原位细胞凋亡的荧光、酶免疫化学法检测实验研究

用荧光法和酶免疫化学法末端转移酶介导的ｄＵＴＰ缺口末端标记（ＴＵＮＥＬ）实验分别对小鼠心肌组织、人慢性肝炎肝组织和内膜增生的小型猪血管组织进行了细胞凋亡检测。结果显示：①心肌无凋亡细胞发现，肝组织和血管内膜组织有散在的０～１２个细胞／２５倍物镜视野的细胞标记阳性（凋亡）。②酶免疫化学法ＴＵＮＥＬ实验的检测敏感度较荧光法ＴＵＮＥＬ实验的敏感度高。③过高的末端转移酶可导致假阳性的产生，对荧光法ＴＵＮＥＬ实验，末端转移酶／标记缓冲液比例应为１∶９～１９。

血清胸苷激酶1在中老年人肿瘤免疫监测中的作用

目的:用酶免疫化学发光法检测查体者血清胸苷激酶(S-TK1)的含量,探讨S-TK1在中老年人肿瘤免疫监测中的作用。方法:利用抗胸苷激酶单克隆抗体(anti-TK1mAb)建立酶免疫化学发光法,分析72例来我院查体的中老年人S-TK1的水平。结果:72例来我院查体的中老年人S-TK1的X-=0.450,s=1.149。其中3名查体者的S-TK1浓度分别为7.1、1.8和7.0pmol/L,明显高于其他查体者。经随访得知,此3人患有恶性肿瘤。将这3名患者的数据去除,其余69例S-TK1的X-=0.239,s=0.165。结论:提示S-TK1可以作为中老年人肿瘤免疫监测预警的指标之一。

Inhibition of high-mobility group box 1 expression by siRNA in rat hepatic stellate cells

AIM:To explore the role of high-mobility group box 1 (HMGB1) protein during liver fibrogenesis and investigate the functional effects of HMGB1 gene silencing in hepatic stellate cells (HSCs) using siRNA.METHODS:Hepatic fibrosis in rats was induced through serial subcutaneous injections of dimethylnitrosamine,and expression of HMGB1 was detected by immunohistochemistry.HMGB1 siRNAs were developed and transiently transfected into HSC-T6 cells using Lipofectamine 2000.HMGB1 expression was evaluated by real-time polymerase chain reaction (PCR) and Western blotting analysis.Expression of α-smooth muscle actin (α-SMA) and collagen typesⅠand Ⅲ was evaluated by real-time PCR.Cell proliferation and the cell cycle were determined using the methyl thiazolyl tetrazolium method.Finally,collagen content in HSC supernatant was evaluated by an enzyme-linked immunosorbent assay.RESULTS:The results showed that HMGB1 was upregulated during liver fibrosis and that its expression was closely correlated with the deposition of collagen.siRNA molecules were successfully transfected into HSCs and induced inhibition of HMGB1 expression in a time-dependent manner.Moreover,HMGB1 siRNA treatment inhibited synthesis of α-SMA and collagen types Ⅰ and Ⅲ in transfected HSCs.CONCLUSION:This study suggests a significant functional role for HMGB1 in the development of liver fibrosis.It also demonstrates that downregulation of HMGB1 expression might be a potential strategy to treat liver fibrosis.

Aberrant methylation of the 3q25 tumor suppressor gene PTX3 in human esophageal squamous cell carcinoma

AIM:To identify the novel methylation-silenced gene pentraxin 3(PTX3) in esophageal squamous cell carcinoma(ESCC).METHODS:PTX3 mRNA expression was examined in six human ESCC cell lines,one human immortalized normal esophageal epithelial cell line,primary ESCC tumor tissue,and paired adjacent nontumor tissue using reverse transcription polymerase chain reaction(RTPCR).Semi-quantitative immunohistochemistry was used to examine cellular localisation and protein levels.Methylation specific PCR and bisulphite genomic sequencing were employed to investigate the methylation of the candidate gene.RESULTS:In the majority of ESCC cell lines,we found that PTX3 expression was down-regulated due to gene promoter hypermethylation,which was further confirmed by bisulphite genomic sequencing.Demethylation treatment with 5-aza-2'-deoxycytidine restored PTX3 mRNA expression in ESCC cell lines.Methylation was more common in tumor tissues(85%) than in adjacent nontumor tissues(25%)(P < 0.01).CONCLUSION:PTX3 is down-regulated through promoter hypermethylation in ESCC,and could potentially serve as a biomarker of ESCC.

