Dickkopf-1在胃癌中表达水平及诊断意义研究
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摘要
背景:胃癌是当今严重威胁人类生命健康的重大疾病之一,世界范围内其发病率位居肺癌、乳腺癌和肠癌之后,每年新发病例约934 000人,而其死亡率则位于癌症死因的第二位,每年约为700 000人,我国的胃癌发病人数更占到世界总发病人数的40%以上。近年研究提示,Wnt信号通路成分的突变和表达异常往往会促进肿瘤细胞转化、生长、侵袭转移和血管生成。分泌型蛋白DKK1是Wnt信号通路中一种重要的调节因子,其在肺癌、食管鳞癌、肝癌等多种肿瘤中表达异常,但与胃癌的关系有待进一步研究。本论文从mRNA表达水平和蛋白表达水平研究DKK1在胃癌组织、血清中的表达及含量,有助于进一步了解胃癌发生发展的分子机制,有利于寻找新的胃癌血清学诊断指标。
方法:收集河北大学附属医院2007年3月至2009年6月间对胃癌患者施行的手术切除后标本40例,其中胃癌组织标本25例,癌旁组织标本(癌旁3~5 cm外)15例,标本均取自术前未接受放、化疗的胃癌患者。血清样本由保定市肿瘤医院和河北大学附属医院于2008年1月至2009年8月间采集的血液制备,病人血液在未经手术切除和放化疗前抽取。我们用半定量逆转录-聚合酶链式反应技术(RT-PCR)和蛋白质印迹(Western blot)方法检测胃癌组织及癌旁组织中DKK1 mRNA表达和蛋白表达水平;使用酶联免疫(ELISA)方法分析癌症病人血清和正常人血清中DKK1蛋白含量。并用相应生物统计学软件比较组间差异。
结果:RT-PCR和Western blot结果显示,与对照组癌旁组织相比,DKK1 mRNA和蛋白表达水平总体无差异,但在胃癌组织中表达水平离散程度较高,部分胃癌组织中DKK1表达量明显上升,部分胃癌组织中表达量明显下降。而ELISA结果则显示胃癌患者血清中DKK1含量为4.471μg/L(0.885-23.829μg/L),明显低于正常人血清的5.015μg/L(1.705-8.384μg/L),P<0.01,在95%置信区间内,当取含量值为3.5390μg/L为参考值时,有61.8%的胃癌患者低于此值,而只有15.8%的正常人低于此值,其诊断敏感性为61.8%,特异性为84.2%。
结论:胃癌患者肿瘤组织中DKK1 mRNA及蛋白表达水平与癌旁组织无明显差异,但血清中DKK1含量则比正常人显著降低,提示DKK1可能在胃癌发生发展早期发挥主要作用。同时胃癌患者癌组织中DKK1表达和血清含量水平离散程度高,一些胃癌组织中DKK1 mRNA与蛋白表达量显著上升,一些胃癌组织中表达量明显下降,提示其表达水平的改变可能对肿瘤组织分型、TNM分期、淋巴结转移等胃癌病理指标更加敏感。胃癌患者血清中DKK1含量明显降低,其诊断敏感性为61.8%,特异性为84.2%,从而有成为胃癌血清学诊断指标的潜在价值。
Background: Gastric cancer is one of the most dangerous threats to human health, its incidence is in the fourth place behind cancers of the lung, breast, colon and rectum, with an estimated 934 000 new cases per year. It is the second most common cause of death from cancer (700 000 deaths annually). Almost 42% of the cases occur in China alone. According to recent researches, the mutation and abnormal expression of components involved in Wnt signaling pathway have been implicated in tumor growth, progression, angiogenesis and metastasis. Secreted protein DKK1 is an important regulator of Wnt signaling pathway, and is abnormal expressed in lung cancer, esophageal adenocarcinoma, liver carcinoma, et al, but the relationship with gastric cancer remains to be identified. So we studied the DKK1 mRNA and DKK1 protein expression levels in gastric cancer tissues, also determined sera DKK1 concentration, which is beneficial to understand molecular mechanism of gastric cancer, also give us reference on its potential value as a clinical serological indicator of diagnosis.
