免疫磁珠联合RT-PCR检测胃癌肿瘤标志物α4GnT

目的：研究α4GnT mRNA的转录本在胃癌患者和消化性溃疡患者外周血中的表达,并分析其在胃癌及消化性溃疡两种疾病间的差别及与胃癌预后的关系。方法：免疫磁珠分选25例胃癌患者及10例消化性溃疡患者外周血中上皮来源的细胞联合RT-PCR法检测α4GnT mRNA的转录本。结果：α4GnT mRNA在胃癌与消化性溃疡患者中阳性率分别60%（15/25）和40%（4/10）,两者之间的差异无显著性（P〉0.05）。α4GnT mRNA在早期胃癌与进展期胃癌患者中阳性率分别17%（1/6）和79%（15/19）,两者之间的差异有显著性（P〈0.05）。结论：运用免疫磁珠联合RT-PCR检测外周血肿瘤标志物α4GnT在鉴别胃癌与消化性溃疡方面无意义,但可作为预测胃癌预后的指标之一。     

肝癌患者PBMC中TH1/TH2类细胞因子mRNA表达

目的探讨TH1类细胞因子IL-2、IFN-γ及TH2类细胞因子IL-4mRNA在Ⅰ期和Ⅱ期肝癌患者外周血单个核细胞(PBMC)中的表达模式,并观察IL-2、IFN-γmRNA在手术前后的表达变化。方法采用逆转录聚合酶链反应(RT-PCR)方法测定8例Ⅰ期和10例Ⅱ期原发性肝癌患者手术前后及15例健康人外周血中IL-2、IFN-γ的表达,并用凝胶图像分析系统进行图像分析。结果在18例肝癌患者中,IL-2、IFN-γmRNA的表达率分别为16.7%、5.6%,低于IL-4的表达率88.9%(P<0.05),呈现明显的TH2偏移状态,且IL-2、IFN-γ的表达强度在0.10~0.11之间,也远低于IL-4的0.21~0.22;在8例Ⅰ期肝癌患者中,术前有25.0%(2/8)表达IL-2mRNA,12.5%(1/8)表达IFN-γ,高于对照组的13.3%(2/15)、5.6%(1/15);手术治疗后两者的表达率分别是50.0%、37.5%,高于术前组;术前组及正常对照组两种细胞因子mRNA表达强度(0.10~0.12)明显低于术后组的0.20~0.22(P<0.05)。另外,10例Ⅱ期患者手术前后术均无IFN-γm...     

CD133 mRNA在胃癌患者外周血单核细胞中的表达及意义

[目的]通过检测胃癌患者外周血单核细胞中CD133的表达,探讨其与临床病理特征的关系。[方法]于术前抽取30例胃癌患者、15例胃溃疡患者、15名健康志愿者外周静脉血,密度梯度离心法分离单核细胞。半定量反转录聚合酶链反应检测CD133 mRNA表达水平。分析胃癌患者外周血CD133 mRNA表达与临床病理特征的关系。[结果]胃癌患者外周血中CD133mRNA表达水平明显高于胃溃疡患者和健康志愿者（0.2804±0.1835 vs 0.0984±0.1321,t=6.724,P〈0.001;0.2804±0.1835 vs 0.0282±0.0597,t=-7.327,P〈0.001）。CD133 mRNA表达与肿瘤最大直径、淋巴结转移、TNM分期有关（P〈0.05）,而与性别、分化程度、淋巴管浸润、血管浸润、浸润深度无关。Spearman相关分析显示,CD133 mRNA表达与淋巴结转移呈正相关（P〈0.01）。[结论]胃癌患者外周血中CD133 mRNA的表达与胃癌的发展、转移及预后密切相关。     

尿路移行细胞癌微转移的检测

目的 探讨尿路移行细胞癌患者外周血CK20的表达及临床意义.方法 采用逆转录聚合酶链反应(RT-PCR)检测47例尿路移行细胞癌患者和14例健康志愿者以及18例非肿瘤患者外周血CK20的表达.以GAPDH作为内参照.结果 14例健康志愿者和18例非肿瘤患者外周血CK20的表达均为阴性;47例尿路移行细胞癌患者外周血CK20阳性表达率为21.3%(10/47):T1为0(0/29),T2为20%(2/10),T3为100%(4/4),T4为100%(4/4).随访12个月,6例(T2～T3)外周血CK20阳性的尿路移行细胞癌有3例(T3G2膀胱癌1例,TaG2和T3G2输尿管癌各1例)发生远处转移.结论 检测外周血CK20表达,可以提示癌细胞的血行播散,对判断预后以及指导治疗具有一定临床价值.     

