Objective.To investigate PTEN expression and loss of heterozygosity(LOH)of its epigenetic microsatel-lites in gastric carcinoma and explore their roles in tumorigenesis and progression of gastric carcinoma.Methods.LOH...Objective.To investigate PTEN expression and loss of heterozygosity(LOH)of its epigenetic microsatel-lites in gastric carcinoma and explore their roles in tumorigenesis and progression of gastric carcinoma.Methods.LOH of epigenetic microsatellites of PTEN(D10S541,D10S583and D10S1687)was exam-ined in advanced gastric carcinomas(n=56)by PCR-SSCP.The mRNA and protein expressions of PTEN gene were evaluated in normal mucosa(n=56),early(n=11)and advanced carcinomas(n=56)of the stomach using RT?PCR and immunohistochemoistry respectively.PTEN mRNA and protein expressions were compared with clinicopathological staging and lymph node metastasis of tumors.The relationship be-tween PTEN mRNA expression and LOH of microsatellites was discussed,as well as relationship between PTEN mRNA and protein expression.Results.LOH of D10S541,D10S583and D10S1687was found in28.6%(16/56)of advanced gas-tric carcinomas.The positive rates of PTEN expression were80.4%(45/56),45.5%(5/11)and32.1%(18/56)in normal gastric mucosa,early and advanced gastric carcinomas at mRNA level,while78.6%(44/56),36.4%(4/11)and28.6%(16/56)at protein level.PTEN mRNA and protein were less fre-quently expressed in early and advanced gastric carcinomas than normal gastric mucosa(P<0.05).There was negative correlation between PTEN mRNA expression and LOH of microsatellites(P<0.05).PTEN protein expression paralleled to its mRNA expression(P<0.05).The PTEN mRNA and protein expres-sions were negatively correlated with lymph node metastasis of advanced gastric carcinomas(P<0.05).Conclusion.Down?regulated expression of PTEN and frequent LOH of its epigenetic microsatellites might play an important role in gastric carcinogenesis.Reduced PTEN mRNA expression was closely as-sociated with LOH of its epigenetic microsatellites.Altered expression of PTEN might contribute to lymph node metastasis of gastric carcinoma by decreasing cell adhesion and apoptosis,increasing angiogenesis and cell mobility.展开更多
DNA ploidy of 57 gastric carcinomas with metastases(12 liver,1 adrenal,4 ovary and 48 lymph node) were measured by flow cytometry.DNA anueploidy was significantly related to liver metastases:9 out of 12 gastric carcin...DNA ploidy of 57 gastric carcinomas with metastases(12 liver,1 adrenal,4 ovary and 48 lymph node) were measured by flow cytometry.DNA anueploidy was significantly related to liver metastases:9 out of 12 gastric carcinomas with liver metastases were anueploid(75%) as compared to 13 out of 45(28.8%) of cases without liver metastases(P<0.01);the one gastric carcinoma with adrenal metastasis was also anueploid.DNA ploidy was not related to ovarian or lymph node metastases.Another interesting finding was that all of 3 gastric carcinomas with liver metastases which showed a diploid DNA pattern,expressed p53 protein, while all of 3 carcinomas with liver metastases but no p53 protein expression were anueploid.The expression of p53 protein was not related to ovarian metastases.The results suggested that an anueploid DNA pattern and the expression of p53 protein are both objective markers valuable in predicting high risk potential of metastases to the liver,and that the combined detection of these markers can be a most useful method in the follow-up of Patients with gastric carcinoma in detecting those at high risk of developing metastases following surgical resection.Also the poorer prognosis of Patients with gastric carcinoma showing an anueploid DNA pattern may be related to the development of distant organ metastases through the blood vascular system.Furthermore,the clone of gastric carcinoma cells which accumulate p53protein or show an anueploid DNA pattern may have a causative role in the development of liver(&.adrenal) metastases.展开更多
Gastric cancer is the result of multiple risk factors,including environmental factors,genetic factors and the interaction between them.The environmental factors mainly include dietary,Helicobacter pylori infection and...Gastric cancer is the result of multiple risk factors,including environmental factors,genetic factors and the interaction between them.The environmental factors mainly include dietary,Helicobacter pylori infection and family history of gastric cancer.Genetic factors mainly refer to the susceptible genes that cause epigenetic alterations in oncogenes,tumor suppress genes,cell cycle regulators,DNA repair genes and signaling molecules.This paper summarizes the susceptible genes of gastric cancer and explores the genetic basis of it.展开更多
Objective. To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti - Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for develo...Objective. To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti - Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer.Methods. Balb/c mice were immunized i. p. with MG7 McAb conjugated with keyhole limpet hemocyanin (KLH), and mRNA was isolated from the spleens of the immunized mice. Heavy and light chain (VH and VL) genes of antibody were amplified separately and assembled into ScFv genes with a linker DNA by PCR. The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TGI. The transformants were infected with M13K07 helper phage to yield recombinant phages displaying ScFv on the tips of M13 phage. After 4 rounds of panning with MG7, the MG7-positive clones were selected by ELISA from the enriched phages. The types of the anti-Id ScFv displayed on the selected phage clones were preliminarily identified by competition ELISA.Results. The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. Twenty-four MG7-positive clones were selected from 60 enriched phage clones, among which 5 displayed β or γ type anti-Id ScFv.Conclusion. The anti-Id ScFv to MG7 McAb can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma by using anti-Id ScFv.展开更多
