【作者】 陈萍；

【导师】 刘新兰；

【作者基本信息】 宁夏医学院 ， 肿瘤学， 2008， 硕士

【摘要】 目的检测胃肠道恶性肿瘤患者外周血中细胞角蛋白20(Cytokeratin20, CK20) mRNA和人端粒酶逆转录酶(human telomerase reverse transcriptase, hTERT)mRNA的表达,分析它们与肿瘤临床病理特征及预后的关系,探讨其可否作为诊断胃肠道恶性肿瘤患者外周血微转移和判断预后的分子生物学指标。方法采用RT-PCR技术检测2005年10月至2006年3月在宁夏医学院附属医院和银川市第一人民医院住院治疗的72例胃肠道恶性肿瘤患者(胃癌40例,大肠癌32例)、30例胃肠道良性病变患者和30例健康体检者外周血中CK20 mRNA和hTERT mRNA的定性和半定量表达。以20例胃肠道恶性肿瘤新鲜组织标本和人胃腺癌细胞株SGC-7901作阳性对照。实验数据采用SPSS13.0 for Windows软件包统计分析。结果(1)CK20 mRNA和hTERT mRNA在胃肠道恶性肿瘤患者外周血中的阳性表达率分别为51.39%和61.11%,相对系数分别为0.84±0.17和0.82±0.17(胃癌分别为52.50%和65.00%,相对系数分别为0.81±0.17和0.78±0.17,大肠癌分别为50.00%和56.25%,相对系数分别为0.89±0.17和0.88±0.17);在胃肠道良性病变患者外周血中阳性表达率分别为6.67%和3.33%,相对系数分别为0.11±0.01和0.11;在健康体检者外周血中阳性表达率及相对系数均为0。胃肠道恶性肿瘤患者与胃肠道良性病变患者、健康体检者相比,CK20 mRNA和hTERT mRNA的表达均有统计学差异(P<0.05),胃癌组和大肠癌组相比均无统计学差异。CK20 mRNA和hTERT mRNA在胃肠道恶性肿瘤组织和人胃腺癌细胞株SGC-7901中均阳性表达。(2)CK20 mRNA阳性表达率和相对系数在胃肠道恶性肿瘤中与临床分期、远处转移均有关(P<0.05);在胃癌中其阳性表达率与浸润深度、淋巴结转移有关(P<0.05),在大肠癌中则无关,但相对系数与浸润深度、淋巴结转移均有关(P<0.05)。阳性表达者根治术后复发转移率高于阴性者,生存率低于阴性者(P<0.05)。(3)hTERT mRNA阳性表达率和相对系数在胃肠道恶性肿瘤中与临床分期、远处转移均有关(P<0.05);其阳性表达率与胃肠道恶性肿瘤浸润深度无关,在胃癌中与淋巴结转移有关(P<0.01),在大肠癌中则无关,但相对系数与浸润深度及淋巴结转移均有关(P<0.05)。阳性表达者根治术后复发转移率高于阴性者,生存率低于阴性者(P<0.05)。(4)logistic逐步回归分析显示,临床分期独立地与CK20 mRNA、hTERT mRNA的表达相关。(5)RT-PCR法检测胃肠道恶性肿瘤患者外周血中CK20 mRNA和hTERT mRNA诊断微转移,灵敏度为75.00%~90.00%,特异度为66.67%~88.89%,实验准确度为71.43%~85.71%。两项指标间及在胃癌、大肠癌间的灵敏度、特异度、准确度均无统计学差异。(6)CK20 mRNA、hTERT mRNA与癌胚抗原(carcinoembryonic antigen, CEA)、糖链抗原199(carbohydrate antigen 199, CA199)的表达呈正相关(P<0.05);在胃癌患者中CK20 mRNA、hTERT mRNA阳性表达率高于CA199(P<0.05)。(7)CK20 mRNA与hTERT mRNA在胃肠道恶性肿瘤患者外周血中的表达呈正相关(P<0.01)。结论(1)胃肠道恶性肿瘤患者外周血中存在CK20 mRNA和hTERT mRNA的表达,与临床分期、复发转移及生存有关,提示CK20 mRNA和hTERT mRNA与胃肠道恶性肿瘤的侵袭转移密切相关,可作为独立的预后因素。(2)RT-PCR技术定性和半定量检测外周血CK20 mRNA和hTERT mRNA表达可作为诊断胃肠道恶性肿瘤患者外周血微转移的预测指标。(3)CK20 mRNA、hTERT mRNA与CEA、CA199的表达呈正相关,在胃癌患者中CK20 mRNA、hTERT mRNA诊断外周血微转移优于CA199。(4)CK20 mRNA与hTERT mRNA在胃肠道恶性肿瘤患者外周血中的表达呈正相关。更多还原

