【作者】 仝昕；

【导师】 郭亚军； 赵健； 戴建新； 王皓； 李博华； 寇庚；

【作者基本信息】 第二军医大学 ， 肿瘤学， 2008， 硕士

【摘要】 研究背景:肺癌是当今世界上发病率增长最快和死亡率最高的恶性肿瘤。然而由于肺癌发病隐匿、恶性程度高、发展迅速,80%的肺癌患者就诊时已处于进展期。目前,仅有20%-30%的患者得到早期诊断。尽管采用手术、放疗和化疗等综合治疗措施,由于肿瘤转移和多药耐药,总的5年生存率仍低于15%。因此,深入研究肺癌的转移机制,寻找更加有效的诊断和治疗方法是肺癌研究中迫切需要解决的重要课题。大量研究表明,肺癌的发生、发展和转移是一个多因素、多步骤和多基因参与的复杂过程,这期间基因突变、原癌基因激活、抑癌基因失活及其导致一系列信号转导的异常等分子病理学改变贯穿了肺癌病变的全过程。筛选出高灵敏性,高特异性的肿瘤标记物,从分子水平辅助肺癌的早期诊断,监测肺癌的发生发展及预后,进行靶向性肿瘤生物治疗,具有重要的临床应用价值。TIP30,又称CC3,是一种新发现的肿瘤抑制基因,最早是通过RNA差异显示技术比较高转移性小细胞肺癌(v-SCLC)和低转移性小细胞肺癌(c-SCLC)mRNA时发现的。近年来的研究表明,TIP30在多种正常组织中表达,在黑色素瘤、成神经细胞瘤、恶性胶质细胞瘤、乳腺癌、结肠癌和肝细胞癌等多种肿瘤中表达下调。早期的研究发现TIP30具有抑制肿瘤细胞增殖,抑制肿瘤血管生成,促进肿瘤细胞凋亡等作用。TIP30基因的突变体具有了癌基因的功能,并且TIP30敲除老鼠可自发生成多种肿瘤,并导致乳腺导管细胞的癌前转化。早期的研究表明,TIP30缺失的小细胞肺癌细胞株比表达TIP30的细胞对死亡信号(无生长因子、放化疗药物等)的耐受能力更强,并且将TIP30基因转入高转移性小细胞肺癌细胞株(TIP30表达缺失)明显地抑制了细胞的增殖能力,增加了细胞对死亡刺激信号的敏感性,并抑制肿瘤细胞在裸鼠体内的转移,证明TIP30在小细胞肺癌的发生、发展和转移中发挥重要作用。但是,TIP30在肺癌组织中的表达及其与临床指标的相关性尚未见报道。我们成功制备了高纯度高特异性TIP30多克隆抗体,利用组织芯片结合免疫组化技术检测206例肺癌及癌旁组织和部分转移灶中TIP30的表达,分析TIP30表达与多项临床指标的相关性及其与患者预后的关系,并对其生物学功能在肺癌细胞株中进行了进一步验证。研究方法及结果:1.重组人TIP30基因克隆、蛋白表达、纯化及多克隆抗体的制备:成功克隆人TIP30基因,用含GST(glutathione S-transferase)标签的pGEX-4T-2载体,构建重组pGEX-TIP30表达质粒,转化大肠杆菌BL21(DE3)。用异丙基β-D-硫代半乳糖苷(isopropyl-beta-D-thiogalactopyranaside,IPTG)诱导蛋白表达,并以Glutathione-Sepharose 4B纯化GST-TIP30融合蛋白,免疫新西兰白兔,制备高滴度高特异性抗TIP30多克隆抗体。2.组织芯片技术结合免疫组化检测TIP30在肺癌组织中的表达及其与临床指标的关系:利用Envision法检测TIP30在206例肺癌及相应癌旁组织中的表达情况,按照免疫组化评分,将TIP30在肺癌组织中的表达分为高表达组和低表达组。TIP30蛋白呈棕黄色颗粒分布于细胞浆,未见胞核着色。正常肺癌组织均有着色,主要分布于支气管上皮和肺泡上皮。在非小细胞肺癌中TIP30低表达组占36.5%(72/197);在9例小细胞肺癌中,TIP30均为低表达。在鳞状细胞癌,腺癌,腺鳞癌中的低表达率分别为36.7%(33/90),36.2%(34/94)和38.5%(5/13),组织类型间TIP30表达无显著性差异。比较70例病人原发灶和相应的淋巴转移灶中TIP30表达变化,TIP30在转移灶中表达水平明显下降(P＜0.001)。在肺鳞癌中,高分化及Ⅰ-Ⅱ期癌组织中TIP30的表达高,低分化及Ⅲ-Ⅳ期肺癌组织中其表达降低(P=0.005/P=0.012)。在鳞癌和腺癌中TIP30和p53表达呈负相关(P＜0.001/P=0.01)。对其中2001-2002年74例患者随访,TIP30和病人预后呈正相关(腺癌P=0.0112/鳞癌P=0.0306)。3.TIP30对NSCLC细胞增殖和转移潜能的调节作用:运用实时定量PCR和Western blotting蛋白印迹检测五种肺癌细胞(A549,NCI-H460,SK-MES-1,LTEP-a-2,H1299)内源性TIP30基因的表达情况,选取TIP30高表达细胞株A549和TIP30低表达细胞株H1299。利用慢病毒载体干扰A549内源TIP30表达或将TIP30导入H1299肿瘤细胞,和对照组相比,下调内源性TIP30表达后A549细胞的增殖能力,软琼脂克隆形成能力,对无血清刺激耐受能力及迁移和侵袭能力显著增强;而转染TIP30后,H1299细胞的增殖减慢,软琼脂克隆形成数目少,细胞迁移和侵袭能力显著下降。结论:1.成功制备高纯度高特异性抗人TIP30多克隆抗体,为进一步研究TIP30功能奠定基础;2.约36%的非小细胞肺癌组织中TIP30表达水平下降,TIP30和p53表达呈负相关,TIP30表达水平和鳞癌的临床分期和分化相关,表明TIP30表达失调参与非小细胞肺癌的发生和发展;3.TIP30的表达水平和患者的存活时间密切相关,检测TIP30表达可能成为一项重要的预后判断指标;4.TIP30的表达强度从正常组织,原发性癌组织和转移灶中依次下降,体外实验证明TIP30负向调节肿瘤细胞的迁移和侵袭能力,表明TIP30是一种重要的肿瘤转移抑制基因。更多还原

【Abstract】 BackgroudLung cancer is one of the leading causes of death worldwide with increased incidence in China.The overall survival of patients with lung cancer remains poor because of the advanced stage in most patients at the time of diagnosis and lack of effective therapies. Although about 50%of lung cancer patients respond well to initial chemotherapy and external bean radiation therapy with regression of the primary tumor,the survival of lung cancer patients is dismal because of