原发性甲状旁腺功能亢进症的临床资料分析及相应的分子生物学研究

【作者】 赵建国；

【导师】 廖泉；

【作者基本信息】 北京协和医学院 ， 普通外科学（专业学位）， 2014， 博士

【摘要】 研究背景原发性甲状旁腺功能亢进(primary hyperparathyroidism, PHPT)是由于甲状旁腺病变使甲状旁腺激素(parathyroid hormone, PTH)合成和分泌过多,造成血清PTH升高和钙磷代谢紊乱。随着医学的发展和筛查的推广,目前本病的发现率呈明显上升的趋势,尤其是被诊断为无症状PHPT病人也越来越多。但由于本病临床表现复杂多样,加上广大医务人员对本病的认识相对缺乏,致使对本病的发现率较低,误诊率偏高,一方面使患者遭受长期的病痛折磨,甚至最终使部分患者致残；另一方面也因长期就医,给患者造成很大的经济负担和医疗资源浪费。甲状旁腺癌是引起PHPT的重要原因之一,大多数甲状旁腺癌可引起严重的临床症状。然而,甲状旁腺癌的术前诊断非常困难,即使组织病理学手段也经常很难鉴别甲状旁腺癌与良性肿瘤,由此也给后期治疗带来很大困难和挑战。另外区别认识甲状旁腺癌术后容易局部复发和转移的恶性生物学行为仍然是治疗本病的一个关键,但是目前关于PHPT尤其是甲状旁腺癌的研究不多。本研究结合我院大量的病例和临床病理学及随访资料,在全面分析的基础上进行PHPT基因表达谱的分子生物学研究,争取通过本研究寻找用于可能有助于甲状旁腺癌诊断和鉴别诊断以及判断复发预后的相关基因。目的：1.分析北京协和医院外科诊治PHPT的临床病理资料,揭示不同年代和不同年龄段的PHPT病人之间临床病理的变化情况,并探讨不同病理类型PHPT之间的差异。2.通过基因表达谱筛选不同病理类型PHPT之间的差异基因,对差异基因进行分子生物学验证研究,寻找用于甲状旁腺癌临床诊断和鉴别诊断的相关基因。3.研究候选差异基因与甲状旁腺癌病人临床病理特点及复发预后之间的关系,为甲状旁腺癌的复发预后判断和寻找治疗靶点提供理论基础和依据。方法1.回顾性分析了1975年1月到2013年6月之间在中国医学科学院北京协和医院进行外科诊治的临床资料完整的490例PHPT病人,统计分析该疾病的人口性别特征、临床生化特点、诊断及外科治疗方面的情况,从这些方面对比不同年代和不同年龄段病例临床病理特点的变化情况；并进一步将不同病理分类的PHPT的病人做比较,分析有关临床生化指标的差异情况及原因。2.将组织标本分为正常甲状旁腺组,甲状旁腺增生组,甲状旁腺腺瘤组,不典型腺瘤组和甲状旁腺癌组,在此基础上进行基因表达谱筛查研究,进一步从基因功能和信号通路方面利用Go-analysis和Pathway-analysis进行分析,筛选出甲状旁腺良恶性肿瘤的差异基因,构建目标差异基因(参与显著性GO和显著性Pathway的基因)的信号转导网络图(Signal-Net),最后筛选出关键候选差异基因。将组织标本分为甲状旁腺癌组织和甲状旁腺良性肿瘤(由甲状旁腺腺瘤、增生和不典型腺瘤组成)两组,首先利用qRT-PCR方法验证已筛选出的候选差异基因的表达情况。再对甲状旁腺癌和甲状旁腺良性肿瘤组织切片进行候选差异基因免疫组化染色,利用免疫组化对候选基因进行验证。最后收集甲状旁腺癌和甲状旁腺良性肿瘤病人标本,提取组织标本蛋白,对候选差异基因进行Western-blot验证。3.选取甲状旁腺癌组织切片；利用免疫组化分析甲状旁腺癌标本中的候选差异基因表达变化。将候选差异基因的表达改变与甲状旁腺癌病人的临床病理学特征及随访情况进行统计学相关分析,确定候选基因表达改变与甲状旁腺癌的哪些临床和病理指标相关,进一步研究这些基因的表达与甲状旁腺癌复发及预后之间的意义。结果1.第一部分结果：(1)PHPT病例随着时间推移明显增加,尤其是从2010年后明显增多。(2)病例以女性为主,男女之比约为1：2.40。将>50岁和≤50岁的病例相比,两组的性别之间存在显著差异,>50岁女性病例数明显增加。(3)将2010年以前和以后两组病例的病程相比,2010年以后病程明显缩短。(4)将2010年以前和以后两组病例的临床表现相比,混合型的比例明显下降,肾型和无症状型的比例明显上升,各组间均有显著性差异。(5)入院时PTH水平与入院时血钙、ALP、肌酐及肿瘤最大直径呈明显正相关：血钙与ALP、血肌酐、肿瘤最大直径及术后血钙呈明显正相关。(6)将不同病理类型组之间的临床生化指标进行比较,结果显示癌组和其他组相比,年龄偏小,病程相对较长。在术前和术后血PTH、术前和术后血钙、血肌酐、肿瘤最大直径及肿瘤体积方面,各组之间存在显著性差异。癌组和其他组相比,血PTH、血钙,肿瘤大小和血肌酐的均值明显升高。2.第二部分结果：(1)通过对PHPT组织标本进行基因表达谱分析,Go analysis和Pathway-analysis,然后绘制了差异基因的Signal-net网络图,选取出4个核心差异基因,分别为CD24, HMOX1, VCAM1和KCNA3基因。(2) RT-PCR验证结果显示：CD24在甲状旁腺癌和甲状旁腺腺瘤组织中的表达水平高于其他组织,尤其是在甲状旁腺癌组织中更高。HMOX1在甲状旁腺癌和不典型甲状旁腺腺瘤组织中的表达水平高于其他组织,在甲状旁腺癌组表达水平更高。VCAM1在甲状旁腺癌和不典型甲状旁腺腺瘤中的表达水平高于其他组织,但在不典型甲状旁腺腺瘤中的表达水平更高。KCNA3在甲状旁腺癌组织的表达水平明显高于其他组织。(3)免疫组化验证结果显示：CD24在甲状旁腺癌中的表达明显高于其他组织,主要表达于细胞核,部分胞浆可见阳性染色。HMOX1在甲状旁腺癌中的表达较其他组织高,主要表达于细胞浆,胞核也有部分表达。VCAM1在甲状旁腺癌和增生组织中的表达均明显强于腺瘤和不典型腺瘤组织,主要表达于细胞浆,部分胞核可见阳性染色。KCNA3在甲状旁腺癌和不典型腺瘤组组织中的表达强于其他组织,主要表达于细胞核。(4) Western-blotting结果显示：CD24在不同组织中均有表达,在甲状旁腺癌和腺瘤组织中的表达水平明显高于其他组织。HMOX1在所有组织中表达均较弱,在甲状旁腺不典型腺瘤中的表达较其他组织稍强。VCAM1在甲状旁腺癌和增生组织中表达明显强于其他组织,而在癌组织中的表达更为显著。KCNA3在甲状旁腺增生,不典型腺瘤和癌组织中表达高于其他组织,而在不典型腺瘤组织中的表达相对更明显。3.第三部分结果：(1)在甲状旁腺癌组织中,CD24的蛋白表达与术后首次复发时间相关(P=0.043)；VCAM1的蛋白表达水平与复发次数明显相关(P=0.006); HMOX1的蛋白表达不仅与术后首次复发时间(P=0.015)和复发次数(P=0.038)明显相关,而且与肿瘤直径大小也明显相关(P=0.002)；但KCNA3的蛋白表达与临床病理特点无明显相关性。(2)通过生存分析也发现,HMOX1的表达与患者的总生存期和无病生存期均明显相关(P=0.026和P=0.022)。