【作者】 张艳红；

【导师】 王桂琴；

【作者基本信息】 山西医科大学 ， 免疫学， 2011， 硕士

【摘要】 背景近年甲状腺癌(thyroid carcinoma, TC)在全球发病率逐年上升,每年增长约4%,成为备受关注的肿瘤之一,也是内分泌系统肿瘤领域的研究热点。多年来,常规放化疗在传统“手术+碘131+甲状腺素”方案治疗无效的甲状腺癌治疗中的作用显得十分有限,包括阿霉素(美国FDA批准)在内的单一或联合化疗方案治疗难治性甲状腺癌步履维艰。随着甲状腺癌分子靶向治疗时代的悄然而至,难治性甲状腺癌治疗模式的转变已经在不经意间拉开帷幕。自噬(Autophagy)被称为Ⅱ型程序性死亡,是细胞适应环境变化、防御病原微生物侵袭、维持内环境稳定、维持人类和其他真核生物健康的重要机制,越来越多的研究证明在多种人类肿瘤细胞中存在有自噬活性的改变。Beclin1基因也称BECN1基因,是哺乳动物参加自噬的双等位抑癌基因,位于人染色体17q21上,其杂合性缺失是细胞发生恶性转化的原因之一。很多研究表明,Beclin1通过参与自噬体的形成和调节自噬水平在肿瘤的发生、发展中发挥要作用。PTEN是迄今发现的第一个具有双特异磷酸酶活性的抑癌基因,也是人类肿瘤中最常发生突变的抑癌基因,位于人类染色体10q23.3。PTEN可抑制PI3K/AKT (PKB)通路,使细胞自噬开启,进而促进自噬体的形成而抑制肿瘤的发展。很多研究提示,Beclin1和PTEN可能共同参与调节恶性肿瘤细胞的自噬活性。但从Beclin1蛋白表达水平研究自噬在甲状腺癌的表达目前文献报道很少,PTEN在甲状腺癌的蛋白表达情况也鲜有报道,Beclin1和PTEN是否共同调节甲状腺癌细胞的自噬活性尚未见报道。目的本研究检测自噬相关基因Beclin1和PTEN在甲状腺癌、甲状腺良性病变、正常甲状腺组织的表达,分析两者间的相关性,探讨二者在甲状腺癌发生、发展中的作用以及相互间的关系；分析甲状腺癌组织Beclin1和PTEN的表达与手术病理分期、组织类型、细胞分化程度、淋巴结转移、包膜浸润等临床病理参数的相关性,揭示二者的表达在甲状腺癌中的临床意义。旨在为临床寻求新的有效地甲状腺癌治疗靶点和新的治疗策略提供依据。方法1.搜集选取2008年1月～2010年6月忻州市人民医院病理科存档蜡块标本,其中79例甲状腺癌和34例甲状腺良性病变。并取同期手术切除正常甲状腺组织15例作为正常组织对照。2.记录所有研究对象的肿瘤快大小、组织类型、手术病理分期、淋巴结转移、包膜浸润等临床病理记录；所有患者手术前均未做过任何化疗和放疗。3.采用免疫组化法测定甲状腺癌组织、甲状腺良性病变、正常甲状腺组织Beclinl和PTEN蛋白表达,并将其与临床病理参数进行相关性分析。4.所有数据采用SPSS13.0软件包分析。结果1.Beclin1蛋白的表达情况：Beclin1在正常甲状腺组织(80.00%)、甲状腺良性病变组织(58.82%)、甲状腺癌组织(34.18%)中表达逐渐下降,差异有显著性(χ2=13.67,p=0.001)。2.Beclin1表达与甲状腺癌临床病理参数间的关系：Beclin1表达强度与甲状腺癌的淋巴结转移有关(χ2=7.202,p=0.007),与年龄、性别、分化程度、肿瘤大小、手术病理分期及包膜侵犯程无关(χ2=0.084,p=0.773；χ2=0.046,p=0.831；χ2=0.402,p=0.940；χ2=1.241, p=0.265；χ2=0.018,p=0.894；χ2=2.011,p=0.156)。3.PTEN的蛋白表达情况：PTEN在正常甲状腺组织(86.67%)、甲状腺良性病变组织(64.71%)、甲状腺癌组织(32.91%中)表达逐渐下降,差异有显著性(χ2=31.75,p=0.000)。4.PTEN表达与甲状腺癌临床病理参数间的关系：PTEN与甲状腺癌的淋巴结转移、手术病理分期有关(χ2=15.39,p=0.000；χ2=5.462,p=0.012)；与年龄、性别、分化程度、肿瘤大小、包膜侵犯程度无关(χ2=0.342,p=0.555；χ2=0.012,p=0.914；χ2=1.247,p=0.742；χ2=0.200,p=0.655；χ2=0.059,p=0.808).5.Beclin1与PTEN表达的相关性：Beclin1与PTEN在79例甲状腺癌中的表达呈正相关关系(rs=0.5929,p=0.000)。结论1.从正常甲状腺组织、甲状腺良性病变组织到甲状腺癌组织,Beclin1蛋白表达以及PTEN蛋白表达均逐渐下降,提示自噬相关基因Beclin1和PTEN在甲状腺癌的发生、发展中可能起一定的作用。2.Beclin1蛋白表达阳性率与淋巴结转移有关,提示自噬活性的下调可能与甲状腺癌的预后有一定的关系。3.PTEN蛋白表达阳性率与手术病理分期、淋巴结转移有关,表明在甲状腺癌的发展过程PTEN蛋白表达随恶性程度升高而呈进行性下降。提示PTEN可能被视为甲状腺癌恶性程度的潜在标志物。4.Becin1、PTEN蛋白表达具有良好的相关性,提示Beclin1、PTEN可能相互作用,共同参与了甲状腺癌的发生、发展。更多还原

【Abstract】 Background:Recently the incidence of thyroid carcinoma rises every year all over the world, with its annual growth of about 4%, which has attracted particular attention to most people. Meanwhile, TC has become one of the hottest research fields in endocrine system. Over the past years, conventional chemoradiation has been used for the treatment of ineffective thyroid carcinoma with the traditional treatment method of "Surgery+Iodine 131+Thyroxin". But its effect appears quite limited, including the single or combined chemotherapy in the treatment of refractory thyroid cancer with the Adriamycin approved by the U.S. FDA. However, with the appearance of Molecular targeted therapy for TC, it has brought some changes to the treatment model for refractory