人乳腺癌外周血中hMAM mRNA表达及临床意义

目的：研究人乳腺癌外周血珠蛋白（hMAM）mRNA表达及临床意义。方法：应用nested-RT-PCR技术检测50例乳腺癌外周血hMAM mRNA的表达和化疗前后外周血hMAM mRNA的变化,并取20例乳腺纤维腺瘤、10例健康志愿者作阴性对照。结果：50例乳腺癌外周血hMAM mRNA的阳性表达率为34.0%;20例乳腺纤维腺瘤及10例健康志愿者外周血中均未检出hMAM mRNA的表达。hMAM mRNA的阳性与淋巴结转移状况、肿瘤TNM分期之间差异有统计学意义;50例乳腺癌化疗前检测17例hMAM mRNA阳性,化疗2～3周期后13例hMAM mRNA转为阴性,转阴率76.5%（13/17）,化疗前后差异非常显著（P=0.001）。结论：乳腺癌外周血中hMAM mRNA表达可以反映术后化疗对乳腺癌血液微转移的影响,有望成为乳腺癌血道微转移标志物和一个重要的预后指标。     

Glycosylation of MUC6 by α1,4‐linked N‐acetylglucosamine enhances suppression of pancreatic cancer malignancy

Biomarkers for early diagnosis of pancreatic cancer are greatly needed, as the high fatality of this cancer is in part due to delayed detection. α1,4‐linked N‐acetylglucosamine (αGlcNAc), a unique O‐glycan specific to gastric gland mucus, is biosynthesized by α1,4‐N‐acetylglucosaminyltransferase (α4GnT) and primarily bound at the terminal glycosylated residue to scaffold protein MUC6. We previously reported that αGlcNAc expression decreases at early stages of neoplastic pancreatic lesions, followed by decreased MUC6 expression, although functional effects of these outcomes were unknown. Here, we ectopically expressed α4GnT, the αGlcNAc biosynthetic enzyme, together with MUC6 in the human pancreatic cancer cell lines MIA PaCa‐2 and PANC‐1, neither of which expresses α4GnT and MUC6. We observed significantly suppressed proliferation in both lines following coexpression of α4GnT and MUC6. Moreover, cellular motility decreased following MUC6 ectopic expression, an effect enhanced by cotransduction with α4GnT. MUC6 expression also attenuated invasiveness of both lines relative to controls, and this effect was also enhanced by additional α4GnT expression. We found αGlcNAc‐bound MUC6 formed a complex with trefoil factor 2. Furthermore, analysis of survival curves of patients with pancreatic ductal adenocarcinoma using a gene expression database showed that samples marked by higher A4GNT or MUC6 mRNA levels were associated with relatively favorable prognosis. These results strongly suggest that αGlcNAc and MUC6 function as tumor suppressors in pancreatic cancer and that decreased expression of both may serve as a biomarker of tumor progression to pancreatic cancer.     

Overexpression of beta 1,4N-acetylgalactosaminyl- transferase mRNA as a molecular marker for various types of cancers

OBJECTIVE: To determine GalNAcT mRNA expression in human carcinoma cell lines and primary tumor tissues. Assessment of the potential use of GalNAcT mRNA as a molecular marker for detection of metastatic cancer cells in the peripheral blood of patients with hepatocellular carcinoma. METHODS/RESULTS: We investigated GalNAcT mRNA expression in various human cancer cell lines and primary cancer tissues using RT-PCR assay for GalNAcT mRNA. The expression of GalNAcT mRNA was detected in 25 of 26 cancer cell lines tested and in the majority of primary tumors from different organs: 8 of 10 colon cancers, 9 of 9 breast cancers, 11 of 12 esophageal cancers, 14 of 14 gastric cancers, 4 of 18 pancreatic cancers, 6 of 12 biliary tract cancers, 17 of 18 hepatocellular carcinomas and 13 of 14 lung cancers. Semi-quantitative analysis with duplex RT-PCR showed that the amount of the GalNAcT mRNA was enhanced in cancer tissues as compared to the surrounding cancer-free tissues. Blood specimens of 5 of 14 patients with hepatocellular carcinoma were positive for GalNAcT mRNA, all of whom developed recurrent disease in less than 24 months. Peripheral blood samples of 30 normal subjects were negative for GalNAcT mRNA. CONCLUSION: Our results suggest that the RT-PCR assay for GalNAcT mRNA could be a potentially useful molecular marker for detecting cancer dissemination in blood circulation of patients with malignancy.     

