本文選題：膽管癌 + B7-H4　； 參考：《第三軍醫(yī)大學(xué)》2015年博士論文

【摘要】：一、研究背景膽管癌作為一種常見的惡性腫瘤,發(fā)病率在我國消化系統(tǒng)腫瘤中排名第五,其發(fā)病率每年仍有增高的趨勢。流行病學(xué)調(diào)查顯示膽管癌患者發(fā)病年齡多為50-60歲,男性多于女性。膽管癌的發(fā)病率具有一定民族和區(qū)域分布差異,多發(fā)生亞洲,特別是東南亞以及中東國家,我國主要是華南以及東南沿海發(fā)病率較高。近些年,隨著環(huán)境不斷惡化以及臨床診斷技術(shù)的發(fā)展進(jìn)步,世界范圍內(nèi)膽管癌發(fā)病率的呈逐年上升趨勢。盡管膽管癌的發(fā)病率仍低于消化系統(tǒng)其他惡性腫瘤,但是患者的死亡率卻一直處于較高水平。膽管癌所處的特殊解剖部位以及高侵襲轉(zhuǎn)移能力,是造成高死亡率的主要原因。目前針對(duì)膽管癌的治療方法中,膽管癌根治性切除術(shù)是提高患者預(yù)后降低死亡率的主要手段。但是由于膽管癌起病隱匿、臨床表現(xiàn)不典型以及缺乏早期診斷標(biāo)志物,部分患者就診時(shí)腫瘤已經(jīng)進(jìn)入晚期,有些甚至已經(jīng)發(fā)生轉(zhuǎn)移,因而失去了手術(shù)切除腫瘤的最佳時(shí)機(jī)。部分膽管癌患者即使接受根治性手術(shù)切除,腫瘤復(fù)發(fā)以及轉(zhuǎn)移的風(fēng)險(xiǎn)仍然偏高。以上這些因素嚴(yán)重影響了膽管癌根治性切除術(shù)的遠(yuǎn)期療效。近年來,針對(duì)膽管癌發(fā)生、發(fā)展的相關(guān)分子機(jī)制研究已經(jīng)取得一定的進(jìn)展,但是涉及到膽管癌的免疫逃逸以及侵襲轉(zhuǎn)移的相關(guān)研究還不是很完善。所以我們把研究的重點(diǎn)放在了膽管癌的腫瘤微環(huán)境,特別是免疫逃逸以及侵襲轉(zhuǎn)移的相關(guān)研究上。通過相關(guān)內(nèi)容的研究,我們嘗試發(fā)現(xiàn)新的分子以及相關(guān)通路作為診斷和治療靶點(diǎn),并且能夠?yàn)閯?chuàng)新臨床診斷和治療的方法提供理論依據(jù)。T細(xì)胞免疫作為腫瘤微環(huán)境中抗腫瘤免疫的重要組成部分,在腫瘤的發(fā)生、發(fā)展中起到十分關(guān)鍵的作用。其中T細(xì)胞的活化和增殖需兩種信號(hào)途徑。第一條信號(hào)途徑是抗原遞呈細(xì)胞通過呈遞抗原肽并且與T細(xì)胞受體識(shí)別,傳遞活化信號(hào)。第二條信號(hào)途徑是通過協(xié)同刺激信號(hào)分子活化T細(xì)胞。協(xié)同刺激分子決定了T細(xì)胞是被激活并且增殖還是轉(zhuǎn)為抑制或者無應(yīng)答狀態(tài)。目前所知的協(xié)同刺激分子主要分為三類:腫瘤壞死因子家族、細(xì)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家族作為重要的協(xié)同刺激分子,它們不僅能起到促進(jìn)T細(xì)胞增殖活化以及產(chǎn)生細(xì)胞因子的作用,同時(shí)也能起到抑制、限制或者減弱T細(xì)胞反應(yīng)的作用。B7-H4作為協(xié)同刺激分子B7家族中的成員,主要提供負(fù)性調(diào)節(jié)信號(hào),通過抑制T細(xì)胞增殖、分泌相關(guān)細(xì)胞因子以及降低T細(xì)胞殺傷腫瘤細(xì)胞能力,從而促進(jìn)腫瘤細(xì)胞逃過宿主免疫監(jiān)視進(jìn)而發(fā)生免疫逃逸現(xiàn)象;同時(shí),B7-H4在部分腫瘤中特異性表達(dá),還可以增強(qiáng)腫瘤細(xì)胞侵襲轉(zhuǎn)移能力,促進(jìn)腫瘤細(xì)胞惡性轉(zhuǎn)化。