微流控技术在癌细胞标记中的应用

针对癌症的早期诊断是其治疗的一大突破口,大量研究结果表明,早期诊断能大幅提高癌症的治愈率。由于早期肿瘤体积较小和发病位置较隐蔽,导致常规检测难度上升。近年来,随着微流控技术的发展,其在生物标记领域有着越来越重要的作用。文章主要对现阶段几种癌症早期诊断的标记技术进行阐述,通过对比重点介绍了微流控技术在癌细胞标记中的应用。

PCR和癌细胞标记物分析

近年来 PCR 技术研究进展颇快,已初步应用于肿瘤临床、分子生物学、诊断和流行病学究研中,被认为是肿瘤研究中带有革命性的技术。本文结合 PCR 技术检测 t(14;18)易位和 ERmRNA 表达的研究实例,将 PCR 技术与传统的方法比较,认为 PCR 技术最终将取代现有的许多试验方法,同时对 PCR 结果评价和注意事项作了评估,并对 PCR 在肿瘤其它方面的研究进展作了简介。本文对了解PCR 的最新研究进展,开阔肿瘤基础及临床研究视野有重要参考价值。

^(131)I标记抗卵巢癌细胞膜相关抗体体外杀伤活性的实验研究

目的:研究131I标记抗卵巢癌细胞膜相关抗体体外对卵巢癌细胞株细胞的杀伤能力。方法:采用补体介导细胞毒性试验检测131I标记抗卵巢癌细胞膜相关抗体不同剂量以及不同时间段对卵巢癌细胞细胞毒作用的表现。结果:131I标记抗卵巢癌细胞膜相关抗体对靶细胞的杀伤率明显高于131I加抗体混合组、单纯131I、单纯抗体;最佳剂量为3.18×1011Bq/g,最佳作用时间为24~36h。结论:131I标记抗卵巢癌细胞膜相关抗体对卵巢癌细胞有明显的体外杀伤作用且有一定的时间、剂量依赖性。

The prognostic molecular markers in hepatocellular carcinoma

The prognosis of hepatocellular carcinoma (HCC) still remains dismal, although many advances in its clinical study have been made. It is important for tumor control to identify the factors that predispose patients to death. With new discoveries in cancer biology, the pathological and biological prognostic factors of HCC have been studied quite extensively. Analyzing molecular markers (biomarkers) with prognostic significance is a complementary method. A large number of molecular factors have been shown to associate with the invasiveness of HCC, and have potential prognostic significance. One important aspect is the analysis of molecular markers for the cellular malignancy phenotype. These include alterations in DNA ploidy, cellular proliferation markers (PCNA, Ki-67, Mcm2, MIB1, MIA, and CSE1L/CAS protein), nuclear morphology, the p53 gene and its related molecule MD M2, other cell cycle regulators (cyclin A, cyclin D, cyclin E, cdc2, p27, p73), oncogenes and their receptors (such as ras, c-myc, c-fms, HGF, c-met, and erb-B receptor family members), apoptosis related factors (Fas and FasL), as well as telomerase activity. Another important aspect is the analysis of molecular markers involved in the process of cancer invasion and metastasis. Adhesion molecules (E-cadherin, catenins, serum intercellular adhesion molecule-1, CD44 variants), proteinases involved in the degradation of extracellular matrix (MMP-2, MMP-9, uPA, uPAR, PAI), as well as other molecules have been regarded as biomarkers for the malignant phenotype of HCC, and are related to prognosis and therapeutic outcomes. Tumor angiogenesis is critical to both the growth and metastasis of cancers including HCC, and has drawn much attention in recent years. Many angiogenesis-related markers, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived endothelial cell growth factor (PD-ECGF), thrombospondin (TSP), angiogenin, pleiotrophin, and endostatin (ES) levels, as well as intratumor microvessel density (MVD) have been evaluated and found to be of prognostic significance. Body fluid (particularly blood and urinary) testing for biomarkers is easily accessible and useful in clinical patients. The prognostic significance of circulating DNA in plasma or serum, and its genetic alterations in HCC are other important trends. More attention should be paid to these two areas in future. As the progress of the human genome project advances, so does a clearer understanding of tumor biology, and more and more new prognostic markers with high sensitivity and specificity will be found and used in clinical assays. However, the combination of some items, i.e., the pathological features and some biomarkers mentioned above, seems to be more practical for now.