metastases and the development of resistance to chemotherapy.Transformation of a normal phenotype into a malignant phenotype requires accumulation of multiple genetic and epigenetic changes,which result in growth and/or cellular survival advantage.Loss of tumor suppressor genes is one of the critical alterations in this multi-step process.Therefore,identification of novel molecular biomarkers to distinguish tumor cells from normal cells and predict tumor behavior such as pathologic stage,response to chemotherapy and potential to metastasis,is of great importance for improving the outcomes of lung cancer patients.TIP30,also called CC3,is a tumor suppressor with pro-apoptotic and anti-metastasis properties.It was originally identified by a differential display analysis of messenger RNA from the highly metastatic human variant small cell lung cancer（v-SCLC） versus less metastatic classic small cell lung cancer（c-SCLC） cell lines.The expression of TIP30 has been detected in many human normal tissues and was down-regulated in certain tumor cells such as melanoma,breast cancer,neuroblastoma,glioblastoma,colon cancer,and hepatocellular carcinoma.TIP30 was considered to have tumor suppressor activity by inhibiting tumor growth,invasion,and angiogenesis and inducing apoptosis.TIP30 mutants not only abrogated the tumor suppressor potential but also gained oncogenic activities.The depletion of TIP30 predisposed mammary epithelial cells to neoplastic transformation and TIP30-deficient mice spontaneously developed various tumors.It has been reported that v-SCLC lacking endogenous TIP30 expression showed more resistance to death-inducing signals than c-SCLC with high TIP30 expression. Furthermore,over-expression of TIP30 in highly metastatic v-SCLC can significantly inhibited cell proliferation,sensitized cells to cytotoxic drugs,promoted apoptosis and inhibited metastasis. Taken together,these data demonstrated the importance of TIP30 in suppressing the progression and metastasis of SCLC.In this study,we generate the polyclonal antibody against human TIP30 protein with high-purity and high-specificity.The expression profile of TIP30 in clinical specimens is evaluated by immunohistochemistry in 206 lung carcinoma tissues using a high-density tissue microarray.We also assess the effects of TIP30 on tumor cell metastasis potential and proliferation in vitro.Results:1.The gene encoding TIP30 was amplified from the plasmid and inserted into the prokaryotic expression vector pGEX-4T-2 with tag GST（glutathione S-transferase）.The E.coli BL21（DE3） transformed with pGEX-TIP30 was induced by IPTG（isopropylbeta-D-thiogalactopyranaside） for expression of TIP30 fusion protein which was recovered in the supernatants and purified via glutathione-Sepharose 4B affinity column.The rabbits were subcutaneously immunized with purified protein and bled.The antibody purification was