既往文獻(xiàn)報(bào)道,B7-H4在多種腫瘤中存在異常表達(dá),并且與腫瘤臨床病理指標(biāo)以及病人預(yù)后密切相關(guān)。由于B7-H4在協(xié)助腫瘤細(xì)胞逃避機(jī)體免疫監(jiān)視以及腫瘤發(fā)生發(fā)展中起到了十分重要的作用。根據(jù)既往研究成果以及前期研究結(jié)果,我們對(duì)B7-H4在膽管癌免疫逃逸中的作用和臨床意義進(jìn)行初步研究,并提出研究假設(shè):B7-H4在膽管癌患者中特異性表達(dá)增高,并且通過抑制腫瘤微環(huán)境中的免疫淋巴細(xì)胞,降低對(duì)腫瘤細(xì)胞的殺傷能力,從而促進(jìn)腫瘤細(xì)胞發(fā)生免疫逃逸。同時(shí)B7-H4還可促進(jìn)膽管癌細(xì)胞發(fā)生惡性轉(zhuǎn)化,增強(qiáng)癌細(xì)胞自身侵襲轉(zhuǎn)移能力。二、研究方法為了驗(yàn)證上述假設(shè),首先,我們通過免疫組化檢測統(tǒng)計(jì)B7-H4在膽管癌病人中的表達(dá)情況,分析其表達(dá)水平與臨床病理特征以及患者預(yù)后的關(guān)系,并分析腫瘤組織和非腫瘤組織中B7-H4的差異表達(dá)的情況;其次,我們通過免疫組化分別統(tǒng)計(jì)腫瘤實(shí)質(zhì)和間質(zhì)中CD4,CD8陽性T淋巴細(xì)胞浸潤情況與B7-H4陽性表達(dá)之間的聯(lián)系,分析B7-H4是否抑制T淋巴細(xì)胞在腫瘤微環(huán)境中浸潤的情況;再次,我們在體外試驗(yàn)中干擾B7-H4在腫瘤細(xì)胞中的表達(dá),分析B7-H4表達(dá)水平的改變對(duì)細(xì)胞毒性淋巴細(xì)胞的殺傷作用和相關(guān)細(xì)胞因子分泌情況的影響。最后,通過下調(diào)B7-H4在膽管癌細(xì)胞中表達(dá)水平,觀察膽管癌細(xì)胞侵襲和遷移能力的變化情況。本項(xiàng)研究目的在于探討B(tài)7-H4在膽管癌中的表達(dá)情況以及相關(guān)臨床意義,以及它如何促進(jìn)膽管癌細(xì)胞在腫瘤微環(huán)境中發(fā)生免疫逃逸以及惡性轉(zhuǎn)化,尤其是增強(qiáng)癌細(xì)胞侵襲遷移能力的可能機(jī)制,開拓膽管癌發(fā)生腫瘤免疫逃逸以及侵襲轉(zhuǎn)移的研究思路,為發(fā)現(xiàn)新的膽管癌預(yù)后指標(biāo)以及新的免疫治療靶點(diǎn)提供科學(xué)研究依據(jù)。1.收集從2005年至2012年第三軍醫(yī)大學(xué)西南醫(yī)院肝膽外科研究所進(jìn)行根治性切除并經(jīng)病理診斷明確為膽管癌的病例。上述收集的病例均未進(jìn)行放療或者化療。同時(shí)收集19例慢性膽管炎、8例膽管腺瘤標(biāo)本和18例膽管癌淋巴結(jié)轉(zhuǎn)移組織標(biāo)本。收集的臨床資料包括病人性別、年齡、腫瘤位置、大小、分化程度、分期分級(jí)、淋巴結(jié)轉(zhuǎn)移以及遠(yuǎn)處轉(zhuǎn)移的情況。采用免疫組化的方法檢測B7-H4在各種組織中的表達(dá)情況,并對(duì)比腫瘤組織和非腫瘤組織中B7-H4的表達(dá)差異;同時(shí)分析B7-H4與患者臨床病理特征的相關(guān)性。2.