Objective: To investigate the methylation status of the PCDH8 (Protocadherin-8) gene in gastric cancer tissues and find out the relationship between methylation status of the PCDH8 and clinicopathological features in ...Objective: To investigate the methylation status of the PCDH8 (Protocadherin-8) gene in gastric cancer tissues and find out the relationship between methylation status of the PCDH8 and clinicopathological features in gastric cancer patients. Methods: We first investigated the methylation status of the PCDH8 (Protocadherin-8) gene in 65 gastric cancer and detected aberrant promoter methylation in gastric cancers; and then analyzed he relationship between methylation status of the PCDH8 and clinicopathological status with SPSS 13.0 software. Results: We first investigated the methylation status of the PCDH8 (Protocadherin-8) gene in 65 gastric cancer and detected aberrant promoter methylation in 36 of 65 (55.4%) gastric cancers. There was no significant difference in the distribution of patients with methylation or unmethylation of PCDH8 in terms of age, sex, tumor size, distant metastasis, or TNM stage. Methylation of PCDH8 was significantly correlated to negative pathological lymph node metastasis (P=0.038) and tumor differentiation (P=0.01). These two factors were proved to be of prognostic importance. Conclusion: Methylated PCDH8 seems to have a trend for worse prognosis in gastric cancer. However, a further large series of tumor samples and a longer follow-up period are required to elucidate its potential role.展开更多
To construct ScFv and Fab from murine anti gastric cancer monoclonal antibody(mAb)3H11. Methods.At first,3H11 ScFv and Fab were constructed with V genes PCR amplified by degenerate primers for FR1.The bacterial expres...To construct ScFv and Fab from murine anti gastric cancer monoclonal antibody(mAb)3H11. Methods.At first,3H11 ScFv and Fab were constructed with V genes PCR amplified by degenerate primers for FR1.The bacterial expressed 3H11 Ab fragments showed no antigen binding activity.Then,phage antibody library and random mutated library were constructed from 3H11 hybridoma cells and panning selection was performed.Again the identification of positive clone was failed.Finally the N terminal sequences of V regions were resumed to 3H11 original sequences by site directed mutagenesis via PCR. Results.Binding activity to gastric cancer cells was detected only from N terminal sequence corrected 3H11 ScFv and Fab,though the expression of the Ab fragments was not affected.Correction of either VL or VH N terminal sequences could partially resume the antigen binding activity. Conclusion.Sequence changes of V region N terminal introduced by PCR may seriously affect antigen binding without affecting the expression of antibody.展开更多
文摘Objective.To investigate PTEN expression and loss of heterozygosity(LOH)of its epigenetic microsatel-lites in gastric carcinoma and explore their roles in tumorigenesis and progression of gastric carcinoma.Methods.LOH of epigenetic microsatellites of PTEN(D10S541,D10S583and D10S1687)was exam-ined in advanced gastric carcinomas(n=56)by PCR-SSCP.The mRNA and protein expressions of PTEN gene were evaluated in normal mucosa(n=56),early(n=11)and advanced carcinomas(n=56)of the stomach using RT?PCR and immunohistochemoistry respectively.PTEN mRNA and protein expressions were compared with clinicopathological staging and lymph node metastasis of tumors.The relationship be-tween PTEN mRNA expression and LOH of microsatellites was discussed,as well as relationship between PTEN mRNA and protein expression.Results.LOH of D10S541,D10S583and D10S1687was found in28.6%(16/56)of advanced gas-tric carcinomas.The positive rates of PTEN expression were80.4%(45/56),45.5%(5/11)and32.1%(18/56)in normal gastric mucosa,early and advanced gastric carcinomas at mRNA level,while78.6%(44/56),36.4%(4/11)and28.6%(16/56)at protein level.PTEN mRNA and protein were less fre-quently expressed in early and advanced gastric carcinomas than normal gastric mucosa(P<0.05).There was negative correlation between PTEN mRNA expression and LOH of microsatellites(P<0.05).PTEN protein expression paralleled to its mRNA expression(P<0.05).The PTEN mRNA and protein expres-sions were negatively correlated with lymph node metastasis of advanced gastric carcinomas(P<0.05).Conclusion.Down?regulated expression of PTEN and frequent LOH of its epigenetic microsatellites might play an important role in gastric carcinogenesis.Reduced PTEN mRNA expression was closely as-sociated with LOH of its epigenetic microsatellites.Altered expression of PTEN might contribute to lymph node metastasis of gastric carcinoma by decreasing cell adhesion and apoptosis,increasing angiogenesis and cell mobility.
