肺癌患者血浆及支气管肺泡灌洗液游离RNA测定与临床意义
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摘要
目的:最近研究证实游离RNA的存在,其具有肿瘤细胞来源,使之成为了肿瘤研究的热点。GAPDH mRNA是编码磷酸甘油醛脱氢酶的看家基因,在所有类型的细胞内呈组成性表达, GAPDH mRNA是体液中表达最为丰富的游离RNA之一,测定体液中该基因的拷贝数可以反映总游离RNA水平。为此,我们采用实时定量逆转录聚合酶链式反应(PCR)方法测定GAPDH mRNA拷贝数,探讨肺癌患者支气管肺泡灌洗液(BALF)和血浆游离RNA的表达及其临床意义。
方法:以确诊的43例肺癌作为实验组,17例肺部良性病变作为对照组,采用实时定量PCR方法测定支气管肺泡灌洗液上清和血浆中游离GAPDH mRNA的拷贝数。常规细胞学方法检测肺泡灌洗液脱落细胞。应用受试者工作特征(ROC)曲线分析方法评价游离RNA诊断价值。采用CA-1500型全自动血凝分析仪测定两组血液的凝血指标。
结果:(1)肺癌及良性疾病组BALF和血浆中均可检测到游离RNA的存在。(2)肺癌患者BALF中游离RNA拷贝数明显高于肺良性病变组(t=3.457,P=0.001),但血浆中游离RNA两组比较差别无统计学意义。(3)肺泡灌洗液细胞学检查肿瘤细胞阳性组游离RNA拷贝数明显高于阴性组(t=2.094,P=0.024)。(4)肺癌游离RNA拷贝数在不同的病理类型、临床分期中无差异(P>0.05)。(5) ROC曲线分析肺泡灌洗液游离RNA对肺癌的诊断价值: ROC曲线最佳工作点对应的游离RNA拷贝数对数、敏感性和特异性分别为4.433、86.0%、76.5%,ROC曲线下面积0.882。当特异性为100%时,诊断的敏感性为53.5%。(6)肺癌组血浆纤维蛋白原含量明显高于肺良性疾病组(t= 2.427,P= 0.018)。(7)肺癌血浆游离RNA拷贝数的对数与纤维蛋白含量呈正相关(r=0.668,P<0.05)。
结论:应用荧光定量PCR方法检测BALF中游离RNA诊断肺癌具有较高的特异性、敏感性,对肺癌的诊断具有一定的价值。肺癌患者血液处于高凝状态,游离RNA可能参与了肺癌患者高凝状态形成。
Objective: Recently the studies confirmed the existence of cell-free RNA which was of tumor origin. GAPDH mRNA which is one of housegroup genes encodes glyceraldehyde phosphate dehydrogenase proteins.GAPDH mRNA is one of most copiousest gene in the body fluid. Base on some promising data, we established real-time reverse transcription polymerase chain reaction(RT-PCR) with primers specific for GAPDH) gene to investigate the clinical value of cell-free RNA in bronchoalveolar lavage fluid supernatant (BALF) and plasma in patients with lung cancer.
Methods:The copies of cell-free RNA in the plasma and BALF were measured by real time quantitative PCR in 43 patients with lung cancer and 17 patients with benign lung diseases. The sediment of BALF were examined by cytologists. The cell-free RNA copies in two groups were analyzed by receiver operating characteristic (ROC) curve. Sysmex CA -1500 automated coagulation analyzer was used to detect blood coagulation index in 60 patients.
Results:(1) Cell-free RNA were dectected in the BALF and plasma of two group.(2)The copies of BALF RNA in lung cancer group were much higher than those in benign disease group (t=3.457, P =0.001) , But the difference of cell free RNA level in the plasma between cancer patients and benign group was not significant. (3)It was also found that the copies of RNA in the BALF with positive cancer cells were higher than those with the negative cancer cells(t=2.094, P=0.024). (4)The correlation between the clinical pathological datas and cell free RNA level was not found. (5) ROC of cell-free RNA in BALF analysis showed the best diagnostic cut-off point is 4.433,which generated diagnostic sensitivity of 86.0% and specificity of 76.3%. When the sensitivity was 53.5%,the specificity could reach 100%, and area under curve (AUC)of 0.882. (6) Fibrinogen in lung cancer patients were significant higher than benign disease group (t=2.427,P=0.018) (7) There were positive correlation between the fibrinogen and the Log of RNA copies in plasma of lung cancer patients(r=0.669,P<0.05).