VCAM1的表达与患者的总生存期也明显相关(P=0.047),而VCAM1表达与患者无病生存时间无明显相关(P=0.243)。结论1.我国的PHPT的发病率和发现率明显上升,以女性为主,为本病高发人群年龄大于50岁。2010年以前和以后两组病例相比,2010年以后病程明显缩短,混合型的比例明显下降,肾型和无症状型的比例明显上升。癌组和良性肿瘤组相比,趋向于年轻化,在血PTH、血钙、血肌酐、肿瘤最大直径及肿瘤体积指标方面存在显著性差异。2.首次通过基因表达谱技术对中国人群PHPT进行研究,筛选出CD24, HMOX-1, VCAM-1和KCNA3候选差异基因。CD24, HMOX-1, VCAM-1和KCNA3在甲状旁腺癌组织中表达较其他组织高,可能对甲状旁腺癌的诊断及甲状旁腺良恶性肿瘤的鉴别诊断具有重要作用。3. CD24, HMOX-1和VCAM-1可作为判断甲状旁腺癌术后复发的生物学指标,HMOX-1和VCAM-1与甲状旁腺癌的预后有一定关系,有望发展为甲状旁腺癌个体化治疗的潜在靶点。更多还原

【Abstract】 Backgroundprimary hyperparathyroidism (PHPT) is characterized by an increased secretion of parathyroid hormone (PTH) together with hypercalcemia caused by the increase of functional parathyroid tissue due to tumor or hyperplasia. With the medical development, this disease was increasing. Most patients in the developed world are now diagnosed on routine screening at an asymptomatic stage. Because of the clinical manifestation diversity and lack of understanding, the misdiagnostic condition of this disease often occurs, which resulted in patients suffering and high medical costs. Parathyroid cancer can cause the severe clinical symptoms and is an important cause of death in the group of patients with PHPT. However, parathyroid tumors are heterogeneous and the diagnosis of parathyroid tumor subtypes remains challenging. The histopathological diagnosis of parathyroid carcinoma can be a problem. Differentiation of parathyroid carcinoma from parathyroid adenoma especially atypical adenoma is very difficult in some cases Once diagnosed, parathyroid carcinoma is usually related with a high incidence of local recurrence and distant metastasis. On the basis of our hospital patients, we performed the gene expression profiles and molecular biology study of PHPT.Objective:1.To analyze the clinical characteristics of PHPT patients diagnosed in Peking Union Medical College Hospital from1975to2013. To investigate the change of PHPT clinical manifestation in different periods, age and pathological classification.2. To screen the differential genes between benign and malignant parathyroid tumor by gene expression profiles and validate these genes through modern molecular biology techniques. Then to find the genes used for the diagnosis of parathyroid cancer.3. To study the relevance between candidate genes and the clinicopathological features of patients in parathyroid cancer and provide the theoretical supports for the recurrence and prognosis of this disease.Methods:1. We conducted a retrospective study in Peking Union Medical College Hospital in China.490patients who were diagnosed with PHPT between January1975and June2010were enrolled in this study. All patients underwent operations and possessed complete medical datas. Medical records of patients were systematically reviewed, including demographic information, clinical and biological data, surgical and postsurgical data. The changes in different period, age