【Abstract】 Objective To analyze the relationship of cytokeratin 20 (CK20) mRNA and human telomerase reverse transcriptase (hTERT) mRNA to clinicopathologic characteristics and prognosis of gastrointestinal carcinoma, the expression of CK20 mRNA and hTERT mRNA were detected in peripheral blood of patients with gastrointestinal carcinoma. To discuss whether they can be used as molecular biological markers of predicting micrometastasis in peripheral blood and evaluating prognosis.Methods Peripheral blood of 72 gastrointestinal carcinoma patients in the affiliated hospital of Ningxia medical college and the first hospital of Yinchuan from Oct 2005 to Mar 2006 were detected by reverse transcriptase polymerase chain reaction (RT-PCR) technique for qualitative and semi-quantitative analysis of CK20 mRNA and hTERT mRNA, including 40 patients with gastric carcinoma and 32 patients with colorectal carcinoma; 30 patients with benign gastrointestinal lesion, 30 cases from healthy examination. 20 gastrointestinal carcinoma fresh tissues and human SGC-7901 gastric adenocarcinoma cell line were used as positive controls. The results were analyzed by SPSS13.0 software.Results (1) The positive expression rate of CK20 mRNA and hTERT mRNA in peripheral blood of patients with gastrointestinal carcinoma was 51.39% and 61.11%, relative coefficient was 0.84±0.17 and 0.82±0.17 (52.50% and 65.00% in gastric carcinoma patients, relative coefficient was 0.81±0.17 and 0.78±0.17, 50.00% and 56.25% in colorectal carcinoma patients, relative coefficient was 0.89±0.17 and 0.88±0.17); 6.67% and 3.33% in benign gastrointestinal lesion, relative coefficient was 0.11±0.01 and 0.11, respectively. CK20 mRNA and hTERT mRNA were not detected in peripheral blood of healthy people. Compared the expressions of CK20 mRNA and hTERT mRNA in gastrointestinal carcinoma patients with benign gastrointestinal lesion patients and healthy people, there were all significant differences (P<0.05); there were no significant differences between the expressions of gastric carcinoma patients and colorectal carcinoma patients. CK20 mRNA and hTERT mRNA were positive in gastrointestinal carcinoma tissues and human SGC-7901 gastric adenocarcinoma cell line. (2) The positive expression rates and relative coefficients of CK20 mRNA were significantly correlated with clinical stage, distant metastasis in gastrointestinal carcinoma patients (P<0.05); the positive expression rate was also correlated with depth of tumor invasion, lymph node metastasis in gastric carcinoma patients (P<0.05), no correlation in colorectal carcinoma patients, but relative coefficients were all correlated with them (P<0.05). The patients who had radical resection with positive expression have higher recurrence or metastasis rates, and lower survival rates compared with negative expression patients (P<0.05). (3) The positive expression rates and relative coefficients of hTERT mRNA were significantly correlated with clinical stage, distant metastasis in gastrointestinal carcinoma patients (P<0.05); the positive expression rates were not correlated with depth of tumor invasion in gastrointestinal carcinoma, correlated with lymph node metastasis in gastric carcinoma patients (P<0.01), no correlation in colorectal carcinoma patients, but relative coefficients were all correlated with depth of tumor invasion and lymph node metastasis (P<0.05). The patients who had radical resection with positive expression have higher recurrence or metastasis rates, and lower survival rates compared with negative expression patients (P<0.05). (4) Logistic stepwise regression analysis revealed that clinical stage is independently related to the expression of CK20 mRNA and hTERT mRNA. (5) The expression of CK20 mRNA and hTERT mRNA which were detected by RT-PCR technique diagnose micrometastasis in peripheral blood of gastrointestinal carcinoma patients with sensitivity of 75.00%~90.00%, specificity of 66.67%~88.89%, accuracy of 71.43%~ 85.71%. There were no significant differences between CK20 mRNA and hTERT mRNA, gastric carcinoma patients and colorectal carcinoma patients. (6) The positive expression rates and relative coefficients of CK20 mRNA and hTERT mRNA were positively correlated with the expression of serum carcinoembryonic antigen (CEA) and carbohydrate antigen 199 (CA199) level in gastrointestinal carcinoma patients. The positive expression rates of CK20 mRNA and hTERT mRNA were all higher than that of CA199 in gastric carcinoma patients (P<0.05). (7) The positive expression rates and relative coefficients of CK20 mRNA were positively correlated with hTERT mRNA in peripheral blood of patients with gastrointestinal carcinoma (P<0.01).Conclution (1) CK20 mRNA and hTERT mRNA were positive expressed in peripheral blood of patients with gastrointestinal carcinoma. We predict that the two markers were independent prognostic markers in gastrointestinal carcinoma patients because the expression of CK20 mRNA and hTERT mRNA had closely correlation with clinical stage, recurrence, metastasis and survival. (2) The expression of CK20 mRNA and hTERT mRNA which were detected by RT-PCR technique for qualitative and semi-quantitative analysis can be used as predictive markers for diagnosing micrometastasis in peripheral blood of patients with gastrointestinal carcinoma. (3) The positive expression rates and relative coefficients of CK20 mRNA and hTERT mRNA were positively correlated with the expression of CEA and CA199 level in gastrointestinal carcinoma patients. CK20 mRNA and hTERT mRNA were better than CA199 for diagnosing micrometastasis in gastric carcinoma patients. (4) The positive expression rates and relative coefficients of CK20 mRNA were positively correlated with hTERT mRNA in peripheral blood of patients with gastrointestinal carcinoma.更多还原