Clinicopathological Features of Non-familial Colorectal Cancer with High-frequency Microsatellite Instability

Objective To explore the clinicopathological features of non-familial colorectal cancer with high-frequency microsatellite instability (MSI-H). Methods One hundred and fifty patients with colorectal cancer who had no family history were enrolled in this study from June 2006 to June 2008. Five standard microsatellite loci including BAT25, BAT26, D2S123, D5S346, and D17S250 were amplified with immunofluorescent polymerase chain reaction. The patient information including age, sex, and tumor location was recorded. Pathological features including differentiation, mucinous differentiation, histological heterogeneity, and Crohn's-like reaction were observed under light microscope. The presence of tumor-infiltrating lymphocytes (TLs, CD4+ and CD8+) was detected by means of immunohistochemistry. A regression equation was obtained by stepwise logistic regression analysis to evaluate the relationship between MSI-H phenotype in colorectal cancer ands pathological features. Results MSI-H phenotype occurred in 13.33% of the 150 patients with non-familial colorectal cancer. Poor differentiation, histological heterogeneity, Crohn's-like reaction, and presence of TLs were found to be independent factors to identify MSI-H non-familial colorectal cancer. Logistic regression equation showed an overall sensitivity of 70.0%, specificity of 99.2%, and accuracy of 95.3% in identifying MSI-H non-familial colorectal cancer. Conclusion MSI-H non-familial colorectal cancer manifests specific pathological features, which may be relied upon for effective identification of that disease.

Study of the Correlation of EGFR and K-RAS Gene Mutations with Its Protein Expression in Non-small Cell Lung Cancer

OBJECTIVE To investigate gene mutations of epidermal growth factor receptor （EGFR） and K-RAS （Kirsten rat sarcoma viral oncogene） in Chinese patients with non-small cell lung cancer （NSCLC）, and study the correlation with its protein expression and its clinical significance on gefitinib.METHODS Detect the EGFR and K-RAS gene mutations status by gene sequencing and use the method of immunohistochemistry to detect EGFR and K-RAS protein expression.RESULTS The frequency of EGFR mutations was 33%, mainly located in exon 19 and exon 21. The frequency of K-RAS mutations was 5.5%, mainly located in codon 12. There was no case which both had EGFR and K-RAS mutations, suggesting a mutually exclusive relationship between the two. EGFR mutations are more common in adenocarcinomas （particularly those with bronchioloalveolar features）, nonsmokers and females. 16% were detected EGFR positive expression and had no correlation with EGFR mutation （P 〉 0.05）, but had significant correlation with mutation in exon 19 （P 〈 0.05）. The frequency of K-RAS positive expression was 52.5% and had no correlation with K-RAS mutation （P 〉 0.05）. Twelve （8 cases were protein-negative） out of 15 gefitinib-treated NSCLC patients with disease control carry EGFR mutations.CONCLUSION EGFR protein expression has some correlation with exon 19 mutations. Combined detection of EGFR and K-RAS gene mutations can help clinicians to choose patients who may benefit from EGFR tyrosine kinase inhibitor （EGFR-TKI） and to predict the response and prognosis of gefitinib.