奈达铂+5-氟尿嘧啶联合放疗对食管鳞癌的临床疗效及其作用机制

目的探讨奈达铂+5-氟尿嘧啶(5-Fu)联合放疗对食管鳞癌的临床疗效及对患者外周血中热休克蛋白90a(HSP90a)、癌胚抗原(CEA)及CK20蛋白表达的影响。方法回顾性分析2014年1月至2017年1月在海安医院肿瘤科接受放化疗的130例中晚期食管鳞癌患者的临床资料。按照给药方案不同,分为试验组(67例)和对照组(63例),试验组采用奈达铂+5-Fu方案化疗联合放疗;对照组采用顺铂+5-Fu方案化疗联合放疗。观察并比较6个疗程后两组患者临床疗效、副反应及患者外周血中热休克蛋白90a、CEA、CK20蛋白水平变化。结果试验组临床疗效高于对照组,差异有统计学意义(P <0. 05);试验组患者胃肠道反应和肝肾功能损伤发生率(22. 00%,5. 00%)均低于对照组(43. 00%,24. 00%),差异有统计学意义(P <0. 05);试验组治疗后HSP90a、CEA及CK20蛋白水平低于对照组,差异有统计学意义(P <0. 05)。结论奈达铂+5-Fu联合放疗治疗食管癌疗效优于顺铂+5-Fu联合方案,可降低患者外周血HSP90a、CEA及CK20表达水平。

Assessment of KL-6 as a tumor marker in patients with hepatocellular carcinoma

AIM： To investigate the clinical significance of KL-6 as a tumor marker of HCC in two different ethnic groups with chronic liver disease consecutively encountered at outpatient clinics. METHODS： Serum KL-6 was measured by the sandwich enzyme immunoassay method using the KL-6 antibody （Ab） as both the capture and tracer Ab according to the manufacturer＇s instructions （Eisai, Tokyo, Japan）. Assessment of alpha fetoprotein （AFP） and protein induced vitamin K deficiency or absence （PIVKA-II） was performed in both groups using commercially available kits. RESULTS： A significantly higher mean serum KL-6 （556±467 U/L） was found in HCC in comparison with non-HCC groups either with （391±176 U/L; P〈0.001） or without （361±161 U/L; P〈0.001） liver cirrhosis （LC）. Serum KL-6 level did not correlate with either AFP or PIVKA-II serU/Levels. Using rec：eiver operating curve analysis for KL-6 as a predictor for HCC showed that the area under the curve was 0.574 （95%CI = 0.50-0.64） and the KL-6 level that gave the best sensitivity （61%） was found to be 334 U/L but according to the manufacturer＇s instructions; a cut-off point of 500 U/L was used that showed the highest specificity （80%） in comparison with AFP and PIVKA-II （78% vs 72% respectively）. Combining the values of the three markersimproved specificity of AFP for HCC diagnosis from 78% for AFP alone; 93% for AFP plus PIVKA-II to 99% for both plus KL-6 value （P〈0.001）. Mean serum alkaline phosphatase level was significantly higher in KL-6 positive （564＋475） in comparison with KL-6 negative （505＋469） HCC patients （P = 0.021）, but such a difference was not found among non-HCC corresponding groups. CONCLUSION： KL-6 is suggested as a tumor for HCC. Its positivity may reflect HCC-associated cholestasis and/ or local tumor invasion.

