【作者】 王朝霞；

【导师】 魏枫；

【作者基本信息】 内蒙古科技大学包头医学院 ， 内科学， 2012， 硕士

【摘要】 目的探讨结节性甲状腺疾病患者外周血中死亡相关蛋白激酶(deathassociatedprotein kinase，DAPK)基因，维甲酸受体β2(retinoic acid receptor,RARβ2)基因启动子区的CpG岛甲基化状态及mRNA表达并探讨其临床意义，为甲状腺结节、肿瘤寻找新的肿瘤标记物和抗结节治疗及靶向治疗提供依据。方法选择2007年9月～2011年12确诊的结节性甲状腺疾病患者（结节性甲状腺肿41例，甲状腺腺瘤32例，grave，s病合并结节34例，桥本合并结节40例，甲状腺癌23例）共170例和正常对照组30例，采空腹静脉血，应用甲基化特异性PCR方法（Methylation-specificPCR，MSP）检测DAPK，RARβ2基因甲基化状态；应用逆转录聚合酶链反应(Reversedtranscriptive polymerase chain reaction RT-PCR)方法检测外周血DAPK，RARβ2基因的mRNA表达情况。采用SPSS13.0统计软件，用χ2检验评估组间甲基化状态及其mRNA表达，以P<0.05为差异，有统计学意义。分析外周血DAPK，RARβ2基因甲基化与其基因表达之间关系。结果（1）在甲状腺癌中DAPK，RARβ2基因呈高甲基化率（分别为39.1%，43.5%），与正常对照组比较(P=0.000,0.000，P＜0.05)具显著性差异；（2）在甲状腺腺瘤组中DAPK呈高甲基化率（21.8%），与正常对照组比较（P＝0.033,P＜0.05），有统计学意义；在桥本甲状腺炎组RARβ2基因呈高甲基化率（35%），与正常对照组比较（P=0.001,P＜0.05），具显著性差异；（3）在有钙化点或淋巴结肿大的甲状腺癌中，DAPK，RARβ2基因呈高甲基化率（fisher值＝0.040，0.018；0.029，0.019，P＜0.05）,具统计学意义，在甲状腺癌组DAPK，RARβ2基因的甲基化与患者的年龄，性别，结节大小比较fisher值＞0.05，无统计学意义。（4）DAPK基因在甲状腺癌，甲状腺腺瘤中mRNA表达减低或表达缺失,与正常对照组比较(P＝0.000，0.003，P＜0.05）,有显著性差异；RARβ2基因在甲状腺癌，桥本甲状腺炎合并结节中mRNA表达减低或表达缺失，与正常对照组比较(P＝0.000，0.002，P＜0.05）有显著性差异。（5）DAPK，RARβ2基因的表达减低或缺失与基因启动子区异常甲基化有关；基因甲基化与mRNA表达呈负相关。结论（1）DAPK，RARβ2基因在甲状腺癌外周血中呈高甲基化率，而其mRNA表达减低或表达缺失；DAPK基因在甲状腺腺瘤中呈高甲基化率,甲基化率低于甲状腺癌组，mRNA表达减低；RARβ2基因在桥本甲状腺炎外周血中呈高甲基化率，mRNA表达减低。（2）甲状腺癌中DAPK，RARβ2基因甲基化与性别，年龄，结节大小无显著差异，与结节钙化及颈部淋巴结肿大有关。(3)联合检测DAPK、RARβ2基因甲基化状态能明显提高甲状腺癌的诊断敏感度，有可能成为甲状腺癌早期诊断的有效指标。(4)DAPK，RARβ2基因的表达缺失与基因启动子区异常甲基化有关。更多还原

【Abstract】 Objective To study the gene of deathassociated protein kinase, DAPK in Nodular thyroiddisease in patients peripheral blood, the CpG island state of gene promoter methylation ofretinoic acid receptor, RARβ2and mRNA expression and then discuss its clinicalsignificance: the thesis aims to look for new tumor markers and then provides the basis forresistance to nodules treatment and targeted therapy for thyroid nodule and tumor.MethodsWe collected170cases of clinical patient from First Affiliated Hospital of Baotou medicalcollege between2007.9-2011.12(including41cases of nodular goiter,32cases of thyroidadenoma，34case of Grave，s disease，40case of Thyroid inflammation in hashimotogroup，23cases of thyroid carcinoma and30healthy control subjects who were takenlimosis vein blood. The thease examines gene methylation state of DAPK and RAR betavia application of the method of specific Methylation-specific PCR，MSP. It alsoexamines gene expression of mRNA existing in peripheral blood DAPK, RARβ2viaapplication of the method of Reversed transcriptive polymerase chain reaction RT-PCR. Itanalyzes gene methylation of peripheral blood, RARβ2and the relationship between thegene expression DAPK by using the statistical software SPSS13.0.Results (1)compared with normal control group (P=0.000,0.000, P <0.05), gene has high rate ofmethylation （39.1%，43.5%） in the thyroid cancer DAPK, RAR beta2with a significantdifference;(2) compared with normal (P=0.033, P <0.05), DAPK methylation rate（21.8%） are high among the thyroid gland tumor group, which has a statisticalsignificance; Thyroid inflammation in hashimoto group RARβ2gene has high rate ofmethylation, compared with normal control group (P=0.001, P <0.05), and has thesignificant difference;(3) in the calcification point or lymph node enlargement of thethyroid cancer, DAPK, RARβ2gene has high rate of methylation (fisher value=0.040,0.018,0.029,0.019, P <0.05), a statistical significance, in the thyroid cancer group DAPK,RARβ2gene methylation and of the patient’s age, gender, nodules size comparison fishervalue>0.05, no statistical significance.(4) DAPK gene in the thyroid cancer, thyroidgland tumors mRNA expression or express reduce loss, compared with normal control group (P=0.000,0.003, P <0.05), significant difference; RARβ2gene in the thyroidcancer, thyroid inflammation in merger hashimoto nodules mRNA express reduce orexpress is missing, and normal control group (P=0.000, compared to0.002, P <0.05)have significant differences.(5) DAPK, RARβ2gene expression to reduce or lack andgene promoter area abnormal methylation involved; Gene methylation and mRNAexpression was negatively correlated.Conclusion (1) DAPK, RAR β2gene in the thyroidcancer in peripheral blood are high in methylation rate while the mRNA express is reducedor lost in expression; the lack DAPK gene in the thyroid gland tumors has high rate ofmethylation, methylation rates lower than thyroid cancer group, reduce mRNA expression;RARβ2gene in the thyroid inflammation in the peripheral blood is hashimoto highmethylation state, reduce mRNA expression.(2) In the thyroid cancer, gene methylation ofDAPK, RARβ2has no significant differences with gender, age, size of the nodule, but isrelevant to nodules calcification and neck lymph node enlargement.(3) Joint test RARβ2,DAPK gene methylation state can obviously increase sensitivity of thyroid cancer, whichmay become a thyroid cancer early diagnosis of effective index.(4) The loss of geneexpression in DAPK, RAR beta2is relevant to gene promoter area abnormal methylation.更多还原