收集110例膽管癌患者腫瘤組織,進(jìn)行免疫組化染色,檢測統(tǒng)計(jì)CD4、CD8陽性T淋巴細(xì)胞分別在腫瘤實(shí)質(zhì)以及腫瘤間質(zhì)組織中的浸潤表達(dá)情況,并結(jié)合對(duì)應(yīng)患者的B7-H4表達(dá)情況,分析B7-H4與CD4、CD8陽性T淋巴細(xì)胞表達(dá)情況的相關(guān)性。3.收集110例膽管癌患者臨床隨訪資料(包括患者術(shù)后總存活時(shí)間和無瘤生存時(shí)間)�？偵鏁r(shí)間的計(jì)算方式是從病人手術(shù)時(shí)間到病人死亡時(shí)間或者最后一次聯(lián)系隨訪時(shí)間;無瘤生存時(shí)間的計(jì)算方式是從病人手術(shù)時(shí)間到發(fā)現(xiàn)腫瘤復(fù)發(fā)的時(shí)間。腫瘤的復(fù)發(fā)主要依靠影像學(xué)以及病理診斷為主。單因素生存分析采用KM法(KM method),多因素分析采用Cox多因素逐步回歸(向前,極大似然法)。分析B7-H4在腫瘤組織中的表達(dá)與患者預(yù)后之間的關(guān)系。4.選擇膽管癌細(xì)胞株QBC939和RBE作為研究對(duì)象,利用慢病毒作為載體,構(gòu)建sh RNA-B7-H4序列,下調(diào)兩株細(xì)胞中B7-H4的表達(dá)水平,然后將其與細(xì)胞毒性T淋巴細(xì)胞共培養(yǎng),觀察細(xì)胞毒性T淋巴細(xì)胞對(duì)膽管癌細(xì)胞殺傷作用的變化情況。同時(shí)我們還檢測共培養(yǎng)后培養(yǎng)基上清液相關(guān)細(xì)胞因子分泌水平的變化情況。5.選擇膽管癌細(xì)胞株QBC939和RBE作為研究對(duì)象,利用慢病毒作為載體,構(gòu)建sh RNA-B7-H4序列,下調(diào)兩株細(xì)胞中B7-H4的表達(dá)水平,然后通過觀察兩株膽管癌細(xì)胞的損傷修復(fù)實(shí)驗(yàn)、Transwell侵襲實(shí)驗(yàn),觀察B7-H4表達(dá)對(duì)膽管癌細(xì)胞侵襲和遷移能力的影響。三、研究結(jié)果1.在110例患者的膽管癌組織中,54例呈B7-H4染色陽性,陽性率為49%;8例膽管腺瘤組織中,全部呈B7-H4染色陰性;19例慢性膽管炎組織中,4例呈B7-H4染色陽性,陽性率為21.1%。本部分結(jié)果提示:B7-H4在腫瘤組織中表達(dá)的陽性率明顯高于非腫瘤組織(p=0.023),B7-H4在膽管癌組織中表達(dá)異常增高。通過分析B7-H4表達(dá)情況和患者臨床病理特征的相關(guān)性發(fā)現(xiàn):B7-H4的表達(dá)與腫瘤分期,分級(jí)和淋巴轉(zhuǎn)移有關(guān);而與性別、年齡、部位,遠(yuǎn)處轉(zhuǎn)移無關(guān)。本部分結(jié)果提示:B7-H4的異常表達(dá)可能與腫瘤惡性程度以及侵襲轉(zhuǎn)移相關(guān)。2.在110例患者的膽管癌腫瘤間質(zhì)組織中,CD4陽性T淋巴細(xì)胞輕度浸潤97例,重度浸潤13例;CD8陽性T淋巴細(xì)胞輕度浸潤56例,重度浸潤54例。在110例患者的膽管癌腫瘤實(shí)質(zhì)組織中,CD4陽性T淋巴細(xì)胞輕度浸潤88例,重度浸潤22例;CD8陽性T淋巴細(xì)胞輕度浸潤76例,重度浸潤34例。膽管癌組織中B7-H4的表達(dá)水平與CD8陽性T淋巴細(xì)胞在腫瘤間質(zhì)細(xì)胞浸潤程度有關(guān)。本部分結(jié)果提示:B7-H4分子作為一個(gè)負(fù)性免疫調(diào)控分子,可能通過降低CD8陽性T淋巴細(xì)胞在腫瘤間質(zhì)細(xì)胞浸潤的程度,從而在促進(jìn)膽管癌細(xì)胞發(fā)生免疫逃逸中起到重要作用。