Conclusion:The measurement of cell-free RNA in BALF using real-time PCR has sound sensitivity and specificity in diagnosis of lung cancer, It is suggested that cell-free RNA might have a potential value for detecting lung cancer. Cell-free RNA perhaps result in hypercoagulabale state which mostly existed in patients with lung cancer.
方法:以确诊的43例肺癌作为实验组,17例肺部良性病变作为对照组,采用实时定量PCR方法测定支气管肺泡灌洗液上清和血浆中游离GAPDH mRNA的拷贝数。常规细胞学方法检测肺泡灌洗液脱落细胞。应用受试者工作特征(ROC)曲线分析方法评价游离RNA诊断价值。采用CA-1500型全自动血凝分析仪测定两组血液的凝血指标。
结果:(1)肺癌及良性疾病组BALF和血浆中均可检测到游离RNA的存在。(2)肺癌患者BALF中游离RNA拷贝数明显高于肺良性病变组(t=3.457,P=0.001),但血浆中游离RNA两组比较差别无统计学意义。(3)肺泡灌洗液细胞学检查肿瘤细胞阳性组游离RNA拷贝数明显高于阴性组(t=2.094,P=0.024)。(4)肺癌游离RNA拷贝数在不同的病理类型、临床分期中无差异(P>0.05)。(5) ROC曲线分析肺泡灌洗液游离RNA对肺癌的诊断价值: ROC曲线最佳工作点对应的游离RNA拷贝数对数、敏感性和特异性分别为4.433、86.0%、76.5%,ROC曲线下面积0.882。当特异性为100%时,诊断的敏感性为53.5%。(6)肺癌组血浆纤维蛋白原含量明显高于肺良性疾病组(t= 2.427,P= 0.018)。(7)肺癌血浆游离RNA拷贝数的对数与纤维蛋白含量呈正相关(r=0.668,P<0.05)。
结论:应用荧光定量PCR方法检测BALF中游离RNA诊断肺癌具有较高的特异性、敏感性,对肺癌的诊断具有一定的价值。肺癌患者血液处于高凝状态,游离RNA可能参与了肺癌患者高凝状态形成。
Objective: Recently the studies confirmed the existence of cell-free RNA which was of tumor origin. GAPDH mRNA which is one of housegroup genes encodes glyceraldehyde phosphate dehydrogenase proteins.GAPDH mRNA is one of most copiousest gene in the body fluid. Base on some promising data, we established real-time reverse transcription polymerase chain reaction(RT-PCR) with primers specific for GAPDH) gene to investigate the clinical value of cell-free RNA in bronchoalveolar lavage fluid supernatant (BALF) and plasma in patients with lung cancer.
Methods:The copies of cell-free RNA in the plasma and BALF were measured by real time quantitative PCR in 43 patients with lung cancer and 17 patients with benign lung diseases. The sediment of BALF were examined by cytologists. The cell-free RNA copies in two groups were analyzed by receiver operating characteristic (ROC) curve. Sysmex CA -1500 automated coagulation analyzer was used to detect blood coagulation index in 60 patients.
Results:(1) Cell-free RNA were dectected in the BALF and plasma of two group.(2)The copies of BALF RNA in lung cancer group were much higher than those in benign disease group (t=3.457, P =0.001) , But the difference of cell free RNA level in the plasma between cancer patients and benign group was not significant. (3)It was also found that the copies of RNA in the BALF with positive cancer cells were higher than those with the negative cancer cells(t=2.094, P=0.024). (4)The correlation between the clinical pathological datas and cell free RNA level was not found. (5) ROC of cell-free RNA in BALF analysis showed the best diagnostic cut-off point is 4.433,which generated diagnostic sensitivity of 86.0% and specificity of 76.3%. When the sensitivity was 53.5%,the specificity could reach 100%, and area under curve (AUC)of 0.882. (6) Fibrinogen in lung cancer patients were significant higher than benign disease group (t=2.427,P=0.018) (7) There were positive correlation between the fibrinogen and the Log of RNA copies in plasma of lung cancer patients(r=0.669,P<0.05).
Conclusion:The measurement of cell-free RNA in BALF using real-time PCR has sound sensitivity and specificity in diagnosis of lung cancer, It is suggested that cell-free RNA might have a potential value for detecting lung cancer. Cell-free RNA perhaps result in hypercoagulabale state which mostly existed in patients with lung cancer.
引文
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