Objective To prepare a polyclonal antibody against gastric cancer-related protein GCRG224.Methods The thioredoxin/GCRG224 fusion protein was expressed in E.coli.A polyclonal antibody against GCRG224 was obtained by immunizing a rabbit with the purified GCRG224 protein.The titer and specificity of the antibody were determined by ELISA and Western-blot,respectively.Results The thioredoxin/GCRG224 fusion protein with relative molecular mass of 16.8kD was over-expressed in E.coli.The purity of expressed products directly purified from a denaturing polyacrylamide gel was about 100%.The polyclonal antibody against GCRG224 was obtained.The ELISA titer of antiserum against GCRG224 was about 1∶256 000.Western blot analysis showed that the antiserum could bind to the expressed fusion protein specifically.Conclusion The polyclonal antibody against GCRG224 has been successfully prepared,which lays the foundation for further study on the biological function and the possible role of the GCRG224 in the development of gastric carcinoma.

Objective To express gastric cancer related gene GCRG213 by using thioredoxin fusion expression system, and to prepare human GCRG213 fusion protein. Methods GCRG213 cDNA with complete open reading frame was amplified by PCR from plasmid pGEM-T, and then was cloned into thioredoxin fusion expression vector pET102/D-TOPO. The recombinant plasmid was further transformed into E.coli BL21 strain. After induction with IPTG, the thioredoxin/GCRG213 fusion protein was expressed in E.coli. The product was obtained by means of direct purification from a denaturing polyacrylamide gel. Results SDS-PAGE analysis showed the thioredoxin/GCRG213 fusion protein with relative molecule mass of 29.4kDa was highly expressed. The thin layer gel scanning analysis showed that the yield of GCRG213 fusion protein was 28.7% of the total bacterial protein. The product was obtained with a purity of about 100% by means of direct purification from a denaturing polyacrylamide gel. Conclusion The thioredoxin/GCRG213 fusion protein was successfully expressed in E.coli and the product with high purity was obtained, which laid the foundation for the function research and antibody preparation hereafter.

Objective To investigate the effect of gene GCRG213 transfection (sense, anti-sense) on growth of gastric cancer cell MKN45. Methods The sense and anti-sense fragment of GCRG213 were obtained by PCR. They were cloned into eukaryotic expression vector pcDNA3.1(+). The recombinant plasmid pcDNA3.1-a, pcDNA3.1-b and the vector pcDNA3.1 were transfected separately into MKN45 cells conducted by lipofectamine~ TM 2000. Expression of GCRG213 was assessed with semi-quantitative RT-PCR and Western Blot. The growth graph was plotted by the methods of cell counting of three steady transfected cells. FACS was used to determine the cell cycle, and Annexin V FITC/PI bi-labeling method was used to determine the cell apoptosis. Results By sequencing, sense GCRG213 and anti-sense GCRG213 were proved to be successfully cloned into pcDNA3.1. Transfecting the sense vector (pcDNA3.1-a) into the MKN45 significantly increased the expression of GCRG213, both in mRNA level and protein level. Transfecting the anti-sense vector (pcDNA3.1-b) into the MKN45 significantly decreased the expression of GCRG213, both in mRNA level and protein level. The growth of pcDNA3.1-a transfected cells was faster than that of vector transfected cells, and the cell apoptosis decreased. But the growth of pcDNA3.1-b transfected cells was slower in multiplication than that of vector transfected cells, and the cell apoptosis was increased. Conclusion Stable transfection showed that GCRG213 promoted cell to grow, inhibited the cell apoptosis. GCRG213 might be a new promoter to tumor.