3.通過對(duì)110膽管癌患者生存數(shù)據(jù)的統(tǒng)計(jì)。Kaplan—Meier模型分析提示:B7-H4陽性組患者生存時(shí)間明顯低于B7-H4陰性組患者(p=0.015)。我們按照相同方法發(fā)現(xiàn):B7-H4陽性組患者復(fù)發(fā)時(shí)間明顯低于B7-H4陰性組患者(p=0.045)。為了排除相關(guān)混雜因素的干擾,我們將患者年齡、性別、腫瘤部位、分化程度以及B7-H4表達(dá)情況帶入Cox模型中進(jìn)行術(shù)后存活時(shí)間以及復(fù)發(fā)時(shí)間的多因素相關(guān)性分析。結(jié)果顯示:B7-H4陽性表達(dá)是判定患者術(shù)后存活時(shí)間和復(fù)發(fā)時(shí)間的一個(gè)相對(duì)獨(dú)立危險(xiǎn)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)。本部分結(jié)果提示:B7-H4陽性表達(dá)與膽管癌患者預(yù)后較差顯著相關(guān)而且可以作為判斷患者預(yù)后的獨(dú)立分子標(biāo)志物。4.我們通過Western-blot方法檢測,發(fā)現(xiàn):兩株膽管癌細(xì)胞株QBC939和RBE均有B7-H4蛋白的表達(dá)。我們采用慢病毒轉(zhuǎn)染sh RNA-B7-H4進(jìn)入細(xì)胞株,成功下調(diào)B7-H4表達(dá)水平;在效靶比為(20:1或者10:1)時(shí),特異性CD8+CTL對(duì)B7-H4表達(dá)水平下調(diào)的膽管癌細(xì)胞殺傷能力明顯高于對(duì)照組的殺傷能力。淋巴細(xì)胞和B7-H4表達(dá)水平下調(diào)的膽管癌細(xì)胞株混合培養(yǎng)后,TGF-β,IL-6細(xì)胞因子分泌水平明顯低于對(duì)照組。本部分結(jié)果提示B7-H4有可通過能增強(qiáng)細(xì)胞因子TGF-β,IL-6的分泌水平,誘導(dǎo)CD8陽性T分化為不具有細(xì)胞毒性Th17,從而降低CD8+CTL對(duì)腫瘤細(xì)胞的殺傷能力。5.采用慢病毒轉(zhuǎn)染sh RNA-B7-H4進(jìn)入細(xì)胞株,成功下調(diào)B7-H4表達(dá)水平;通過觀察損傷修復(fù)實(shí)驗(yàn)、Transwell侵襲實(shí)驗(yàn)發(fā)現(xiàn)下調(diào)膽管癌細(xì)胞中B7-H4蛋白表達(dá)水平能明顯抑其癌細(xì)胞侵襲和遷移能力。本部分結(jié)果提示:B7-H4明顯促進(jìn)了膽管癌的侵襲和轉(zhuǎn)移。四、研究結(jié)論膽管癌中存在B7-H4異常表達(dá),其高表達(dá)提示患者預(yù)后不良。分析原因可能是B7-H4有可能通過促進(jìn)腫瘤侵襲遷移,或者通過增強(qiáng)TGF-β,IL-6分泌水平,降低細(xì)胞毒性T淋巴細(xì)胞對(duì)腫瘤細(xì)胞的殺傷作用,從而促進(jìn)膽管癌細(xì)胞發(fā)生免疫逃逸,進(jìn)而影響患者預(yù)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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.8

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本文編號(hào)：1914650