Hepatocellular carcinoma (HCC) is one of the most common malignancies in China. To date, surgery is still the best solution to it. However, metastatic recurrences after curative hepatic resections are very common. Tan...Hepatocellular carcinoma (HCC) is one of the most common malignancies in China. To date, surgery is still the best solution to it. However, metastatic recurrences after curative hepatic resections are very common. Tang et al have reported that recurrence rate within 5 years of curative hepatic resection is 61.5% [1]. As curative hepatic resection has a high tendency for metastatic recurrence, therapeutic interventions such as transarterial embolization and antiangiogenesis have been tried to further improve prognosis of HCC patients. Therefore, establishing a dependable, sensitive, easy, and economical method to predict metastatic recurrence following curative hepatic resection is of clinical urgency.展开更多
AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cass...AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cassette in the reverse orientation transcribing small antisense RNA which could specifically interact with VEGF165, and VEGF121 mRNA. Construct the retroviral vector containing this antisense VEGF U6 cassette and package the replication-deficient recombinant retrovirus. SMMC-7721 cells were transduced with these virus and positive clones were selected with G418. PCR and Southern blot analysis were performed to determine if U6 cassette integrated into the genomic DNA of positive clone. Transfected tumor cells were evaluated for RNA expression by ribonuclease protection assays. The VEGF protein in the supernatant of parental tumor cells and genetically modified tumor cells was determined with ELISA. In vitro and in vivo growth properties of antisense VEGF cell clone in nude mice were analyzed. RESULTS: Restriction enzyme digestion and PCR sequencing verified that the antisense VEGF RNA retroviral vector was successfully constructed.After G418 selection, resistant SMMC-7721 cell clone was picked up. PCR and Southern blot analysis suggested that U6 cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transduced with U6 antisense RNA cassette could express 200 bp small antisense VEGF RNA and secrete reduced levels of VEGF in culture condition. Production of VEGF by antisense transgene-expressing cells was 65+/-10 ng/L per 10(6) cells, 42045 ng/L per 10(6) cells in sense group and 485+/-30 ng/L per 10(6) cells in the negative control group, (P【 0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S.C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with control cells. CONCLUSION: Expression of antisense VEGF RNA in SMMC-7721 cells could decrease the tumorigenicity, and antisense-VEGF gene therapy may be an adjuvant treatment for hepatoma.展开更多
Circulating vascular endothelial growth factor-C （VEGF-C） and vascular endothelial growth factor （VEGF） levels in patients with colorectal carcinoma were determined in order to assess their clinical significance a...Circulating vascular endothelial growth factor-C （VEGF-C） and vascular endothelial growth factor （VEGF） levels in patients with colorectal carcinoma were determined in order to assess their clinical significance as a diagnostic tool for monitoring lymph node metastasis. In 66 patients with colorectal carcinoma and 30 healthy controls, circulating VEGF-C and VEGF levels were assessed by using enzyme-linked immunosorbent assay （ELISA）. Serum VEGF-C and VEGF levels were higher in patients with colorectal carcinoma than in healthy controls. Patients with lymph node metastasis had higher serum VEGF-C and VEGF levels than those without lymph node metastasis. The levels of VEGF-C and VEGF were higher in the invasion group than in the non-invasion group. Serum VEGF-C levels reached a sensitivity of 81% and a specificity of 76 % with a cutoff value of 1438.0 pg/mL, whereas VEGF levels reached 72 % sensitivity and 74 % specificity at 240.2 pg/ mL. If 66 patients were divided into 4 groups according to the combined determination of VEGF-C and VEGF levels, the positive predictive value was 85.3 %, the negative predictive value was 94.6 %, and accuracy was 93.7 %. It was suggested that circulating VEGF-C levels might provide additional information for distinguishing the absence from presence of lymph node metastasis in patients with colorectal carcinoma. The combined determination of VEGF-C and VEGF levels could be used as an important index for preoperatively clinical stage of colorectal carcinoma.展开更多