Objective To investigate the influence of FBG2 on the growth, proliferation, apoptosis, infiltration and cell cycle of the gastric cancer line MKN45. Methods A critical component ubiquitin-protein ligase complex, FBG2 cDNA, was subcloned into a constitutive vector pcDNA3.1, followed by transfection in MKN45 by using liposome. Then stable expression clones were selected and appraised. The apoptosis and cell cycles were detected using flow cytometry. The growth and proliferation were analyzed by plotting cell growth curves and colony formation assay respectively. The ability of infiltration was tested using cancer cell migration assay. The MKN-FBG2 group and two control groups were included in the study. Results MKN-FBG2 grew faster than MKN45 and MKN-PC. The cell counts of MKN-FBG2 on the fourth, fifth, sixth and seventh day were significantly larger than that of others (P

Objective To detect the expressions of PCNA, Cyclin D1 and P27, which were related to cell proliferation and cell cycle in gastric carcinoma and precancerous lesion of gastric mucosa. Methods Tissue microarray technique was applied to construct a tissue microarray containing gastric carcinoma, precancerous lesions and normal gastric mucosa. Diagnosis of the tissue in the microarray was pathologically confirmed. Immunohistochemical method was used to assess the expression of PCNA, Cyclin D1 and P27 in all the samples of tissue microarray. Results The expressions of PCNA and Cyclin D1 in gastric carcinoma, gastric hyperplasia, and intestinal metaplasia tissues were higher than that in normal gastric mucosa. The expressions of P27 in gastric hyperplasia, intestinal metaplasia and gastric carcinoma tissues were lower than that in normal gastric mucosa. Conclusion Gastric carcinoma, precancerous lesion and normal gastric mucosa tissues present different degrees of expression of PCNA, Cyclin D1 and P27, either with a increasing trend or a decreasing trend. These changes in expression are all closely related to gastric carcinogenesis.

Objectives To prepare the gastric adenocarcinoma related cDNA microarray and to screen differentially expressed genes between human gastric adenocarcinoma and normal gastric mucosa. Methods A set of cDNA microarray slides containing a duplicate set of 576 cDNA spots. These selected clones have been verified in sequence. The PCR products of human genes were spotted onto a chemical-material-coated glass plates in array. DNAs were fixed on the glass plate after series of treatment. Both RNAs from the gastric adenocarcinoma and normal gastric mucosa were reversely transcribed to the cDNAs with the incorporation of fluorescent dUTP to prepare the hybridization probes. The mixed probes were hybridized to the cDNA microarray. After high-stringent washing, the cDNA microarray was scanned for fluorescent signals to display differences between two tissues. Results Gastric adenocarcinoma related cDNA microarray slides contained 111 differentially expressed genes of gastric adenocarcinoma, 37 known genes related to gastric carcinogenesis (such as p21ras, p53, etc.) and control genes. The results of 10 couples of hybridizations to the gastric adenocarcinoma related cDNA microarray slides with Cy3-labeled cDNA probe from normal gastric tissue and Cy5-labeled cDNA probe from gastric adenocarcinoma showed analyzed spots uniformity background and clear signal. Among the 576 target genes, the expression level of 48 genes differed between gastric adenocarcinoma and normal mucosa in 5 or more samples (eight genes differed in all samples). There were 39 down-regulated and 9 up-regulated genes in gastric adenocarcinoma. Conclusion Gastric adenocarcinoma related cDNA microarray slides were successfully developed. The results of the experiments showed that gastric adenocarcinoma related cDNA microarray slides are reliable, sensitive and reproducible.

2cm) and undifferentiated type were significantly related to lymph node metastasis in submucosal cancer. No lymph node metastasis was observed in 25 patients with submucosal invasion who showed none of the three risk factors, whereas 72.7% (8/11) of patients with all the three factors had lymph node metastasis. Conclusion The tumor size and lymphatic vessel involvement are related with lymph node metastasis in mucosal cancer. Poor differentiation, tumor size and lymphatic vessel involvement are related with lymph node metastasis in submucosal cancer.

