本文選題：宮頸癌 + B7-H3　； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：研究背景宮頸癌是全球范圍內(nèi)發(fā)病率較高的婦科惡性腫瘤之一,嚴(yán)重威脅女性身心健康。據(jù)報道,80%的宮頸癌發(fā)生在發(fā)展中國家,而高危型人乳頭瘤病毒(human papillomavirus,HPV)持續(xù)感染是宮頸癌發(fā)生的主要危險因素。然而,并非所有高危型HPV感染者都發(fā)展為宮頸癌,只有少數(shù)持續(xù)感染者發(fā)展為宮頸上皮內(nèi)瘤變(cervical intraepithelial neoplasia,CIN),進(jìn)而發(fā)展為宮頸癌。人免疫缺陷病毒(Human Immunodeficiency Virus,HIV)感染者或長期應(yīng)用免疫抑制劑的女性發(fā)生CIN或?qū)m頸癌的概率顯著增加。這提示宮頸癌的發(fā)生不僅與HPV持續(xù)感染有關(guān),還與機(jī)體的免疫因素有關(guān)。B7家族屬于免疫共刺激分子,可提供T細(xì)胞免疫應(yīng)答的相關(guān)刺激信號,也可提供限制和減弱T細(xì)胞免疫反應(yīng)的相關(guān)抑制信號,在腫瘤疾病、器官移植和自身免疫病中發(fā)揮重要作用。B7家族已知成員包括B7-1(CD80),B7-2(CD86),B7-H1(PD-L1),B7-H2(ICOSL),B7-DC(PD-L2),B7-H3(CD276),B7-H4(B7X)。B7-H3是于2001年被克隆的B7家族成員,目前發(fā)現(xiàn)其有兩種存在形式:2Ig-B7-H3和4Ig-B7-H3。2Ig-B7-H3表達(dá)于鼠與人的細(xì)胞中,其有細(xì)胞外的IgV-IgC結(jié)構(gòu);4Ig-B7-H3僅表達(dá)于人細(xì)胞中,由串聯(lián)重復(fù)的IgV-IgC-IgV-IgC結(jié)構(gòu)組成。B7-H3廣泛表達(dá)于各種器官及細(xì)胞中,例如肺,乳腺,肝臟,胎盤,前列腺以及樹突細(xì)胞等。研究顯示,B7-H3異常高表達(dá)于多種癌細(xì)胞或組織中,包括前列腺癌、胰腺癌、乳腺癌、子宮內(nèi)膜癌、肝癌、結(jié)腸癌、骨肉瘤以及血液系統(tǒng)惡性疾病。B7-H3與B7-H1(PD-L1)有相似的分子結(jié)構(gòu),現(xiàn)已證實腫瘤細(xì)胞表達(dá)的B7-H1(PD-L1)與T細(xì)胞表面的PD-1結(jié)合,可以下調(diào)T細(xì)胞功能,從而促進(jìn)腫瘤細(xì)胞的免疫逃逸,目前針對PD-1/PD-L1(B7-H1)的腫瘤免疫治療藥物已應(yīng)用于黑色素瘤等的臨床治療。但是,B7-H3在腫瘤免疫方面的功能尚未明確,有文獻(xiàn)報道,B7-H3可下調(diào)T輔助1型介導(dǎo)的免疫反應(yīng),抑制CD4+T細(xì)胞活化,并抑制細(xì)胞因子的產(chǎn)生,因而可能發(fā)揮促進(jìn)癌細(xì)胞免疫逃逸的作用。除了可能在腫瘤免疫逃逸中發(fā)揮作用外,B7-H3還可能影響腫瘤的增殖、侵襲和轉(zhuǎn)移等生物學(xué)特征,并與部分腫瘤的預(yù)后不良相關(guān)。臨床腫瘤研究顯示,B7-H3分子在肺癌、大腸癌、胰腺癌等癌組織中存在高表達(dá),其高表達(dá)與腫瘤的擴(kuò)散或預(yù)后較差具有相關(guān)性。細(xì)胞學(xué)水平的研究顯示,B7-H3在胰腺癌、前列腺癌、黑色素瘤等腫瘤細(xì)胞系中的高表達(dá)可促進(jìn)腫瘤細(xì)胞的侵襲和轉(zhuǎn)移。B7-H3影響腫瘤細(xì)胞生物學(xué)特征的原因尚不明確。目前,B7-H3分子在宮頸癌中的表達(dá)及其作用尚缺乏研究。本研究擬探討B(tài)7-H3在宮頸癌中的表達(dá)情況及其與宮頸癌患者病情和預(yù)后的關(guān)系,并探討B(tài)7-H3在宮頸癌細(xì)胞增殖、侵襲及遷移中的作用。研究方法1.臨床病理研究:納入宮頸癌患者和正常對照者,通過免疫組織化學(xué)方法檢測B7-H3分子在宮頸癌患者組織標(biāo)本和正常宮頸組織標(biāo)本中的表達(dá)情況并進(jìn)行對比,分析其表達(dá)水平與患者病理改變、臨床分期和疾病預(yù)后的關(guān)系;2.B7-H3在血液中表達(dá)情況的研究:運(yùn)用ELISA檢測B7-H3在宮頸癌患者血液標(biāo)本和正常對照組患者血液標(biāo)本中的表達(dá)情況,并進(jìn)行對比;3.細(xì)胞學(xué)試驗:(1)構(gòu)建細(xì)胞系:構(gòu)建質(zhì)粒,轉(zhuǎn)染宮頸癌細(xì)胞系,上調(diào)或下調(diào)Caski、Siha和Hela宮頸癌細(xì)胞系中B7-H3分子表達(dá);運(yùn)用Western Blot驗證細(xì)胞系構(gòu)建是否成功;(2)探討B(tài)7-H3表達(dá)對宮頸癌細(xì)胞增殖和侵襲能力的影響:通過MTT技術(shù)檢測細(xì)胞增殖情況;通過Transwell體系檢測細(xì)胞侵襲及遷移情況。研究結(jié)果:1.B7-H3在宮頸癌組織中的表達(dá)顯著高于正常宮頸組織(p0.05),其表達(dá)強(qiáng)度與 TNM 分期相關(guān)(r=0.509,p0.001);2.B7-H3表達(dá)強(qiáng)陽性的患者5年生存率顯著低于B7-H3表達(dá)弱陽性或陰性的患者,Cox分析顯示B7-H3高表達(dá)是宮頸癌患者死亡的危險因素(HR=4.463,p=0.035);3.B7-H3在宮頸癌患者血液中的表達(dá)顯著高于對照組血液中的表達(dá)。4.Western Blot結(jié)果顯示:B7-H3表達(dá)上調(diào)及下調(diào)宮頸癌細(xì)胞系構(gòu)建成功。5.MTT結(jié)果顯示:B7-H3表達(dá)上調(diào)宮頸癌細(xì)胞增殖能力高于正常對照組及表達(dá)下調(diào)組(p0.05);B7-H3表達(dá)下調(diào)宮頸癌細(xì)胞增殖能力低于正常對照組及表達(dá)上調(diào)組(p0.05)。6.Transwell結(jié)果顯示:B7-H3表達(dá)上調(diào)宮頸癌細(xì)胞遷移及侵襲能力高于正常對照組(p0.05);B7-H3表達(dá)下調(diào)宮頸癌細(xì)胞遷移及侵襲能力低于正常對照組(p0.05)。研究結(jié)論:1.B7-H3在宮頸癌組織及血液中表達(dá)升高,其在宮頸癌組織的表達(dá)與患者臨床分期和預(yù)后相關(guān);2.B7-H3能夠增強(qiáng)宮頸癌細(xì)胞的增殖及遷移、侵襲能力,從而有望成為宮頸癌治療的潛在靶點。