thyroid carcinoma.Autophagy is called TypeⅡapotosis, which is the important mechanism for the cells adapting to the environment changes and defending the pathogenic microorganisms from invading. Moreover, it can maintain homeostasis and maintain the health of humans and other eukaryotes. More and more studies have shown that changes in autophagic activity in a variety of human tumor cells exist.Beclinl gene also can be called BECN1 gene, it is the double alleles tumor-suppressor gene which the mammals use in autophagy. It is located on human chromosome 17q21, and the loss of heterozygosity is one of the reasons for the cells’ deterioration and transformation. It has shown that it plays an important role in the occurrence and development of cancer by participating in the autophagy body’s formation and autophagy levels’ regulation.Till now PTEN is the first but also the most frequently mutated cancer suppressor gene with double specific phosphatase activites, which is located on human chromosome 10q23.3. PTEN can inhibit PI3K/PKB pathway and make the cell autophagy open so that it can promote the formation of autophagy and inhibit the development of tumor.In many researches, it is suggested that both the Beclinl and PTEN can be involved in the regulation of autophagic activity in malignant cells. By now there are few reports about the expression of autophagy protein level in TC, and the protein expression about PTEN in thyroid cancer has been reported rarely. Regarding to the regulation of thyroid cancer cells’s autophagy activity with both the Beclinl and PTEN are not mentioned before.Objects:In this present study, the writer not only examined both the Beclinl and PTEN’s expression in the thyroid cancer, the benign thyroid and the normal thyroid tissue, but also analyzed the correlation between each other. Besides, its effects and relationships during the occurrence and development of thyroid cancer were introduced. The writer also made an analysis of thyroid cancer tissue Beclinl and PTEN’s expression and relevancy of clinical parameters, such as clinical stages, tissue type, and grade of cell differentiation, lymph node metastasis, and capsular infiltration and so on. Finally, the writer revealed the clinical significance of Beclinl and PTEN in the thyroid cancer. It is hoped that this research can provide the basis for the search of the effective therapeutic target and for the new therapeutic approaches.Method:1.The writer selected pathology archived paraffin specimens from 2008 January to 2010 June in Xinzhou City People’s Hospital, in which all together 113 people are chosen,79 subjects with thyroid cancer and another 34 with benign thyroid. At the same time,15 people were picked out with surgical resection of thyroid tissue over the same period for comparison.2.The writer recorded the tumor size, tissue type, clinical stage, lymph node metastasis, capsular infiltration and other clinical