外周血单个核细胞端粒酶hTERT mRNA与葡萄胎恶变的临床研究

目的：了解外周血单个核细胞hTERT mRNA在葡萄胎自然转归及恶变中的表达变化,探讨其在判断葡萄胎预后中的应用价值。方法：FQ-RT-PCR法检测葡萄胎患者外周血单个核细胞hTERT mRNA的表达水平;将hTERT与GAPDH拷贝数之比的100倍作为标准化hTERTNhTERT）;随访每一例葡萄胎患者,根据预后将其分为试验组8例（恶变组）和对照组22例（自然转归组）;比较两组葡萄胎患者外周血单个核细胞hTERT mRNA的表达水平。结果：葡萄胎实验组和对照组外周血单个核细胞hTERT mRNA,分别为6.31±0.32和1.21±0.65,两者比较有显著差异（P〈0.01）。结论：FQ-RT-PCR方法能对端粒酶hTERT mRNA进行准确高效的定量检测;外周血单个核细胞端粒酶hTERT mRNA的表达水平可能用于早期判断葡萄胎的预后。     

Clinicopathological significance of core 2 beta1,6-N-acetylglucosaminyltransferase messenger RNA expressed in the pulmonary adenocarcinoma determined by in situ hybridization

Cell surface carbohydrates of epithelial cells play important roles in tumor progression. Previously, we have shown that expression of core 2 branched O-glycans in colorectal cancer is closely correlated with the vessel invasion and depth of invasion (K. Shimodaira et al., Cancer Res., 57: 5201-5206, 1997). To test whether this is also the case in human lung cancer, we have examined the expression pattern of core 2 beta1,6-N-acetylglucosaminyltransferase (C2GnT) mRNA responsible for the biosynthesis of core 2 branched O-glycans in 41 cases of lung cancer. Using in situ hybridization, C2GnT mRNA was detected in 73.2% of the lung cancer cells, irrespective of the histopathological type; whereas in normal lung tissues, its expression was restricted to the basal cells of bronchial mucosa. These results indicate that the expression level of C2GnT mRNA was significantly enhanced in association with malignant transformation. Statistical analysis between the C2GnT mRNA expressed in pulmonary adenocarcinoma and clinicopathological variables revealed that the expression of C2GnT was correlated with vessel invasion and lymph node metastasis with significant difference (P < 0.05), but expression of sialyl Le(x), which is frequently expressed in the adenocarcinoma, was not significantly correlated with lymph node metastasis. These results indicate that C2GnT mRNA detected by in situ hybridization reflects the malignant potentials of pulmonary adenocarcinoma, because lymph node metastasis is the most affecting factor to the patients' prognosis.     

单纯检测外周血CD44v6不宜作为筛选肿瘤转移的标志物

目的通过检测CD44v6剪接体在正常人外周血的表达情况,进而评价其作为肿瘤转移分子标志物的价值.方法提取50例健康志愿者的外周血单个核细胞,应用RT-PCR的办法检测CD44v6基因的表达,并对其产物进行DNA测序.结果在适当的对照控制条件下,50例外周血中有29例(58%)CD44v6 mRNA阳性.结论在普通人群外周血样本中应用RT-PCR的方法CD44v6检出率较高,因此单纯检测CD44v6不适宜作为筛选肿瘤转移的分子标记物.     

外周血单个核细胞中Nrf2mRNA表达水平与化疗后骨髓抑制程度的相关性分析

目的:探讨肿瘤患者化疗前外周血有核细胞中核因子E2相关因子2(nuclear factor erythroid-2-related factor 2,Nrf2)mRNA表达水平的个体差异与化疗所致骨髓抑制程度的相关性.方法:采用半定量RT-PCR法检测30例肿瘤患者化疗前外周血有核细胞中Nrf2 mRNA的表达水平,并分析其与肿瘤患者化疗后骨髓抑制程度的相关性.结果:肿瘤患者化疗前外周血单个核细胞中Nrf2 mRNA表达水平存在明显的个体差异;其在化疗后发生0～1度白细胞或中性粒细胞减少患者中的表达水平明显高于2～4度的患者(P＜0.05),但与患者的年龄、性别、治疗方案及肿瘤类型等无相关性.相关性分析表明,外周血单个核细胞中Nrf2 mRNA的表达水平与白细胞减少(r=-0.448,P=0.013)和中性粒细胞减少(r=-0.493,P=0.006)呈显著负相关.结论:化疗前外周血单个核细胞中Nrf2 mRNA表达水平与化疗所致骨髓抑制具有相关性,因此,其可能成为一种预测化疗所致骨髓抑制程度的理想标志物,用于指导临床方案的选择及骨髓抑制的早期防治.     

