【作者】 姚晓峰；

【导师】 张仑； 郑红；

【作者基本信息】 天津医科大学 ， 肿瘤学， 2008， 硕士

【摘要】 目的通过逆转录-聚合酶链反应研究TMPRSS4 mRNA和onfFN mRNA在甲状腺癌、甲状腺良性组织及癌旁组织中的表达情况,分析它们与甲状腺癌的相关性,不同病理临床参数之间的表达差异,进一步通过荧光定量逆转录-聚合酶链反应检测两种分子标记物在良恶性甲状腺结节细针吸组织中的表达情况。方法收集新鲜无菌手术标本80例,其中包括甲状腺乳头状癌23例,甲状腺滤泡癌4例,甲状腺髓样癌5例,甲状腺良性病变组织30例,甲状腺癌旁组织18例。标本收集严格按照无菌操作,在手术中收集的标本马上放入液氮中,迅速冰冻,然后将标本做好标记存入组织库内,待实验所需。另外,收集甲状腺细针吸标本64例,其中术后证实为甲状腺癌的42例,甲状腺良性病变的22例,所有病例均经过了超声引导下的细针吸细胞学检查,其中一部分标本用于涂片后细胞学检测,另一部分放入装有TRIzol试剂试管内,然后在-80℃的冰箱内冻存。同时,收集所有患者的资料,将完整的资料存放等待最后分析实验结果。首先分离提取所有手术组织标本的总RNA,将总RNA利用RT-PCR逆转录为cDNA,在PCR反应体系中用特异性TMPRSS4和onfFN引物实现它们的大量扩增,经琼脂糖电泳来分析各组织标本TMPRSS4和onfFN的表达情况。然后,通过荧光定量逆转录-聚合酶链反定量检测细针吸甲状腺结节组织标本中两种分子标记物的表达情况。最后,根据所得数据分析两种分子标记物与甲状腺癌的关系及其不同临床病例参数之间的差异。结果TMPRSS4 mRNA在甲状腺癌组织中的表达与甲状腺良性病变组织中的表达差异有统计学意义(p=0.000),与甲状腺癌旁组织中的表达差异也具有统计学意义(p=0.000),在甲状腺良性病变组织和癌旁组织中的表达差异无统计学意义(p=1.000)。onfFN mRNA在甲状腺癌中的表达与甲状腺良性病变组织中表达差异具有统计学意义(p=0.000),与甲状腺癌旁组织中的表达差异也具有统计学意义(p=0.001),而甲状腺良性病变组织和癌旁组织中表达差异无统计学意义(p=1.000)。TMPRSS4 mRNA和onfFN mRNA在甲状腺癌中的表达呈正相关(r=0.413,p=0.019)。在不同的临床病理参数之间,TMPRSS4 mRNA的表达在性别、年龄、肿瘤大小和不同病理类型的甲状腺癌组织中差异无统计学意义(p=0.703,p=0.472,p=1.000,p=0.315)。在上述参数中onfFN mRNA的表达也无统计学意义(p=0.637,p=0.0.161,p=0.138,p=0.207)。在有无淋巴结转移和不同TNM分期的组织中TMPRSS4 mRNA的表达差异具有统计学意义(分别p=0.003和p=0.008),onfFN mRNA的表达同样具有统计学差异(分别p=0.015和p=0.05)。定量结果表明,可以通过荧光定量RT-PCR可以检测到细针吸组织中的微量mRNA的表达,t检验分析显示TMPRSS4和onfFN mRNA在恶性组织中的表达要高于良性组织中(分别t=6.301和7.570,均p=0.000),受试者工作曲线(ROC)分析后得到两分子标志物的曲线下面积分别为0.939和0.909(均p=0.000),敏感性和特异性分别为92.9%、72.7%和88.1%、77.3%。结论在甲状腺癌病变组织中TMPRSS4 mRNA和onfFN mRNA的表达显著高于甲状腺良性病变组织和甲状腺癌旁组织,荧光定量RT-PCR可以检测到甲状腺结节细针吸组织标本中mRNA的表达,因此可以通过检测两种分子标记物的表达联合细针吸细胞学检查,从而提高甲状腺癌的术前诊断和鉴别诊断,提高甲状腺癌术前诊断的准确率,为临床制定治疗方案提供准确的信息。在有无淋巴结转移和不同TNM分期的甲状腺癌组织中,TMPRSS4 mRNA和onfFN mRNA的表达也存在着差异,说明两者在甲状腺癌的发生、发展和转移过程中起到了重要作用。同时,研究发现,在甲状腺癌组织中TMPRSS4 mRNA和onfFN mRNA的表达呈正相关,表明两者在甲状腺癌的发生和发展过程中具有协同作用。更多还原