performed by using Protein A Sepharose^TM CL-4B.2.TIP30 expression was assessed by an immunohistochemic approach on 206 paired lung cancers and adjacent non-tumor tissues,as well as 70 lymph node metastases.The correlation of TIP30 expression with the clinicopathological features of the patients was analysed.In non-tumor lung tissues,TIP30 was preferentially expressed in the cytoplasm of bronchial epithelia cells and alveolar epithelia cells.TIP30 expression was found decreased in all the nine cases of SCLC and in 72 of 197（36.5%） cases of non-small cell lung cancer（NSCLC）,with 36.2%（34/94） of adenocarcinoma（AC）,36.7%（33/90） of squamous cell carcimona（SCC）,and 38.5%（5/13） of adenosquamous carcinoma. Decreased TIP30 expression was correlated with the poor differentiation of tumor and the advanced stage of disease in squamous cell carcinoma（SCC）.TIP30 expression in the metastatic lesion was decreased in comparison with that in the primary lung lesions in 22 out of 70（31.4%） NSCLC.There was an inverse association between TIP30 expression and abnormal p53 accumulation in lung cancer（P＜0.001）.Importantly,the survival rate of patients who had high TIP30 expression in primary tumor was significantly higher than that of patients with low TIP30 expression in AC（log-rank test,P=0.0112）,and in SCC （log-rank test,P=0.0306）.3.The expression of TIP30 mRNA and protein in five lung cancer cell lines were analysed. The expression of TIP30 was knockdowned in A549 which contains high level of endogenous TIP30 or introduced TIP30 gene into H1299 which contains low level of endogenous TIP30 by lentivirus.LV-shTIP30 significantly promoted cell growth of A549, inhibited its apoptosis,enhanced its motility and invasion through matrigel as compared with LV-None or mock treatment.However,introduction of TIP30 in H1299 cells dramatically retarded its growth and reduced its invasive and migration properties.Conclusions:1.Obtain the polyclonal antibody against TIP30 protein with high-purity and high-specificity which paves the way for future study.2.Decreased TIP30 expression is correlated with the poor differentiation of tumor and the advanced stage of disease in SCC and the abnormal p53 accumulation in NSCLC,which indicates the importance of down-regulation of TIP30 in the development and progression of non-small cell lung cancer.3.Down-regulation of TIP30 is statistically related to poor prognosis in patients with NSCLC.The determination of the immunohistochemical expression status of TIP30 might be a valuable prognosticator for patients with this disease.4.TIP30 expression in lung cancer shows a sequential decreased pattern from normal tissue,tumor tissue to metastatic carcinoma.The importance of TIP30 in suppression of metastasis is further proved by wound-healing and matrigel invasion assays in vitro.TIP30 serves as a tumor metastasis suppressor gene in lung cancer.更多还原