parameters of each subject. One point should be stressed is that all subjects did not do any chemotherapy and radiotherapy before the surgery.3.Both the protein expression of Belinl and PTEN in thyroid cancer, benign thyroid and normal thyroid tissue were analyzed by Immunohistochemistry Method. And the correlation analysis with clinicapathological parameters was carried out.4.All the data was analyzed with the SPAA 13.0 packages.Result:1.Expression of Beclinl protein:Beclinl espression fell off from normal thyroid organization, thyroid benign lesionsto thyroid cancer tissue,(χ2=13.67, p=0.001).Beclinl negative rate was significantly lower in normal thyroid organization(80.00%) than in thyroid benign lesions(58.82%) and thyroid cancer tissue(34.18%),respectively.2.Relationship between Beclinl and cliicopathologic:Beclinl expression was associated with the lymphnode metastasis(χ2=7.202, p=0.007),but with age, gender differentiation degree, tumor size,pathologic, coated infringe(χ2=0.084, p=0.773;χ2=0.046, p=0.831;χ2=0.402, p=0.940;χ2=1.241, p=0.265;χ2=0.018, p=0.894;χ2=2.011, p=0.156).3.Expression of PTEN protein:PTEN espression fell off from normal thyroid organization, thyroid benign lesionsto thyroid cancer tissue,(χ2=31.75, p=0.000).PTEN negative rate was significantly lower in normal thyroid organization(86.67%) than in thyroid benign lesions(64.71%) and thyroid cancer tissue(32.91%),respectively.4.Relationship between pten and cliicopathologic:PTEN espression was associated with the lymphnode metastasis(χ2=15.39, p=0.000)and differentiation degree (χ2=5.462, p=0.012), but with age,gender,umor size,pathologic, coated infringe(χ2=0.342, p=0.555;χ2=0.012, p=0.914; χ2=1.247, p=0.742;χ2=0.200, p=0.655;χ2=0.059, p=0.808).5. Correlation between Beclinl and PTEN:Beclinl espression has a postive corrlation with PTEN in 79 specimens of thyroid cancer(rs=0.5929, p=0.000).Conclusion1.From the normal thyroid tissue, benign thyroid lesions to thyroid cancer, the protein expression of Belinl and PTEN gradually decreases, which suggests that autophagy-related genes Beclinl and PTEN may play a role in the occurrence and development of thyroid cancer.2.As shown, the positive rate of Beclinl protein has something to do with the lymph node metastatsis, which suggests that autophagy activity decline may have certain relation with the prognosis of thyroid cancer.3.The positive rate of PTEN protein is relevant to clinical stage, lymph node metastatsis, which suggests that PTEN expression protein progressively decreases with the increasing degree of malignancy during the development of thyroid cancer. It can be taken as the potential marker to show the degree of malignancy of thyroid cancer.4.The relativity of the Belinl and PTEN protein is quite good, which hints that the Belinl and PTEN may interact with each other and jointly participate in the thyroid cancer and development of papillary thyroid cancer.更多还原