[Abstract]:Background cervical cancer is one of the high incidence of gynecologic malignancies worldwide and is a serious threat to women's physical and mental health. It is reported that 80% of cervical cancer occurs in developing countries, and the persistent infection of high risk human papillomavirus (HPV) is the major risk factor for cervical cancer. However, it is not all high-risk type. HPV infected people develop to cervical cancer, and only a few persistent infections develop into cervical intraepithelial neoplasia (cervical intraepithelial neoplasia, CIN), and then to cervical cancer. The incidence of CIN or cervical cancer in women with human immunodeficiency virus (Human Immunodeficiency Virus, HIV) or for long-term application of anti epidemic inhibitors is significantly increased. This suggests that the occurrence of cervical cancer is not only related to the persistent infection of HPV, but also related to the immune factors of the body, the.B7 family belongs to the immuno stimulator, which can provide the related stimulation signals of the T cell immune response, and also provide the related restraining signals to restrict and weaken the immune response of the T cells, in the tumor, organ transplantation and autoimmune disease. The known members of the.B7 family, including B7-1 (CD80), B7-2 (CD86), B7-H1 (PD-L1), B7-H2 (ICOSL), B7-DC (PD-L2), are cloned in 2001, and are currently found to have two forms of existence: they are expressed in the cells of mice and humans, and they have an extracellular matrix. Structure; 4Ig-B7-H3 is expressed only in human cells and is expressed in a variety of organs and cells, such as lung, mammary gland, liver, placenta, prostate, and dendritic cells, such as lung, breast, liver, placenta, and dendritic cells, which are composed of a series of repeated IgV-IgC-IgV-IgC structures, such as the lung, the breast, the liver, the placenta, the prostate, and the dendritic cells. The study shows that the abnormal expression of the.B7-H3 is in a variety of cancer cells or tissues, including prostate, pancreatic, and breast cancer. Endometrial carcinoma, liver cancer, colon cancer, osteosarcoma and malignant hematological diseases.B7-H3 and B7-H1 (PD-L1) have similar molecular structures. It has been proved that the B7-H1 (PD-L1) expressed by tumor cells is combined with PD-1 on the surface of T cells, which can reduce the function of T cells and thus promote the immune escape of the tumor cells. At present, the tumor of PD-1/PD-L1 (B7-H1) is a tumor of PD-1/PD-L1 (B7-H1). Immunotherapy drugs have been used in the clinical treatment of melanoma. However, the function of B7-H3 in tumor immunity is not clear. It has been reported that B7-H3 can downregulate the immune response of T assisted type 1, inhibit the activation of CD4+T cells and inhibit the production of cytokines, thus can play a role in promoting cancer cell immune escape. B7-H3 can also play a role in tumor immune escape, and it may also affect the biological characteristics of tumor proliferation, invasion and metastasis, and is associated with poor prognosis of some tumors. Clinical tumor studies show that high expression of B7-H3 molecules in lung cancer, colorectal cancer, pancreatic cancer and other cancer tissues, and its high expression and tumor diffusion or poor prognosis The high expression of B7-H3 in pancreatic cancer, prostate cancer, melanoma and other tumor cell lines can promote the invasion of tumor cells and the transfer of.B7-H3 to the biological characteristics of tumor cells. At present, the expression and role of B7-H3 in cervical cancer