DEC1 nuclear expression:A marker of differentiation grade in hepatocellular carcinoma

AIM: To investigate the expression patterns of human differentiated embryo chondrocyte 1 (DEC1) in hepatocellular carcinoma (HCC) and corresponding adjacent non-tumor and the normal liver tissues, the association between DEC1 expression and histopathological variables and the role of DEC1 in hepatocarcinogenesis. METHODS: The expression of DEC1 was detected immunohistochemically in 176 paraffin-embedded sections from 63 patients with HCC and 50 subjects with normal liver tissues. RESULTS: DEC1 protein was persistently expressed in the cytoplasm of hepatocytes in normal liver and HCC tissues. Compared with adjacent non-tumor liver tissues, HCC tissues showed high nuclear expression of DEC1 protein. However, high DEC1 nuclear expression was more frequently detected in well-differentiated (83.3%) than in moderately (27.3%) and poorly differentiated HCC (16.7%). Low DEC1 expression was associated with poor histological differentiation and malignancy progression. A correlation was found between the nuclear expression of DEC1 protein and histological differentiation (r = 0.376, P = 0.024). CONCLUSION: DEC1 is expressed in the cytoplasm of hepatocytes and because nuclear DEC1 expression is decreased with decreasing differentiation status of HCC, nuclear DEC1 might be a marker of HCC differentiation.

Role of CD97^(stalk) and CD55 as molecular markers for prognosis and therapy of gastric carcinoma patients

Objectives： To explore the mechanism of development and aggressiveness in gastric carcinomas by investigating the expression and role of CD97 and its cellular ligand CD55 in gastric carcinomas. Methods： Tumor and corresponding normal mucosal tissue, collected from 39 gastric carcinoma patients, were examined by immunohistochemistry and RT-PCR for the expression of CD97 and CD55. Results： CD97^stalk was strongly stained on scattered tumor cells or small tumor cell clusters at the invasion front of gastric carcinomas. The expression of CD97^stalk was frequently observed in tumors of stage Ⅰ and T1 gastric carcinoma patients. The expression of CD97^stalk between Stage Ⅰ and Stage Ⅱ, Ⅲ, Ⅳ specimens showed significant difference （P〈0.05）, between T1 and T2, T3, T4 specimens also showed significant difference （P〈0.05）. Specimens with tumor invasion depth limited in mucosa of T1 specimens showed higher positive CD55 expression than specimens with the same tumor invasion depth in T2, T3, T4 specimens, the expression of CD55 between T1 and T2, T3, T4 specimens was significantly different （P〈0.05）. There was strong correlation between the distribution patterns of CD97^stalk and CD55 on tumor tissues （r=0.73, P〈0.05）. Signet ring cell carcinomas frequently contained strong CD97^stalk and CD55-staining. Conclusions： Our results suggest that CD97^stalk is probably involved in the growth, invasion and aggressiveness of gastric carcinomas by binding its cellular ligand CD55. CD97^stalk and CD55 could be useful as molecular markers for prognosis and therapy of gastric carcinoma patients.

Vascular architecture:is it a helpful histopathological biomarker for hepatocellular carcinoma?

Hepatocellular carcinoma(HCC) remains one of the major public health problems throughout the world.Although originally associated with tumorigenic processes,liver angiogenesis has also been observed in the context of different liver in-flammatory,fibrotic,and ischemic conditions.Here we investigate the fractal dimension as a quantitator of non-Euclidean two-dimensional vascular geometry in a series of paired specimens of primary HCC and surrounding non-tumoral tissue,and discuss why this parameter might provide additional information regarding cancer behavior.The application of fractal geometry to the measurement of liver vascularity and the availability of a computer-aided quantitative method can eliminate errors in visual interpretation,and make it possible to obtain closer-to-reality numerals that are compulsory for any measurement process.

