【作者】 赵华栋；

【导师】 马庆久； 药立波； 张健；

【作者基本信息】 第四军医大学 ， 外科学， 2008， 博士

【摘要】 甲状腺肿瘤是临床上的常见病和多发病。虽然甲状腺恶性肿瘤占人类所有癌的1%左右,但仍是内分泌系统最常见的恶性病变,严重威胁着人类的生命健康。除乳头状甲状腺癌经包括手术切除、内分泌治疗及放射性核素治疗在内的临床综合治疗后,可望获得较好的预后外,其他病理类型的甲状腺恶性肿瘤,预后仍然较差。而继续对甲状腺肿瘤进行深入研究,进一步取得对甲状腺恶性肿瘤发生、发展的分子机制的更深入的认识,包括寻找与其密切相关的新基因,仍显得十分必要。人NDRG2是我校生物化学与分子生物学教研室首先克隆并报道的一个新基因。对其进行深入研究结果表明,其在多种正常组织均有表达,而在结肠癌、胶质瘤、白血病、淋巴瘤和腮腺癌等10余种肿瘤组织或细胞系中无表达或低表达,是多种肿瘤及相应正常组织细胞的差异表达基因,因此推测该基因可能和肿瘤的发生、发展有关,被列为一新的抑癌候选基因。并且最近的研究结果证实,NDRG2的表达是受到Myc的调控。NDRG2与人甲状腺肿瘤的关系先前并无明确报道。NDRG2在人甲状腺肿瘤中有无表达及其表达模式如何,是首先需要明确的问题。本实验的第一部分就围绕着这一问题进行展开。我们首先运用组织芯片技术对NDRG2在人甲状腺肿瘤中的表达进行了免疫组织化学染色检测,结果发现,NDRG2在正常甲状腺组织中呈高表达,在甲状腺腺瘤组织中呈弱表达或中等强度表达,而在多种病理类型的甲状腺癌组织中呈不表达或极低表达。其次通过收集临床标本进行免疫组织化学染色和western印迹检测,得到了相似结果,这就说明NDRG2在蛋白表达水平上呈差异性表达。此发现也鼓励我们进一步对NDRG2在甲状腺肿瘤中的RNA表达水平进行了检测,通过RT-PCR、real-time PCR方法对收集来的临床肿瘤标本的检测,结果和上述实验结果一致。从而证明NDRG2在正常甲状腺组织及甲状腺癌组织中是一明显差异性表达性基因。为进一步了解NDRG2在甲状腺肿瘤发生、发展中所起的作用。我们运用NDRG2的过表达腺病毒载体,感染甲状腺滤泡状癌细胞株FTC-133从而实现NDRG2的过表达,通过RT-PCR和Western-blot检测感染成功后,对甲状腺癌细胞株进行了MTT、平板克隆形成检测及细胞周期测定,结果表明,过表达NDRG2可以明显抑制甲状腺癌细胞株的增殖。通过Trans-well和细胞划痕实验检测,结果显示,过表达NDRG2可以明显抑制甲状腺癌细胞株的侵袭能力。我们的实验结果证明了NDRG2在正常甲状腺组织及甲状腺癌组织中是一差异性表达性基因,过表达的NDRG2对甲状腺癌细胞株的增殖、侵袭能力呈现明显的抑制作用。由此推断,NDRG2可能参与了甲状腺肿瘤的发生、发展。丰富了对NDRG2和甲状腺肿瘤的认识,从而为以后对甲状腺肿瘤的深入研究奠定了实验基础。更多还原

【Abstract】 Thyroid cancer is one of the common and frequent encountered diseases. Although the occurrence of thyroid cancer constitutes only about 1%of the total human cancers,it is the most common malignant disease in endocrine system and threats human health seriously.Except for the relative better prognosis of the surgical operation,the combined treatment including endocrine hormone and radioactive isotope therapy to papillary thyroid carcinoma,the prognosis of other pathological types is still bad.So,it is very impending to deeply investigate the carcinogenesis mechanism of thyroid tumor.And the discovery and functional research of the thyroid cancer related genes are very intriguing.Human NDRG2(N-Myc downstream-regulated gene) is the novel gene cloned by the department of biochemsitry and molecular chemistry of our university.The primary research work about NDRG2 shows that Ndrg2 expresses ubiquitously in almost all normal tissues.However,it expresses very low or undetectable in nearly 10 tumor tissues or cell lines including colon cancers, glioma,leukemia,lymphoma and parotid cancers etc.It is presumptive that human NDRG2 is close to tumor progression and we believe NDRG2 is one novel candidate of tumor supressor gene.Especially,it is confirmed that NDRG2 is one of the downstream regulated target gene of Myc,the famous oncogene.Untill now,there isn’t the research report about NDRG2 with thyroid cancer. So,firstly,we should elucidate the expression profiling of human NDRG2 in thyroid tumors.The first part of our work to answer this question.The preliminary detection of Ndrg2 expression in thyroid tumor is examined using the tissue microarray.With the immunostainning analysis,the conspicuous different expression pattern of Ndrg2 was detected between thyroid carcinoma and the normal tissues.We find that Ndrg2 expresses very abundant in the normal thyroid and lower or undetectable in much types of thyroid cancers statistically significant.Further,we get the similar results by immunochemistry and western blotting analysis in the collected thyroid cancer samples.Our results demonstrate that the protein level of Ndrg2 differentiate between the nornal and tumor thyroid tissues.Based on the detection of Ndrg2 protein expression in thyroid cancer tissues,real-time PCR was used to investigate NDRG2 mRNA level in thyroid tissue with carcinoma.Expectably,the expression of NDRG2 mRNA in thyroid cancer tissues is relatively low than the normal group,which is accordant with the protein level.Thus,we believe that human NDRG2 is the differentiated expression gene between normal thyroid tissues and tumors.To further explore the potential function of human NDRG2 during the progression of thyroid cancer,we overexpress NDRG2 in papillary thyroid cancer cells FTC-13 3 by the infection of NDRG2 adenovirus.RT-PCR and Western-blot methods are used to confirm the expression status MTT,flat clone formation and cell cycle analysis demonstrate that overexpression of NDRG2 could inhibit the proliferation of thyroid cancer cells significantly.Trans-well and scratch test show that NDRG2 overexpression also causes the decrease of cell metastasis ablity.Our present results demonstrate that NDRG2 is the differentiated expression gene between normal thyroid tissues and tumors.The overexpression of human NDRG2 can suppress the proliferation and metastasis of thyroid cancer cells.It is presumable that NDRG2 participates the carcinogenesis and development of thyroid tumor.The data enriches the work of human NDRG2 with thyroid cancer and lay the foundation for the research of thyroid tumor.更多还原