B7-H3在宫颈癌发展中作用的研究

发布时间：2018-05-03 09:44

本文选题：宫颈癌 + B7-H3　；参考：《山东大学》2017年硕士论文

【摘要】：研究背景宫颈癌是全球范围内发病率较高的妇科恶性肿瘤之一,严重威胁女性身心健康。据报道,80%的宫颈癌发生在发展中国家,而高危型人乳头瘤病毒(human papillomavirus,HPV)持续感染是宫颈癌发生的主要危险因素。然而,并非所有高危型HPV感染者都发展为宫颈癌,只有少数持续感染者发展为宫颈上皮内瘤变(cervical intraepithelial neoplasia,CIN),进而发展为宫颈癌。人免疫缺陷病毒(Human Immunodeficiency Virus,HIV)感染者或长期应用免疫抑制剂的女性发生CIN或宫颈癌的概率显著增加。这提示宫颈癌的发生不仅与HPV持续感染有关,还与机体的免疫因素有关。B7家族属于免疫共刺激分子,可提供T细胞免疫应答的相关刺激信号,也可提供限制和减弱T细胞免疫反应的相关抑制信号,在肿瘤疾病、器官移植和自身免疫病中发挥重要作用。B7家族已知成员包括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家族成员,目前发现其有两种存在形式:2Ig-B7-H3和4Ig-B7-H3。2Ig-B7-H3表达于鼠与人的细胞中,其有细胞外的IgV-IgC结构;4Ig-B7-H3仅表达于人细胞中,由串联重复的IgV-IgC-IgV-IgC结构组成。B7-H3广泛表达于各种器官及细胞中,例如肺,乳腺,肝脏,胎盘,前列腺以及树突细胞等。研究显示,B7-H3异常高表达于多种癌细胞或组织中,包括前列腺癌、胰腺癌、乳腺癌、子宫内膜癌、肝癌、结肠癌、骨肉瘤以及血液系统恶性疾病。B7-H3与B7-H1(PD-L1)有相似的分子结构,现已证实肿瘤细胞表达的B7-H1(PD-L1)与T细胞表面的PD-1结合,可以下调T细胞功能,从而促进肿瘤细胞的免疫逃逸,目前针对PD-1/PD-L1(B7-H1)的肿瘤免疫治疗药物已应用于黑色素瘤等的临床治疗。但是,B7-H3在肿瘤免疫方面的功能尚未明确,有文献报道,B7-H3可下调T辅助1型介导的免疫反应,抑制CD4+T细胞活化,并抑制细胞因子的产生,因而可能发挥促进癌细胞免疫逃逸的作用。除了可能在肿瘤免疫逃逸中发挥作用外,B7-H3还可能影响肿瘤的增殖、侵袭和转移等生物学特征,并与部分肿瘤的预后不良相关。临床肿瘤研究显示,B7-H3分子在肺癌、大肠癌、胰腺癌等癌组织中存在高表达,其高表达与肿瘤的扩散或预后较差具有相关性。细胞学水平的研究显示,B7-H3在胰腺癌、前列腺癌、黑色素瘤等肿瘤细胞系中的高表达可促进肿瘤细胞的侵袭和转移。B7-H3影响肿瘤细胞生物学特征的原因尚不明确。目前,B7-H3分子在宫颈癌中的表达及其作用尚缺乏研究。本研究拟探讨B7-H3在宫颈癌中的表达情况及其与宫颈癌患者病情和预后的关系,并探讨B7-H3在宫颈癌细胞增殖、侵袭及迁移中的作用。研究方法1.临床病理研究:纳入宫颈癌患者和正常对照者,通过免疫组织化学方法检测B7-H3分子在宫颈癌患者组织标本和正常宫颈组织标本中的表达情况并进行对比,分析其表达水平与患者病理改变、临床分期和疾病预后的关系;2.B7-H3在血液中表达情况的研究:运用ELISA检测B7-H3在宫颈癌患者血液标本和正常对照组患者血液标本中的表达情况,并进行对比;3.细胞学试验:(1)构建细胞系:构建质粒,转染宫颈癌细胞系,上调或下调Caski、Siha和Hela宫颈癌细胞系中B7-H3分子表达;运用Western Blot验证细胞系构建是否成功;(2)探讨B7-H3表达对宫颈癌细胞增殖和侵袭能力的影响:通过MTT技术检测细胞增殖情况;通过Transwell体系检测细胞侵袭及迁移情况。研究结果:1.B7-H3在宫颈癌组织中的表达显著高于正常宫颈组织(p0.05),其表达强度与 TNM 分期相关(r=0.509,p0.001);2.B7-H3表达强阳性的患者5年生存率显著低于B7-H3表达弱阳性或阴性的患者,Cox分析显示B7-H3高表达是宫颈癌患者死亡的危险因素(HR=4.463,p=0.035);3.B7-H3在宫颈癌患者血液中的表达显著高于对照组血液中的表达。4.Western Blot结果显示:B7-H3表达上调及下调宫颈癌细胞系构建成功。5.MTT结果显示:B7-H3表达上调宫颈癌细胞增殖能力高于正常对照组及表达下调组(p0.05);B7-H3表达下调宫颈癌细胞增殖能力低于正常对照组及表达上调组(p0.05)。6.Transwell结果显示:B7-H3表达上调宫颈癌细胞迁移及侵袭能力高于正常对照组(p0.05);B7-H3表达下调宫颈癌细胞迁移及侵袭能力低于正常对照组(p0.05)。研究结论:1.B7-H3在宫颈癌组织及血液中表达升高,其在宫颈癌组织的表达与患者临床分期和预后相关;2.B7-H3能够增强宫颈癌细胞的增殖及迁移、侵袭能力,从而有望成为宫颈癌治疗的潜在靶点。
[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.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33

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