Methods: 25 tumor and 15 adjacent non-tumor tissues of stomach were obtained by surgical resection in Affiliated Hospital of Hebei University, between March 2007 and June 2009, and the tissues were resected before malignancies were treated. Serum samples of gastric cancer patients and healthy people were prepared with bloods supplied by Baoding Tumor Hospital and Affiliated Hospital of Hebei University between January 2008 and August 2009, all cancer bloods is collected before surgical removal, chemotherapy or radiation therapy. DKK1 mRNA and DKK1 protein expression levels in gastric cancer tissues were investigated using semi-quantitative RT-PCR and Western blot. Sera DKK1 concentration were determined using ELISA. Differences between groups were tested using SPSS and other biological softwares.
Results: According to RT-PCR and Western blot analysis, there were no significant differences in DKK1 mRNA and DKK1 protein expression levels in gastric tumor tissues compared with adjacent non-tumor tissues, but we observed great up- and down-regulation of expression levels of DKK1 in some cancer tissues. Serum concentration of DKK1 were decreased significantly in gastric cancer patients with 4.471μg/L(0.885-23.829μg/L) than healthy donors with 5.015μg/L(1.705-8.384μg/L), P<0.01. At the concentration of 3.5390μg/L, 61.8% gastric cancer serum samples can be differentiated, whereas only 15.8% of healthy volunteers were under this value. So, the diagnostic sensitivity is 61.8%, specificity is 84.2%.
Conclusions: The differences of DKK1 mRNA and protein expression between tumor tissues and adjacent non-tumor tissues are not significant, but there was a significant decrease of DKK1 expression level in cancer serum samples compared with normal healthy samples, hint us DKK1 may plays an important role at the early stage of gastric cancer carcinogenesis. The great up- and down-regulation of expression levels of DKK1 mRNA and protein in some cancer tissues clue to that it may be sensitive to histologic subtypes, TNM stage, lymph node metastasis and other pathological variables in patients with gastric cancer. Serum concentration of DKK1 were decreased significantly in gastric cancer patients with diagnostic sensitivity of 61.8% and specificity of 84.2%, approves DKK1 may be an index for diagnosis of gastric cancer.
方法:收集河北大学附属医院2007年3月至2009年6月间对胃癌患者施行的手术切除后标本40例,其中胃癌组织标本25例,癌旁组织标本(癌旁3~5 cm外)15例,标本均取自术前未接受放、化疗的胃癌患者。血清样本由保定市肿瘤医院和河北大学附属医院于2008年1月至2009年8月间采集的血液制备,病人血液在未经手术切除和放化疗前抽取。我们用半定量逆转录-聚合酶链式反应技术(RT-PCR)和蛋白质印迹(Western blot)方法检测胃癌组织及癌旁组织中DKK1 mRNA表达和蛋白表达水平;使用酶联免疫(ELISA)方法分析癌症病人血清和正常人血清中DKK1蛋白含量。并用相应生物统计学软件比较组间差异。
结果:RT-PCR和Western blot结果显示,与对照组癌旁组织相比,DKK1 mRNA和蛋白表达水平总体无差异,但在胃癌组织中表达水平离散程度较高,部分胃癌组织中DKK1表达量明显上升,部分胃癌组织中表达量明显下降。而ELISA结果则显示胃癌患者血清中DKK1含量为4.471μg/L(0.885-23.829μg/L),明显低于正常人血清的5.015μg/L(1.705-8.384μg/L),P<0.01,在95%置信区间内,当取含量值为3.5390μg/L为参考值时,有61.8%的胃癌患者低于此值,而只有15.8%的正常人低于此值,其诊断敏感性为61.8%,特异性为84.2%。
结论:胃癌患者肿瘤组织中DKK1 mRNA及蛋白表达水平与癌旁组织无明显差异,但血清中DKK1含量则比正常人显著降低,提示DKK1可能在胃癌发生发展早期发挥主要作用。同时胃癌患者癌组织中DKK1表达和血清含量水平离散程度高,一些胃癌组织中DKK1 mRNA与蛋白表达量显著上升,一些胃癌组织中表达量明显下降,提示其表达水平的改变可能对肿瘤组织分型、TNM分期、淋巴结转移等胃癌病理指标更加敏感。胃癌患者血清中DKK1含量明显降低,其诊断敏感性为61.8%,特异性为84.2%,从而有成为胃癌血清学诊断指标的潜在价值。