Alpha-fetoprotein expression is a potential prognostic marker in hepatocellular carcinoma

AIM： To characterize the alpha-fetoprotein （AFP） positive and negative hepatocellular carcinoma （HCC） samples. METHODS： Thirty-seven paraffin-embedded human HCC samples were analyzed by immunohistochemistry for the following antigens： AFP,β-catenin, p53, CD44, MSH-2, MLH-1, and HNF-4. The tumors were divided into two groups based on the AFP expression. The immunophenotypic data and important clinical parameters were studied between the two groups. RESULTS： Twenty-one of the thirty-seven examined HCCs were AFP positive. Seven with nudear p53 staining were AFP positive, while seven tumors with nuclear β-catenin staining were AFP negative. CD44 staining and high histological tumor grade were more frequent among the AFP-positive HCCs. The other immunophenotypical and dinical parameters did not show statistically significant difference in their distribution between the AFP positive and negative samples. CONCLUSION： AFP expression in HCC correlates with unfavorable prognostic factors, while nuclear β-catenin positivity is more common among the AFP-negative liver tumors. This observation supports the microarray data on in vivo human tumors.

Prognostic value of cancer stem cell markers CD133, ALDH1 and nuclear β-catenin in colon cancer

Objective： Colon cancer is one of the most common human malignancies. Cancer stem cells （CSCs）, despite being only a small subset of cancer cells, have the capability to self-renew and sustain the tumor. They also have the ability to proliferate. Multiple CSCs-associated markers have been identified in colon cancer including CD133, ALDH1 and β-catenin. The aim of the work was to study the prognostic value of CSCs markers （CD133, ALDH 1 and β-catenin）, as well as their rela- tionship to clinicopathological features of colon cancer. Methods： CD133, ALDH1 and β-catenin proteins expression was as- sessed immunohistochemically in a series of colon cancers and their prognostic significance was evaluated. Results： CD133 expression showed significant relationship to tumor stage and lymph node metastasis （P-value 0.004 ＆ 〈 0.001 respectively）, and near significant relationship to liver metastasis （P-value 0.092）. ALDH1 was significantly associated with tumor grade, stage and nodal metastasis （P-value 0.021,0.001 and 0.026 respectively）, but its relationship to liver metastasis was near sig- nificant （P-value 0.068）. Nuclear β-catenin was significantly related to tumor grade, stage, nodal and liver metastasis （P-value 0.001, 〈 0.001, 〈 0.001 and 0.008 respectively）. Overall survival （OS） was associated inversely with CD133, ALDH1 positivity, and directly with nuclear 13-catenin posiUvity （P-value 〈 0.001,0.0001 and 〈 0.001 respectively）. Also recurrence free survival （RFS） was associated inversely with CD133, ALDH1 and directly with nuclearβ-catenin positivity （P-value 0.0001,0.001 and 〈 0.001 respectively）. Conclusion： CD133, ALDH1 and β-catenin expressions of tumor cells have significant impact upon malignant progression of colon cancer and thus patient survival and tumor recurrence. Hence they can be used to predict outcome of colon cancer patients.

Establishment of a real-time PCR for quantifying transforming growth factor beta1 in blood of hepatocellular carcinoma patients

Background: The carcinogenesis of hepatocellular carcinoma (HCC) is a multi-factorial, multistep and complex process. Its prognosis is poor and early detection is of the utmost importance. Transforming growth factor β1 (TGF-β1) message RNA (mRNA) has been reported to be elevated in HCC patients using Northern blotting. However, little work has been done about the detection of TGF-β1 mRNA levels in peripheral blood of patients with HCC using the real-time polymerase chain reactions (PCR) method. Objective: To assess the prognostic value of quantitative levels of TGF-β1 mRNA in peripheral blood of patients with HCC, and to investigate the relationship between the expression of TGF-β1 mRNA in peripheral blood and many diagnostic and pathological factors. Methods: We developed an optimized Taqman real-time PCR to quantify TGF-β1 mRNA in peripheral blood of 53 patients with HCC and 44 healthy volunteers. In addition, blood was collected from patients with HCC for measuring levels of total bilirubin (TBil), prealbumin, albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyltranspeptidase (GGT), alpha-L-fucosidase (AFU), alpha fetoprotein (AFP), carcino-embryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), viral load and platelet counts. Statistical analysis was performed using the SPSS software system (SPSS 10.0). Results: In real-time PCR, fluorescence was detectable in all blood specimens from patients with HCC and healthy volunteers. The levels of TGF-β1 mRNA expression in patients with HCC were significantly higher compared to that in healthy volunteers (P<0.000 1), suggesting an association of the activated TGF-β1 gene transcription with hepato- carcinogenesis. Patients with HCC were divided into 2 groups according to their TGF-β1 mRNA above (group A, n=28)or below (group B, n=25) the mean level. Statistical results demonstrated that TGF-β1 mRNA expression level was correlated with patients age, serum levels of CEA, CA19-9 and viral copy number (P<0.05). Conclusion: Although this is a small sample size pilot study these findings imply that quantitative measurement of TGF-β1 mRNA level in peripheral blood may be a complementary serologic marker of HCC.