【Abstract】 Objective:To study the expression of Transmembrane Protease Serine4(TMPRSS4) and Oncofetal Fibronectins(onfFN)mRNA in thyroid carcinoma,adenoma and paraneoplastic tissue by reverse transcriptase- polymerase chain reaction,analysis their correlation with thyroid carcinoma and the different expression in different clinicopathologic parameter,then a further studay was performed to detect the expression of the two marker in FNAB tissue of thyroid nodule by real-time quantitative polymerase chain reaction.Method:80 fresh thyroid samples were collected after operation include 32 malignant and 30 benign,the samples were put into liquid nitrogen as soon as quickly when obtained from operation room,and saved in tissue bank for the next experiment. And 78 FANB samples of thyroid nodule were collected include 45malignant and 33 benign,the aspiration of those samples was guided by ultrasound,some samples put into test tube with TRIzol,others were smeared for cytology check.At the same time collected the case file material of the patients for the resules analysis.First,extract the RNA of fresh samples,and transcribed the RNA into cDNA by RT-PCR,amplificated the cDNA with specific primer of TMPRSS4 and onfFN,and then analysised the expression of the two markers by agarose electrophoresis experiment.Second, dectected expression of the two markers in FANB sample by real-time quantitative polymerase chain reaction.Final,analysis their correlation with thyroid carcinoma and the different expression in different clinicopathologic parameter according the resules of the experiments.Results:The expression rate of TMPRSS4 in carcinoma,adenoma and paraneoplastic tissue was 78.13%,6.67%,5.56 respectively,and onfFN was 82.37%,16.67%,16.67%respectively.There was a statistically significant higher expression of thyroid carcinoma than adenoma and paraneoplastic(p=0.000),and there was a statistically significant positive correlation between expression of TMPRSS4 and onfFN(r=0.413,p=0.019).The expression of TMPRSS4 and onfFN mRNA in TNMⅢ+Ⅳand lymph node metastasis was higher than TNMⅠ+Ⅱand no lymph node metastasis thyroid carcinoma samples(p=0.003,p=0.008 and p=0.015,p=0.05),but,There was no statistically significant in other clinicopathologic parameters.Quantitative results indicated that the microamount of mRNA expression in FNAB samples can be detected by RT-PCR,t-test result demonstrated that TMPRSS4 and onfFN mRNA expression in malignant samples was higher than that in benign samples(t=6.202 and 7.215 respectively,p=0.000),the area under the receiver operating characteristic(AUC)of TMPRSS4 and onfFN was 0.932 and 0.902 (p=0.000),sensitivity and specificity was 92.9%、72.7%and 88.1%、77.3% respectively.Conclusions:The expression of TMPRSS4 and onfFN mRNA in malignant samples was higher than that in benign and paraneoplastic samples,the microamount of mRNA expression in FNAB samples can be detected by real-time quantitative polymerase chain reaction,so we can combined the two markers and FANB to elevate the diagnosis accurate of thyroid carcinoma before operation,which can offered accurate informations for making treatment perscription.The expression of TMPRSS4 and onfFN mRNA in TNMⅢ+Ⅳand lymph node metastasis was higher than TNMⅠ+Ⅱand no lymph node metastasis thyroid carcinoma samples,which demonstrated that the two markers might play a important role in the carcinogenesis, development and metachoresis of thyroid carcinoma,and there was a statistically significant positive correlation between expression of TMPRSS4 and onfFN in thyroid which displayed that their might be a synergistic effect in those processes.更多还原