and pathological classification were analyzed.2.We performed gene expression profiles study between normal parathyroid and parathyroid tumors including parathyroid hyperplasia, adenoma, atypical adenoma and cancer. Then the differential genes were analyzed through Go-analysis and Pathway-analysis. The candidate genes including CD24, HMOX1, VCAM1and KCNA3were selected through the constructed signal-net. Firstly mRNA level of these genes were examined by RT-PCR technique to evaluate the expression of CD24, HMOX1, VCAM1and KCNA3in normal parathyroid and parathyroid tumors tissue. Then immunohistochemical staining and western blotting were applied to evaluate these genes.3. Expression of CD24, HMOX1, VCAM1and KCNA3in parathyroid cancer tissues was investigated by immunohistochemistry staining; the association between CD24, HMOX1, VCAM1, KCNA3and clinical characteristics of parathyroid cancer was analyzed; the relationship between these genes and prognosis of parathyroid cancer was studied by statistical software.Result1. The results of first part(1) PHPT patents with surgical therapy was increasing and especially obviously after2010.(2) On the whole most patients are female (2.40:1). Compared patients over50years with that below50years, it is mostly seen in people over50years.(3) Compared patients before2010with that after2010, the time from onset of symptoms to diagnosis was obviously shorter.(4) Compared clinical manifestation before2010with that after2010, patients with mixed type was significantly decreasing, patients with renal and asymptomatic type were increasing.(5) Preoperative PTH level was positively correlated to preoperative serum calcium, ALP, serum creatinine and tumor size of patients, but was negatively correlated to postoperative serum calcium. Serum calcium was positively associated with ALP, serum creatinine, tumor size and postoperative serum calcium.(6) Compared with different pathological tumors, the results showed that PTH, serum calcium, serum creatinine and tumor size of patients in cancer group were higher than those in others groups although the age of patients in cancer group was more young.2. The results of second part(1) CD24, HMOX1, VCAM1and KCNA3genes were indentified according to gene expression profiles, go-analysis, pathway-analysis and signal-net.(2) RT-PCR experiment showed the following results. mRNA level of CD24was higher in parathyroid cancer and adenoma tissue than other tissue and especially higher in parathyroid cancer tissue. mRNA level of HMOX1and VCAM1were both higher in parathyroid cancer and atypical adenoma tissue than other tissue and HMOXlwas especially higher in parathyroid cancer tissue but VCAM1was higher in atypical adenoma. mRNA level of KCNA3was higher in parathyroid cancer tissue than other tissue.