Objective To analyze clinicopathological characteristics of recurrence after gastrectomy for early gastric cancer. Methods 308 patients were treated surgically for early gastric cancer from 1983 to 2005. 245 patients were followed up after gastric resection.Clinicopathologic factors were investigated by using univariate methods and multivariate analysis for the possible relationship to recurrence. Results 30 patients developed recurrent disease (median 28 months). The 1-year, 3-year, 5-year, 7-year, 10-year and 15-year recurrent rates were 5.49%, 8.44%, 11.27%, 14.83%, 16.39% and 37.79%, respectively. 13 patients with mucosal gastric cancer developed recurrent disease (median 24 months). The 1-year, 3-year, 5-year, 7-year, 10-year and 15-year recurrent rates were 4.23%, 6.68%, 7.75%, 9.34%, 9.34% and 28.24%, respectively. 17 patients with submucosal gastric cancer developed recurrent disease (median 31 months). The 1-year, 3-year, 5-year, 7-year, 10-year and 15-year recurrent rates were 7.39%, 11.14%, 16.54%, 24.49%, 29.69% and 64.85%, respectively. Cox multivariate analysis showed that submucosal invasion (P=0.044, OR=2.172) was a positive independent risk factor and paracarcinomatous mucosal medium-severe intestinal metaplasia (P=0.047, OR=0.460) was a negative independent risk factor for recurrence. 76.7% (23/30) patients with recurrence did not have indication of resection for a cure, and they did not undergo surgery again. 23.3% (7/30) patients with recurrence had indication for curative resection, and 4 of whom underwent curative resection, but 3 did not because of poor health. Pathological examination after surgery showed that in 3 patients there was early gastric cancer in the remnant stomach without lymph node metastasis, and in one patient there was advanced gastric cancer in remnant stomach with regional lymph node metastasis. The patient with advanced gastric cancer survived for 28 month without detectable tumor. Logistic regression analysis showed paracarcinomatous mucosal medium-severe intestinal metaplasia (P=0.016, OR=17.000) was a positive independent predictor for second radical surgery. The follow-up examinations including endoscopy were performed in 86.7% (26/30) of patients after operation at least every 1-2 years. Conclusion Early gastric cancer patients with submucosal invasion have a high risk of recurrence, and those with paracarcinomatous mucosal medium-severe intestinal metaplasia have a low risk of recurrence. The patients with paracarcinomatous mucosal medium-severe intestinal metaplasia and cancer recurrence are feasible for a curative resection. The follow-up examinations including endoscopy every 1 or 2 years contributed highly to finding an early recurrent cancer in the remnant stomach. But it is not so helpful to increase the possibility of a curative surgery in patients with recurrence and metastasis after gastrectomy.

OBJECTIVE:To evaluate therapeutic efficacy of strontium dichloride (89SrCl2 ) combined with local radiotherapy for pain caused by malignant tumor bone metastasis. METHODS:115 patients with malignant tumor complicated with bone metasta-sis were analyzed retrospectively,and they received therapy regimen of 89SrCl2 combined with local radiotherapy. The amount of 89SrCl2 was calculated according to body weight of patients,and given by intravenous injection manner at the beginning of treat-ment,and then radiotherapy target section was confirmed according to iconography for irradiation. Effect of bone pain control,side effects (myelosuppression) and the effect of treatment on quality of life were evaluated at the end of treatment. RESULTS:After one month of treatment,there were 32 cases of complete remission(CR)and 62 cases of partial remission(PR),and total effec-tive rate was 81.7%;after one month of treatment,6 cases(5.1%)suffered from Ⅲ-Ⅳ degree myelosuppression;but there was none of Ⅲ-Ⅳdegree myelosuppression case after 2 months of treatment. The improvement of overall quality of life was not signifi-cant after treatment. Pain symptoms,general situation and sleep quality all improved significantly. CONCLUSIONS:89SrCl2 com-bined with local radiotherapy can control pain caused by malignant tumor bone metastasis and induce side effect,and improve quali-ty of life to certain extent.

Objective To investigate the expression of vascular endothelial growth factor (VEGF) and platelet drived endothelial cell growth factor (PD-ECGF) and their influence on the clinicopathological characteristics and the prognosis in aged patients with gastric carcinoma. Methods The expression of VEGF and PD-ECGF were examined with immnunohistochemical technique through preoperative biopsied specimens in 92 cases. Results The expression of VEGF and PD-ECGF were significantly higher in gastric carinoma than in chronic atrophic gastritis. VEGF and PD-ECGF levels in advanced carcinoma were higher compared with early carcinoma( P