Detection of cadherin-6 mRNA by nested RT-PCR as a potential marker for circulating cancer cells in renal cell carcinoma

To detect circulating RCC cells, we established a nested RT-PCR system for cadherin-6 mRNA, which is specifically expressed in RCC. A total of 121 samples of peripheral blood (34 healthy volunteers and 87 patients with RCC) were analyzed in this study. Total RNA of the monocyte fraction of the blood was extracted, then nested RT-PCR using specific primers was performed to detect mRNA of N-cadherin or cadherin-6. Nested RT-PCR revealed that expression of cadherin-6 mRNA was not present in the blood of most healthy volunteers (absent in 32/34), but positive expression was observed in the blood at concentrations of 10 cells/ml or greater of the SKRC-33 RCC cell line, which is a strong expresser of cadherin-6. In peripheral blood from patients with metastatic disease, cadherin-6 mRNA was detected in 70.4% (19/27). Messenger RNA of cadherin-6 was detectable in 45.0% (27/60) of patients with localized tumors. The PCR-based detection system for peripheral blood samples from patients with metastatic disease could reveal the presence of circulating RCC cells in the blood. Detection of cadherin-6 mRNA in non-metastatic presurgical RCC patients suggests that careful follow-up study is necessary in these patients.     

RT-PCR方法检测大肠癌患者外周血中组织特异性mRNA的表达及其临床意义

目的：检测大肠癌患者外周血中组织特异性mRNA的表达并分析其临床意义。方法：用RT－PCR方法检测70例大肠癌患者和35例正常健康人外周血中组织特异性mRNA的表达。结果：70例大肠癌患者中有30例外周血中组织特异性mRNA表达阳性，阳性率为42．9％，与健康对照人群相比差异有显著性（P＜0．05），DukesC和Dukes D期大肠癌患者外周血中组织特异性mRNA的表达高于Dukes A期大肠癌患者（P＜0．05），在不同分化程度大肠癌患者外周血中的表达差异无显著性（P＞0．05），手术后比手术前的阳性表达率低，但差异无显著性（P＞0．05），结论：大肠癌患者外周血中组织特异性mRNA的阳性表达同肿瘤的分期有关，可作为大肠癌微转移的监测指标。     

乳腺癌患者外周血SBEM mRNA的检测及其临床意义

背景与目的乳腺癌患者的死亡原因几乎均为肿瘤远处转移。目前乳腺癌诊断尚无公认的特异性标志物,本文旨在探讨特异性的乳腺癌标志物─乳腺小粘蛋白(smallbreastepithelialmucin,SBEM)在乳腺癌外周血的表达及其意义。方法用巢式逆转录聚合酶链反应(Nested-RT-PCR)技术检测67例乳腺癌、16例乳腺良性肿瘤及20例健康志愿者外周静脉血中的SBEMmRNA的表达情况。结果SBEMmRNA在健康志愿者及乳腺良性肿瘤患者外周血中表达均为阴性;67例乳腺癌患者外周血中SBEMmRNA表达率为50.7%(34/67),在乳腺癌患者的临床分期Ⅰ、Ⅱ、Ⅲ和Ⅳ期中SBEMmRNA表达率分别为25.0%(2/8)、45.8%(11/24)、43.8%(7/16)和73.7%(14/19),在Ⅳ期乳腺癌患者外周血中SBEMmRNA的检出率明显高于其他各期(P<0.05)。SBEMmRNA的表达与患者年龄、原发癌灶大小、病理类型、ER和PR的状态无关(P>0.05)。结论SBEMmRNA特异表达于乳腺癌患者的外周血,有可能作为判断乳腺癌血道微小转移的指标之一。     

乳腺癌患者外周血hMAM及SBEM检测及其临床意义

目的研究乳腺癌患者外周血人乳腺珠蛋白（hMAM）及乳腺上皮粘蛋白（SBEM）的表达及临床意义。方法应用逆转录聚合酶链反应（RT-PCR）技术检测58例乳腺癌患者外周血hMAM mRNA及SBEM mRNA的表达，并用健康志愿者及乳腺良性肿瘤患者各10例作为对照。结果hMAM及SBEM在58例乳腺癌患者外周血的阳性表达率分别为34.5%、25.9%，乳腺良性肿瘤和正常对照均无阳性表达，两者的阳性表达与患者的临床分期显著相关（P〈0.05），两者联合检测的总阳性率为41.4%（24/58），与单一标志物相比缺乏统计学意义。结论hMAM及SBEM特异性表达于乳腺癌外周血，可作为检测乳腺癌外周血肿瘤细胞标志物，并有可能在乳腺癌微转移诊断和预后判断中具有重要意义。