【作者】 王帅；

【导师】 刘成玉； 徐文华；

【作者基本信息】 青岛大学 ， 临床检验诊断学， 2021， 硕士

【摘要】 目的评价目前临床常用的胃癌血清肿瘤标志物,并探究外泌体环状RNAs(circular RNAs,circRNAs)作为新型胃癌标志物的潜在价值及临床意义。方法采集260例胃癌患者血清标本作为胃癌组A及140例胃良性疾病患者血清标本作为对照组A,用于评价胃癌血清标志物的诊断价值。采集73例胃癌患者血清标本作为胃癌组B,25例胃良性疾病及30例健康体检者血清标本作为对照组B,用于探究外泌体circRNAs的变化。采用电化学发光法检测血清糖链抗原72-4(carbohydrate antigen72-4,CA72-4)、癌胚抗原(carcinoembryonic antigen,CEA)、糖链抗原19-9(carbohydrate antigen 19-9,CA19-9)、糖链抗原242(carbohydrate antigen 242,CA242)、糖链抗原50(carbohydrate antigen50,CA50)及糖链抗原125(carbohydrate antigen 125,CA125)浓度变化,并分析血清标志物单独检测及联合检测对胃癌的诊断价值;采用实时荧光定量PCR(quantitative-real time PCR,qRT-PCR)技术检测血清外泌体circRNAs的表达水平,应用透射电镜、NTA(Nanoparticle Tracking Analysis)纳米颗粒分析仪和Western blot(蛋白印迹法)对提取的血清外泌体特征进行鉴定。分析血清外泌体circRNAs(circ PVT1和has-circ-0010522)表达水平,及其与胃癌患者临床病理特征的关系,通过建立受试者工作特征曲线(receiver operating characteristic curve,ROC)评价其对胃癌的诊断价值。结果胃癌组A血清CA72-4、CEA、CA19-9、CA242、CA50、CA125浓度均显著升高,与对照组A相比,差异具有显著性(t=5.799～8.826,P<0.05)。血清标志物单项检测的ROC曲线下面积以CEA最高(AUC=0.779),CEA的灵敏度最高(52.31%),特异度以CA242最高(96.43%),准确度以CEA最高(64.75%);联合检测中AUC值最高的为CA72-4+CEA+CA19-9+CA242+CA50+CA125组(0.928);灵敏度最高的为CA72-4+CEA+CA19-9+CA242+CA50组和CA72-4+CEA+CA19-9+CA242+CA50+CA125组(均为85.77%);特异度最高的为CA72-4+CEA+CA19-9组(78.57%);准确度最高的为CA72-4+CEA+CA19-9+CA242+CA50组(82.5%)。胃癌组B血清CA72-4、CEA、CA19-9、CA242、CA50、CA125浓度均显著升高,与对照组B相比,差异具有显著性(t=2.311～5.004,P<0.05)。CA72-4、CEA、CA19-9、CA242、CA50、CA125用于筛查胃癌的AUC值分别为0.748、0.686、0.658、0.701、0.667和0.748,六项血清肿瘤标志物联合检测的AUC值为0.905。经过透射电镜、颗粒分析仪和Western blot,外泌体的特征得到了验证。预实验中,胃癌患者血清外泌体circ PVT1及has-circ-0010522表达水平均显著高于胃良性疾病患者与健康体检者(t=4.571～8.064,P<0.05),而胃良性疾病患者与健康体检者血清外泌体circ PVT1及has-circ-0010522的表达无差异。与对照组B相比,胃癌组B血清外泌体circ PVT1及has-circ-0010522表达水平均显著升高(t=11.364、6.467,P<0.05),外泌体circ PVT1及has-circ-0010522用于筛查胃癌的AUC值分别为0.916、0.798,两种circRNAs联合检测的AUC值为0.933。circ PVT1表达水平与患者性别、肿瘤大小、TNM分期、淋巴结转移以及腹膜转移显著相关(P<0.05);has-circ-0010522的表达水平与年龄、淋巴结转移显著相关(P<0.05);circ PVT1用于区分肿瘤直径是否<5 cm时的AUC值为0.778,circ PVT1用于区分患者处于Ⅰ～Ⅱ期或Ⅲ～Ⅳ期时的AUC值为0.808,circ PVT1用于区分患者是否发生腹膜转移时的AUC值为0.828;circ PVT1及has-circ-0010522用于区分患者是否发生淋巴结转移时的AUC值分别为0.764、0.709,circ PVT1及has-circ-0010522联合检测用于区分患者是否发生淋巴结转移时的AUC值为0.809。结论术前检测临床常用的血清肿瘤标志物,对于胃癌的诊断及鉴别诊断具有重要的价值,探究更高效合理的联合检测组合有助于提高胃癌诊断的灵敏度及准确度。胃癌患者血清外泌体circ PVT1及has-circ-0010522表达均明显增高,且与胃癌临床病理特征相关,对于胃癌的诊断与鉴别诊断具有一定的价值,并且联合检测可明显提高其诊断效能,具有作为新型胃癌肿瘤标志物的可能性。更多还原

