissues was abnormal. The correlation between the expression of B7-H4 and the clinicopathological features of the patients was found to be related to the expression of B7-H4 with tumor staging, classification and lymphatic metastasis. The results suggested that the abnormal expression of B7-H4 may be associated with the malignant degree and invasion and metastasis of the tumor in the interstitial tissue of the 110 patients with cholangiocarcinoma, 97 cases of mild infiltration of CD4 positive T lymphocytes, 13 cases of severe dipping, 56 cases of mild infiltration of CD8 positive T lymphocytes, and severe immersion. In 54 cases, 110 cases of cholangiocarcinoma parenchyma tissue, CD4 positive T lymphocyte mild infiltration in 88 cases, severe infiltration in 22 cases, CD8 positive T lymphocyte infiltration in 76 cases and severe infiltration 34 cases. The expression level of B7-H4 in bile duct carcinoma tissues is related to the degree of CD8 positive T lymphocyte in the infiltration of tumor stromal cells. The results of this part suggest that: B7-H4, as a negative immunomodulating molecule, may play an important role in promoting the immune escape of cholangiocarcinoma cells by reducing the degree of CD8 positive T lymphocyte infiltration in the tumor cells, and.3. through the analysis of.Kaplan Meier model for the survival data of 110 cholangiocarcinoma patients: the B7-H4 positive group patients. The survival time was significantly lower than that of the B7-H4 negative group (p=0.015). We found that the recurrence time of the B7-H4 positive group was significantly lower than that of the B7-H4 negative group (p=0.045). In order to exclude the interference of the related confounding factors, we took the patient's age, sex, the tumor location, the degree of differentiation and the B7-H4 expression into the Cox model. The multifactor correlation analysis of the survival time and the recurrence time after operation showed that B7-H4 positive expression was a relative independent risk factor for determining the survival time and the recurrence time of the patients (HR=1.786,95%CI=1.110-2.872, p=0.017; HR=2.062,95%CI=1.160-3.665, p= 0.014). The results of this part suggest that the positive expression of B7-H4 and the bile duct The poor prognosis of cancer patients is significantly related and can be used as an independent molecular marker for judging the prognosis of patients. We detected by Western-blot method, we found that two cholangiocarcinoma cell lines QBC939 and RBE all have B7-H4 protein expression. We used lentivirus to transfect sh RNA-B7-H4 into the cell line and successfully downregulate the level of B7-H4 expression; in the target target. When compared to (20:1 or 10:1), the killing ability of bile duct cancer cells with down regulation of B7-H4 expression level by specific CD8+CTL was significantly higher than that of the control group. The secretion level of TGF- beta and IL-6 cytokines was significantly lower than that of the control group after the mixed culture of lymphocyte and B7-H4 expression level of bile duct cancer cell lines. The results suggest that B7-H4 has a good effect. By enhancing the level of cytokine TGF- beta, IL-6 secretion, inducing CD8 positive T to differentiate into no cytotoxic Th17, thus reducing the killing ability of CD8+CTL to tumor cells.5.,.5. using lentivirus transfection sh RNA-B7-H4 into the cell line, and successfully downregulating the expression level of B7-H4; by observing injury repair experiment, Transwell invasion experiment found that The expression of B7-H4 protein in the cholangiocarcinoma cells can obviously inhibit the invasion and migration of cancer cells. The results suggest that B7-H4 obviously promotes the invasion and metastasis of cholangiocarcinoma. Four. Conclusion there is abnormal expression of B7-H4 in cholangiocarcinoma. The high expression suggests that the prognosis of the patients is poor. The reason may be that B7-H4 may promote the swelling by promoting the swelling. Tumor invasion and migration, or by enhancing the level of TGF- beta and IL-6 secretion, reduce the cytotoxic T lymphocyte killing effect on tumor cells.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.8

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