Expression of MEK1 in the colorectal cancer and its clinical implication

Objective:The aim of this study was to investigate the relationship between expression of MEK1 protein in the mitogen-activated protein kinase (MAPK) signaling pathway and liver as well as lymph node metastasis in colorectal cancer patients.Methods:Immunohistochemistry was performed to detect the expression of MEK1 protein in primary cancer,normal colonic mucosa,lymph nodes and liver metastatic foci from 86 colorectal cancer patients.Life table analysis was employed to evaluate the association between MEK1 expression and patients' survival.Results:The positive rate of MEK1 expression in the primary cancer,normal colonic mucosa,metastatic lymph nodes and liver metastatic foci was 52.3%,32.6%,71.4% and 78.3%,respectively.The positive rate of MEK1 expression in the primary cancer,metastatic lymph nodes and liver metastatic foci was significantly higher than that in the normal colonic mucosa (P < 0.01).Furthermore,the positive rate of MEK1 expression in stage III and IV colorectal cancer patients was dramatically higher than that in stage I and II colorectal cancer patients (P < 0.01).The positive rate of MEK1 expression in patients with poorly differentiated adenocarcinoma and mucinous adenocarcinoma was significantly higher than patients with well or moderately differentiated adenocarcinoma (P < 0.01).The 3-year disease-free survival rate was 41.3% in MEK1 positive patients and 73.1% in MEK1 negative patients.The survival rate of MEK1 positive patients was significantly lower than that of MEK1 negative patients (P < 0.05).Conclusion:The increased expression of MEK1 was associated with lymph node metastasis and liver metastasis of colorectal cancer.Therefore,detection of MEK1 expression may have important significance in the evaluation of patients' prognosis.

Study on the Mechanism of Xiaotan Sanjie Recipe for Inhibiting Proliferation of Gastric Cancer Cells

Objective:To explore the mechanism of Xiaotan Sanjie Recipe (XtSjR, Recipe for dissolving phlegm to eliminate stagnation) in inhibiting proliferation of gastric cancer cells. Methods: The nude mouse human gastric cancer MKN-45 in situ transplantation tumor model was established by use of OB glue, and 40 model mice were randomized into 5 groups, model group, low-dose XtSjR group, middle-dose XtSjR group, high-dose XtSjR group, and 5-Fu group, 8 rats in each group. Human gastric cancer MKN-45 telomerase reverse transcriptase (hTERT) protein and mRNA expressions were assayed by immunohistochemical method and real-time fluorescence quantitative RT-PCR, and influences of XtSjR on the expressions of hTERT protein and mRNA were investigated in the nude mouse human gastric cancer MKN-45 in situ tumor transplantation model. Results: 1) There were significant differences in the mean tumor weight between the low-, middle-, high-dose XtSjR groups and the model group (all P<0.01); 2) There were significant differences in the hTERT positive expression rate between the middle-and high-dose XtSjR groups and the model group (P<0.05 or P<0.01); 3) There were significant differences in the hTERT mRNA content between the middle-and high-dose XtSjR groups and the model group (P<0.05 or P<0.01). Conclusion: 1) XtSjR has a marked inhibitory effect on the growth of gastric cancer cells; 2) XtSjR inhibits telomerase activity by down-regulating the expressions of hTERT protein and mRNA, shortening the length of cancer cell telomeres gradually, losing the ability to infinitely proliferate, and finally inhibiting the growth and proliferation of tumor cells.

Gold nanolabels and enzymatic recycling dual amplification-based electrochemical immunosensor for the highly sensitive detection of carcinoembryonic antigen

A sensitive electrochemical immunoassay system for the detection of a protein tumor biomarker through a dual amplified strategy was reported. Firstly, this protocol involves in the electropolymerization of o-aminobenzoic acid (o-ABA) on a glass carbon electrode (GCE). Subsequently, capture anti-CEA (Abl) is covalently linked to poly(o-ABA) (PAB) film, via N-(3-dimethylamminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysulfosuccinimid sodium salt (NHS) activation of the carboxyl groups and surface blocking with ethanolamine. Later, the target, carcinoembryonic antigen (CEA), is sandwiched between an electrode surface confined Ab1 and the alkaline phosphatase-labeled signal anti-CEA antibodies conjugated with gold nanoparticles (Ab2-ALP-AuNP bioconjugates). The dual biocatalytic signal amplification for CEA monitoring is achieved by coupling the numerous enzymes loaded on the AuNPs with redox-recycling of the enzymatic products in the presence of the secondary enzyme and the corresponding substrate. The novel dramatic signal amplification strategy, exhibits a good linearity at the studied concentration range from 0.005 to 50 ng mL-1 towards CEA with a detection limit of 2 pg mL-1 (S/N=3). There is a 5-100-fold improvement in detection limit compared to other similar studies. The developed dual signal amplified strategy shows good selectivity, regeneration, stability and acceptable reproducibility. Therefore, the signal amplification approach holds great potential applications in detection of ultra-trace protein biomarkers.