are still lack of research. To investigate the expression of B7-H3 in cervical cancer and its relationship with the condition and prognosis of cervical cancer, and to explore the role of B7-H3 in the proliferation, invasion and migration of cervical cancer cells. Method 1. clinical and pathological studies: cervical cancer patients and normal controls were examined by immunohistochemical method to detect B7-H3 in cervical cancer patients. The expression of the tissue specimen and the normal cervical tissue specimens were compared, and the relationship between the expression level and the pathological changes of the patients, the relationship between the clinical stage and the prognosis of the disease; the study of the expression of 2.B7-H3 in the blood: the expression of B7-H3 in the blood specimens of the patients with cervical cancer and the blood specimens of the normal control group was detected by ELISA. 3. cytological tests: (1) construction of cell lines: Construction of plasmids, transfection of cervical cancer cell lines, up or down the expression of B7-H3 in Caski, Siha and Hela cervical cancer cell lines; using Western Blot to verify the success of cell line construction; (2) explore the effect of B7-H3 expression on the proliferation and invasion of cervical cancer cells: through MTT Technology The results of cell invasion and migration were detected by Transwell system. The results showed that the expression of 1.B7-H3 in cervical cancer tissues was significantly higher than that of normal cervical tissue (P0.05), and the expression intensity was associated with TNM staging (r=0.509, p0.001), and the 5 year survival rate of patients with strong positive expression in 2.B7-H3 was significantly lower than that of weak B7-H3 expression. Cox analysis showed that high expression of B7-H3 was a risk factor for the death of cervical cancer patients (HR=4.463, p=0.035), and the expression of 3.B7-H3 in the blood of cervical cancer patients was significantly higher than that in the control group. The result of.4.Western Blot in the blood of the control group showed that the result of the up regulation of B7-H3 expression and the downregulation of cervical cancer cell lines showed that the B7-H3 table was found. The proliferation ability of cervical cancer cells was higher than that of normal control group and down-regulation group (P0.05); B7-H3 expression down regulation of cervical cancer cells was lower than that of normal control group and up regulation group (P0.05).6.Transwell results showed that B7-H3 expression up regulation of cervical cancer cells migration and invasion ability was higher than normal control group (P0.05); B7-H3 expression was down regulated. The migration and invasion ability of cervical cancer cells is lower than that of the normal control group (P0.05). Conclusion: the expression of 1.B7-H3 in cervical cancer tissues and blood is elevated, the expression of the cervical cancer tissue is related to the clinical stage and prognosis of the patients. 2.B7-H3 can enhance the proliferation, migration and invasion of cervical cancer cells, thus it is expected to be the treatment of cervical cancer. Potential targets.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.33

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