Objective To establish a sandwich ELISA method for detecting vascular endothelial growth factor （VEGF） in sera of population and the patients with hepatocellular carcinoma （HCC）. Methods Full length and two trunca...Objective To establish a sandwich ELISA method for detecting vascular endothelial growth factor （VEGF） in sera of population and the patients with hepatocellular carcinoma （HCC）. Methods Full length and two truncated human VEGF cDNA sequences were amplified from a commercial plasmid pBLAST49-hVEGF by PCR and inserted into the prokaryotic-expression plasmid pET-32a or pGEX-2T. Various VEGF proteins were expressed and purified from E. coli in His-Trx or GST fusion forms. The specific VEGF antibodies were elicited in experimental rabbits and mice by immunization of the full length VEGF fusion protein His-Trx-VEGF1-165. After purification of antibodies with chromatograph of Protein G, a sandwich ELISA technique was established. Serum VEGF levels were evaluated in 229 adults and 291 HCC patients. Results SDS-PAGE displayed that the molecular weights of the expressed full length （His-Trx-VEGF1-165）, N-terminal （His-Trx-VEGF1-100） and C-terminal （GST-VEGF100-165） human VEGF fusion proteins were about 38KD, 31KD, and 33KD, respectively. Western blots confirmed that the prepared antisera were able to recognize both prokaryoticly and eukaryoticly expressed recombinant VEGF proteins. Assays of serially diluted His-Trx-VEGF1-100 by the established sandwich ELISA method showed that the linear range of the standard curve was 0.625-320 ng/mL, with the squared correlation coefficient R^2=0.991. Screening of a serum panel containing 291 serum samples of HCC patients and 229 health adults revealed that the average VEGF level in HCC patients was higher than that in healthy controls, with a statically significant difference. Conclusion The established sandwich ELISA reflects the level of serum VEGF and provide scientific basis for screening metastasis and recurrence of HCC using serum VEGF as an index.展开更多
Background： Hydrothorax, as one of the common complications of malignant tumors, still cannot be sensitively detected in clinical practice, thus requiring a sensitive, specific method for diagnosis. The aim of this s...Background： Hydrothorax, as one of the common complications of malignant tumors, still cannot be sensitively detected in clinical practice, thus requiring a sensitive, specific method for diagnosis. The aim of this study was to analyze the correlation between levels of vascular endothelial growth factor （VEGF） and epidermal growth factor receptor （EGFR） in patients with benign and malignant hydrothorax. Methods： The contents of VEGF in the pleural effusion and serum of the patients with malignant pleural effusion （n = 35） and benign pleural effusion （n = 30） were detected by double antibody sandwich enzyme linked immunosorbent assay. The gene copy number level of EGFR in pleural effusion was detected by fluorescence in situ hybridization （FISH）. The points with the highest sensitivity and specificity were selected as the critical values to calculate the diagnostic value of the VEGF in pleural effusion and serum, and EGFR gene copy number in pleural effusion. Results： The contents of VEGF in pleural effusion and serum of patients with malignant hydrothorax were （384.91 ± 120.18）, and （129.62 ±46.35） ng/L, respectively, which were significantly higher than those of the patients with benign hydrothorax （207.97 ± 64.04）, （63.49 ± 24.58） ng/L （P 〈 0.01 ）. The sensitivity and specificity of detecting VEGF in pleural effusion were 80.0% and 96.7% （the boundary value was 297.06 ng/L）, respectively for diagnosing benign and malignant hydrothorax. The sensitivity and specificity of serum were 74.3% and 96.7%, respectively （the boundary value was 99.21 ng/L） for diagnosing benign and malignant hydrothorax. The diagnostic efficiencies of EGFR and VEGF in hydrothorax were similar. There was a significant correlation between EGFR and VEGF in hydrothorax （P 〈 0.01 ）. Conclusions： VEGF and EGFR play important roles in the formation of pleural effusion. VEGF differed significantly in benign and malignant pleural effusions, which contributed to differential diagnosis results of benign and malignant pleural effusions. It is feasible to detect the gene copy number of the pleural effusion cell mass EGFR by FISH technique. Joint detection can improve the diagnostic sensitivity.展开更多
The immunosuppressive agent FK506 contributes to neural regeneration via growth cone and Schwann cell processes. However, it is difficult to rapidly harvest a large number of Schwann cells, and Schwann cell senescence...The immunosuppressive agent FK506 contributes to neural regeneration via growth cone and Schwann cell processes. However, it is difficult to rapidly harvest a large number of Schwann cells, and Schwann cell senescence is obvious following culture, proliferation, and amplification. The present study investigated the effects of various concentrations of FK506 on proliferation and secretion of cultured Schwann cells. In addition, the suitability of FK506 application as tissue engineered artificial nerves was considered. Bilateral sciatic nerves of 3-5-day old, Sprague Dawley rats were obtained, and primary cultured Schwann cells were identified by S-100 protein staining. Following subculture, Schwann cells purity was 〉 90%. After 3-5 days in culture, Schwann cell proliferation was visible in each group. Results verified that FK506 promoted proliferation of cultured Schwann cells and secretion of nerve growth factor. This effect was significant at a low concentration （1 × 10.8 mol/L）.展开更多