文摘DNA ploidy of 57 gastric carcinomas with metastases(12 liver,1 adrenal,4 ovary and 48 lymph node) were measured by flow cytometry.DNA anueploidy was significantly related to liver metastases:9 out of 12 gastric carcinomas with liver metastases were anueploid(75%) as compared to 13 out of 45(28.8%) of cases without liver metastases(P<0.01);the one gastric carcinoma with adrenal metastasis was also anueploid.DNA ploidy was not related to ovarian or lymph node metastases.Another interesting finding was that all of 3 gastric carcinomas with liver metastases which showed a diploid DNA pattern,expressed p53 protein, while all of 3 carcinomas with liver metastases but no p53 protein expression were anueploid.The expression of p53 protein was not related to ovarian metastases.The results suggested that an anueploid DNA pattern and the expression of p53 protein are both objective markers valuable in predicting high risk potential of metastases to the liver,and that the combined detection of these markers can be a most useful method in the follow-up of Patients with gastric carcinoma in detecting those at high risk of developing metastases following surgical resection.Also the poorer prognosis of Patients with gastric carcinoma showing an anueploid DNA pattern may be related to the development of distant organ metastases through the blood vascular system.Furthermore,the clone of gastric carcinoma cells which accumulate p53protein or show an anueploid DNA pattern may have a causative role in the development of liver(&.adrenal) metastases.
文摘Gastric cancer is the result of multiple risk factors,including environmental factors,genetic factors and the interaction between them.The environmental factors mainly include dietary,Helicobacter pylori infection and family history of gastric cancer.Genetic factors mainly refer to the susceptible genes that cause epigenetic alterations in oncogenes,tumor suppress genes,cell cycle regulators,DNA repair genes and signaling molecules.This paper summarizes the susceptible genes of gastric cancer and explores the genetic basis of it.
基金This work was supported by the National Natural Sciences Founda- tion of China(NSFC, No. 39800057, No. 30200338) the National "863" High-tech Project Foundation (No. 102-10-01 -06) +1 种基金National Distinguished Youth Program of NSFC(No. 39525020) This wor
文摘Objective. To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti - Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer.Methods. Balb/c mice were immunized i. p. with MG7 McAb conjugated with keyhole limpet hemocyanin (KLH), and mRNA was isolated from the spleens of the immunized mice. Heavy and light chain (VH and VL) genes of antibody were amplified separately and assembled into ScFv genes with a linker DNA by PCR. The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TGI. The transformants were infected with M13K07 helper phage to yield recombinant phages displaying ScFv on the tips of M13 phage. After 4 rounds of panning with MG7, the MG7-positive clones were selected by ELISA from the enriched phages. The types of the anti-Id ScFv displayed on the selected phage clones were preliminarily identified by competition ELISA.Results. The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. Twenty-four MG7-positive clones were selected from 60 enriched phage clones, among which 5 displayed β or γ type anti-Id ScFv.Conclusion. The anti-Id ScFv to MG7 McAb can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma by using anti-Id ScFv.
文摘Objective: To investigate the methylation status of the PCDH8 (Protocadherin-8) gene in gastric cancer tissues and find out the relationship between methylation status of the PCDH8 and clinicopathological features in gastric cancer patients. Methods: We first investigated the methylation status of the PCDH8 (Protocadherin-8) gene in 65 gastric cancer and detected aberrant promoter methylation in gastric cancers; and then analyzed he relationship between methylation status of the PCDH8 and clinicopathological status with SPSS 13.0 software. Results: We first investigated the methylation status of the PCDH8 (Protocadherin-8) gene in 65 gastric cancer and detected aberrant promoter methylation in 36 of 65 (55.4%) gastric cancers. There was no significant difference in the distribution of patients with methylation or unmethylation of PCDH8 in terms of age, sex, tumor size, distant metastasis, or TNM stage. Methylation of PCDH8 was significantly correlated to negative pathological lymph node metastasis (P=0.038) and tumor differentiation (P=0.01). These two factors were proved to be of prognostic importance. Conclusion: Methylated PCDH8 seems to have a trend for worse prognosis in gastric cancer. However, a further large series of tumor samples and a longer follow-up period are required to elucidate its potential role.
文摘To construct ScFv and Fab from murine anti gastric cancer monoclonal antibody(mAb)3H11. Methods.At first,3H11 ScFv and Fab were constructed with V genes PCR amplified by degenerate primers for FR1.The bacterial expressed 3H11 Ab fragments showed no antigen binding activity.Then,phage antibody library and random mutated library were constructed from 3H11 hybridoma cells and panning selection was performed.Again the identification of positive clone was failed.Finally the N terminal sequences of V regions were resumed to 3H11 original sequences by site directed mutagenesis via PCR. Results.Binding activity to gastric cancer cells was detected only from N terminal sequence corrected 3H11 ScFv and Fab,though the expression of the Ab fragments was not affected.Correction of either VL or VH N terminal sequences could partially resume the antigen binding activity. Conclusion.Sequence changes of V region N terminal introduced by PCR may seriously affect antigen binding without affecting the expression of antibody.