Background: Gastric cancer is one of the most dangerous threats to human health, its incidence is in the fourth place behind cancers of the lung, breast, colon and rectum, with an estimated 934 000 new cases per year. It is the second most common cause of death from cancer (700 000 deaths annually). Almost 42% of the cases occur in China alone. According to recent researches, the mutation and abnormal expression of components involved in Wnt signaling pathway have been implicated in tumor growth, progression, angiogenesis and metastasis. Secreted protein DKK1 is an important regulator of Wnt signaling pathway, and is abnormal expressed in lung cancer, esophageal adenocarcinoma, liver carcinoma, et al, but the relationship with gastric cancer remains to be identified. So we studied the DKK1 mRNA and DKK1 protein expression levels in gastric cancer tissues, also determined sera DKK1 concentration, which is beneficial to understand molecular mechanism of gastric cancer, also give us reference on its potential value as a clinical serological indicator of diagnosis.
Methods: 25 tumor and 15 adjacent non-tumor tissues of stomach were obtained by surgical resection in Affiliated Hospital of Hebei University, between March 2007 and June 2009, and the tissues were resected before malignancies were treated. Serum samples of gastric cancer patients and healthy people were prepared with bloods supplied by Baoding Tumor Hospital and Affiliated Hospital of Hebei University between January 2008 and August 2009, all cancer bloods is collected before surgical removal, chemotherapy or radiation therapy. DKK1 mRNA and DKK1 protein expression levels in gastric cancer tissues were investigated using semi-quantitative RT-PCR and Western blot. Sera DKK1 concentration were determined using ELISA. Differences between groups were tested using SPSS and other biological softwares.
Results: According to RT-PCR and Western blot analysis, there were no significant differences in DKK1 mRNA and DKK1 protein expression levels in gastric tumor tissues compared with adjacent non-tumor tissues, but we observed great up- and down-regulation of expression levels of DKK1 in some cancer tissues. Serum concentration of DKK1 were decreased significantly in gastric cancer patients with 4.471μg/L(0.885-23.829μg/L) than healthy donors with 5.015μg/L(1.705-8.384μg/L), P<0.01. At the concentration of 3.5390μg/L, 61.8% gastric cancer serum samples can be differentiated, whereas only 15.8% of healthy volunteers were under this value. So, the diagnostic sensitivity is 61.8%, specificity is 84.2%.
Conclusions: The differences of DKK1 mRNA and protein expression between tumor tissues and adjacent non-tumor tissues are not significant, but there was a significant decrease of DKK1 expression level in cancer serum samples compared with normal healthy samples, hint us DKK1 may plays an important role at the early stage of gastric cancer carcinogenesis. The great up- and down-regulation of expression levels of DKK1 mRNA and protein in some cancer tissues clue to that it may be sensitive to histologic subtypes, TNM stage, lymph node metastasis and other pathological variables in patients with gastric cancer. Serum concentration of DKK1 were decreased significantly in gastric cancer patients with diagnostic sensitivity of 61.8% and specificity of 84.2%, approves DKK1 may be an index for diagnosis of gastric cancer.
引文
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[2]李诚,周健,裘炯良,等.胃癌流行病学与分子生物学病因的研究进展[J].肿瘤防治研究, 2004, 31(2): 115-118.
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[4]顾祥仕.不同年龄组胃癌的临床病理特点分析[J].中国医疗前沿, 2008, 3(19): 79-80.
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