Background Measurement of human epidermal growth factor receptor 2 （HER2） protein in the serum of metastatic breast cancer patients has previously been reported, but there are no consistent data to support the clini...Background Measurement of human epidermal growth factor receptor 2 （HER2） protein in the serum of metastatic breast cancer patients has previously been reported, but there are no consistent data to support the clinical utility of serum HER2 extracellular domain for patients with early stage breast cancer. We aimed to evaluate the correlation between serum extracellular domain levels and tissue HER2 expression, and analyzed their relationship with clinico-pathological parameters in patients with early stage disease. Methods A prospective study was conducted on 232 breast cancer patients with stage I-III prior to treatment. Preoperative serum samples were measured by enzyme-linked immunosorbent assay. Tissue HER2 status was analyzed by immunohistochemistry and fluorescence in situ hybridization assays. Results The median serum extracellular domain concentration was 6.8 ng/ml. The best diagnostic cut-off value was 7.4 ng/ml, with 62.9% sensitivity and 85.3% specificity. High serum extracellular domain levels were reported in 89 patients （38.3%）, and HER2-positive expression was observed in 77 patients （33.2%）. Multivariate analysis showed that elevated serum extracellular domain correlated with postmenopausal status （P 〈0.001）, high histological grade （P 〈0.001）, negativity of both estrogen （P=0.012） and progesterone receptors （P 〈0.001）, and high levels of carcinoembryonic antigen 153 （P=-0.048）. Conclusions We recommend that 7.4 ng/ml should be used as the cut-off value when evaluating serum extracellular domain levels in early stage of breast cancer. Patients with high serum extracellular domain levels have a certain clinico- pathological characteristics, may provide a basis for clinical practice.展开更多
基金the Shanghai Leading Medical Subjects Grant(№983001)State Key Basic Research Grant(№G1998051211)for financial supports.
文摘Hepatocellular carcinoma (HCC) is one of the most common malignancies in China. To date, surgery is still the best solution to it. However, metastatic recurrences after curative hepatic resections are very common. Tang et al have reported that recurrence rate within 5 years of curative hepatic resection is 61.5% [1]. As curative hepatic resection has a high tendency for metastatic recurrence, therapeutic interventions such as transarterial embolization and antiangiogenesis have been tried to further improve prognosis of HCC patients. Therefore, establishing a dependable, sensitive, easy, and economical method to predict metastatic recurrence following curative hepatic resection is of clinical urgency.
基金Project supported by National Natural Science Foundation of China,No.863 Z2001-04
文摘AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cassette in the reverse orientation transcribing small antisense RNA which could specifically interact with VEGF165, and VEGF121 mRNA. Construct the retroviral vector containing this antisense VEGF U6 cassette and package the replication-deficient recombinant retrovirus. SMMC-7721 cells were transduced with these virus and positive clones were selected with G418. PCR and Southern blot analysis were performed to determine if U6 cassette integrated into the genomic DNA of positive clone. Transfected tumor cells were evaluated for RNA expression by ribonuclease protection assays. The VEGF protein in the supernatant of parental tumor cells and genetically modified tumor cells was determined with ELISA. In vitro and in vivo growth properties of antisense VEGF cell clone in nude mice were analyzed. RESULTS: Restriction enzyme digestion and PCR sequencing verified that the antisense VEGF RNA retroviral vector was successfully constructed.After G418 selection, resistant SMMC-7721 cell clone was picked up. PCR and Southern blot analysis suggested that U6 cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transduced with U6 antisense RNA cassette could express 200 bp small antisense VEGF RNA and secrete reduced levels of VEGF in culture condition. Production of VEGF by antisense transgene-expressing cells was 65+/-10 ng/L per 10(6) cells, 42045 ng/L per 10(6) cells in sense group and 485+/-30 ng/L per 10(6) cells in the negative control group, (P【 0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S.C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with control cells. CONCLUSION: Expression of antisense VEGF RNA in SMMC-7721 cells could decrease the tumorigenicity, and antisense-VEGF gene therapy may be an adjuvant treatment for hepatoma.