【Abstract】 Objective To evaluate the current clinically commonly used gastric cancer serum tumor markers,and explore the potential value and clinical significance of exosomal circular RNAs(circRNAs)as new gastric cancer markers.Methods Serum samples from 260 patients with gastric cancer were collected as gastric cancer group A,and serum samples from 140 patients with benign gastric diseases were used as control group A to evaluate the traditional gastric cancer serum markers.Serum samples of 73 patients with gastric cancer were collected as gastric cancer group B,25 patients with benign gastric disease and 30 healthy patients were treated as control group B to investigate the changes of exosomal circRNAs.The electrochemiluminescence method was used to detect the changes of serum markers such as CA72-4,CEA,CA19-9,CA242,CA50 and CA125 in serum samples.Analyze the sensitivity and specificity of traditional serum markers such as the sensitivity and specificity of single detection and combined detection.Quantitative-time PCR(q RT-PCR)was used to detect the expression levels of exosomal circRNAs in serum samples.Transmission electron microscopy,nanoparticle analyzer and Western blot were used to identify the characteristics of the extracted serum exosomes.Analyze the expression levels of exosomal circRNAs and their relationship with the clinicopathological characteristics of gastric cancer patients,and evaluate the diagnostic value for gastric cancer by establishing a receiver operating characteristic curve(ROC).Results The concentrations of CA72-4,CEA,CA19-9,CA242,CA50 and CA125 in gastric cancer group A were significantly higher than those in control group A,and the differences were significant(t=5.799～8.826,P<0.05).CEA had the highest area under curve(AUC)value for single test(0.779),CEA had the highest sensitivity(52.31%),CA242 had the highest specificity(96.43%),and CEA had the highest accuracy(64.75%).The highest AUC value in the combined test was the CA72-4 + CEA + CA19-9 + CA242+ CA50 + CA125 group(0.928).The most sensitive were the CA72-4 + CEA + CA19-9 +CA242 + CA50 group and the CA72-4 + CEA + CA19-9 + CA242 + CA50 + CA125group(both 85.77%).The highest specificity was the CA72-4 + CEA + CA19-9 group(78.57%).The highest accuracy was the CA72-4 + CEA + CA19-9 + CA242 + CA50group(82.5%).The concentrations of CA72-4,CEA,CA19-9,CA242,CA50 and CA125 in gastric cancer group B were significantly higher than those in control group B,and the differences were significant(t=2.311～5.004,P<0.05).The AUC values of CA72-4,CEA,CA19-9,CA242,CA50,and CA125 for gastric cancer screening were 0.748,0.686,0.658,0.701,0.667 and 0.748,respectively.The AUC value of the combined detection of six serum tumor markers was 0.905.After transmission electron microscopy,nanoparticle analyzer and Western blot,the characteristics of exosomes have been verified.In preliminary experiments,the expression levels of serum exosomal circ PVT1 and has-circ-0010522 in patients with gastric cancer were significantly higher than those in patients with benign gastric disease and healthy subjects(t = 4.571 ～ 8.064,P < 0.05).There was no difference in the expression level of circ PVT1 and has-circ-0010522 between patients with benign gastric disease and those with healthy physical examination.Compared with the control group B,the expression levels of serum exosomal circ PVT1 and has-circ-0010522 in the gastric cancer group B were significantly increased(t = 11.364,6.467,P < 0.05).The AUC values of exosomal circ PVT1 and has-circ-0010522 for gastric cancer screening were0.916 and 0.798,respectively,and the AUC value of the combined detection of the two circRNAs was 0.933.The expression level of circ PVT1 was significantly correlated with gender,tumor diameter,TNM stage,lymph node metastasis and peritoneal metastasis(P<0.05).The expression level of has-circ-0010522 was significantly correlated with age and lymph node metastasis(P<0.05).The AUC value of circ PVT1 used to distinguish whether the tumor diameter is less than 5 cm is 0.778.The AUC value of circ PVT1 is0.808 when it is used to distinguish patients in stage Ⅰ～Ⅱ or stage Ⅲ～Ⅳ.The AUC value of circ PVT1 is 0.828 when it is used to distinguish whether the patient has peritoneal metastasis.The AUC values of circ PVT1 and has-circ-0010522 used to distinguish whether a patient has lymph node metastasis are 0.764 and 0.709,respectively.The combined detection of circ PVT1 and has-circ-0010522 is used to distinguish whether patients have lymph node metastasis.The AUC value is 0.809.Conclusion Preoperative detection of serum tumor markers commonly used in clinical practice is of great value for the diagnosis and condition judgment of patients with gastric cancer and exploring a more efficient and reasonable combination of detection will help improve the sensitivity and accuracy of diagnosis.Serum exosomal circ PVT1 and has-circ-0010522 were both significantly increased in patients with gastric cancer,which were correlated with the clinicopathological characteristics of gastric cancer,and had certain value for the diagnosis and differential diagnosis of gastric cancer.Moreover,combined detection could significantly improve the diagnostic efficiency,and had the possibility of being used as new tumor markers for gastric cancer.更多还原