Human induced pluripotent stem cells labeled with fluorescent magnetic nanoparticles for targeted imaging and hyperthermia therapy for gastric cancer

Objective: Human induced pluripotent stem(i PS) cells exhibit great potential for generating functional human cells for medical therapies. In this paper, we report for use of human i PS cells labeled with fluorescent magnetic nanoparticles(FMNPs) for targeted imaging and synergistic therapy of gastric cancer cells in vivo. Methods: Human i PS cells were prepared and cultured for 72 h. The culture medium was collected, and then was coincubated with MGC803 cells. Cell viability was analyzed by the MTT method. FMNP-labeled human i PS cells were prepared and injected into gastric cancer-bearing nude mice. The mouse model was observed using a small-animal imaging system. The nude mice were irradiated under an external alternating magnetic field and evaluated using an infrared thermal mapping instrument. Tumor sizes were measured weekly. Results: iP S cells and the collected culture medium inhibited the growth of MGC803 cells. FMNP-labeled human iP S cells targeted and imaged gastric cancer cells in vivo, as well as inhibited cancer growth in vivo through the external magnetic field. Conclusion: FMNP-labeled human i PS cells exhibit considerable potential in applications such as targeted dual-mode imaging and synergistic therapy for early gastric cancer.

Screening study on new tumor marker periplakin for lung cancer

Objective: The aim of this study was to use lung cancer targeting binding polypeptide ZS-9 to screen cDNA library of human lung cancer and obtain ZS-9 specific ligand to confirm tumor marker of non small-cell lung cancer. Methods: Artificially synthesize biotin labeled peptide ZS-9, anchored ZS-9 in the enzyme label plate coupled by avidin, used ZS-9 as probe to screen cDNA library of human lung cancer, after screening, obtained bacteriophage clone specifically binding with anchored polypeptide ZS-9. Extracted plasmid of bacteriophage and performed sequencing after amplified by PCR. Results: It was demonstrated by bioinformatic analysis on the sequence of ligand binded by lung cancer specific peptide ZS-9 that the ligand was the cytoskeletal protein periplakin on the surface of lung cancer cells, suggesting that periplakin might be a new marker for non-small-cell lung cancer in lung cancer. Conclusion: Use specific lung cancer binding peptide to screen new tumor marker periplakin in lung cancer and further studies on its biologic functions in genesis and development of lung cancer are still needed.

Label-free quantification of differentially expressed proteins in mouse liver cancer cells with high and low metastasis rates by a SWATH acquisition method

Label-free quantification is a valuable tool for the analysis of differentially expressed proteins identified by mass spectrometry methods.Herein,we used a new strategy:data-dependent acquisition mode identification combined with label-free quantification by SWATH acquisition mode,to study the differentially expressed proteins in mouse liver cancer metastasis cells.A total of 1528 protein groups were identified,among which 1159 protein groups were quantified and 249 protein groups were observed as differentially expressed proteins(86 proteins up-regulated and 163 down-regulated).This method provides a commendable solution for the identification and quantification of differentially expressed proteins in biological samples.