(3) Immunohistochemical staining confirmed the following results. CD24positive expression was located in nucleus and partly cytoplasm; CD24expression was.higher in parathyroid cancer tissue than other tissue. HMOX1positive expression was located in cytoplasm and partly nucleus; HMOX1expression was also.higher in parathyroid cancer tissue than other tissue. VCAM1positive expression was located in cytoplasm and partly nucleus; VCAM1expression was higher not only.in parathyroid cancer tissue but also in parathyroid hyperplasia tissue. KCNA3positive expression was located in nucleus; KCNA3expression was both higher.in parathyroid cancer and atypical adenoma.(3) Western blotting confirmed the following results. CD24expression was.significantly higher in parathyroid cancer tissue than other tissue. HMOX1expression was. higher in parathyroid atypical adenoma tissue than other tissue. VCAM1expression was both higher.in parathyroid cancer tissue and hyperplasia tissue, especially higher in parathyroid cancer tissue. KCNA3expression was higher.in parathyroid cancer tissue, atypical adenoma and hyperplasia tissue, but higher in atypical adenoma tissue.3. The results of third part(1) There was a relationship between high expression of CD24and the primary recurrence time after surgical operation. High expression of VCAM1was the recurrence times. High expression of HMOX1was associated not only with the primary recurrence time and recurrence times, but also with tumor size. But there was no relationship between high expression of KCNA3and clinicalpathological factors.(2) Kaplan-Meier analysis showed that expression of HMOX1was significantly associated with the overall and disease-free survival time of patients. It was also showed that expression of VCAM1was only related to the overall survival time of patients. Conclusion1. PHPT patents with surgical therapy was obviously increasing. On the whole most patients are female. It is mostly seen in people over50years.Compared patients before2010with that after2010, the time from onset of symptoms to diagnosis was obviously shorter, patients with mixed type was significantly decreasing and patients with renal and asymptomatic type were increasing. Compared with different pathological tumors, the results showed that PTH, serum calcium, serum creatinine and tumor size of patients in cancer group were higher than those in others groups although the age of patients in cancer group was more young.2. CD24, HMOX1, VCAM1and KCNA3genes were selected by gene expression profiles. Expression of CD24, HMOX1, VCAM1and KCNA3was higher in parathyroid cancer tissue than that in other parathyroid tissue. These genes might be useful for the diagnosis of parathyroid cancer and the differentiated diagnosis of parathyroid tumors.3. CD24, HMOX1, VCAM1and KCNA3can provide the theoretical basis for the recurrence and prognosis of parathyroid cancer. CD24, HMOX1, VCAM1and KCNA3may become a promising tool for early monitoring and therapy of parathyroid cancer.更多还原