文摘Circulating vascular endothelial growth factor-C （VEGF-C） and vascular endothelial growth factor （VEGF） levels in patients with colorectal carcinoma were determined in order to assess their clinical significance as a diagnostic tool for monitoring lymph node metastasis. In 66 patients with colorectal carcinoma and 30 healthy controls, circulating VEGF-C and VEGF levels were assessed by using enzyme-linked immunosorbent assay （ELISA）. Serum VEGF-C and VEGF levels were higher in patients with colorectal carcinoma than in healthy controls. Patients with lymph node metastasis had higher serum VEGF-C and VEGF levels than those without lymph node metastasis. The levels of VEGF-C and VEGF were higher in the invasion group than in the non-invasion group. Serum VEGF-C levels reached a sensitivity of 81% and a specificity of 76 % with a cutoff value of 1438.0 pg/mL, whereas VEGF levels reached 72 % sensitivity and 74 % specificity at 240.2 pg/ mL. If 66 patients were divided into 4 groups according to the combined determination of VEGF-C and VEGF levels, the positive predictive value was 85.3 %, the negative predictive value was 94.6 %, and accuracy was 93.7 %. It was suggested that circulating VEGF-C levels might provide additional information for distinguishing the absence from presence of lymph node metastasis in patients with colorectal carcinoma. The combined determination of VEGF-C and VEGF levels could be used as an important index for preoperatively clinical stage of colorectal carcinoma.
基金National High-tech Development Project (863 Project) (No. 2002AA215021)
文摘Objective To establish a sandwich ELISA method for detecting vascular endothelial growth factor （VEGF） in sera of population and the patients with hepatocellular carcinoma （HCC）. Methods Full length and two truncated human VEGF cDNA sequences were amplified from a commercial plasmid pBLAST49-hVEGF by PCR and inserted into the prokaryotic-expression plasmid pET-32a or pGEX-2T. Various VEGF proteins were expressed and purified from E. coli in His-Trx or GST fusion forms. The specific VEGF antibodies were elicited in experimental rabbits and mice by immunization of the full length VEGF fusion protein His-Trx-VEGF1-165. After purification of antibodies with chromatograph of Protein G, a sandwich ELISA technique was established. Serum VEGF levels were evaluated in 229 adults and 291 HCC patients. Results SDS-PAGE displayed that the molecular weights of the expressed full length （His-Trx-VEGF1-165）, N-terminal （His-Trx-VEGF1-100） and C-terminal （GST-VEGF100-165） human VEGF fusion proteins were about 38KD, 31KD, and 33KD, respectively. Western blots confirmed that the prepared antisera were able to recognize both prokaryoticly and eukaryoticly expressed recombinant VEGF proteins. Assays of serially diluted His-Trx-VEGF1-100 by the established sandwich ELISA method showed that the linear range of the standard curve was 0.625-320 ng/mL, with the squared correlation coefficient R^2=0.991. Screening of a serum panel containing 291 serum samples of HCC patients and 229 health adults revealed that the average VEGF level in HCC patients was higher than that in healthy controls, with a statically significant difference. Conclusion The established sandwich ELISA reflects the level of serum VEGF and provide scientific basis for screening metastasis and recurrence of HCC using serum VEGF as an index.
文摘Background： Hydrothorax, as one of the common complications of malignant tumors, still cannot be sensitively detected in clinical practice, thus requiring a sensitive, specific method for diagnosis. The aim of this study was to analyze the correlation between levels of vascular endothelial growth factor （VEGF） and epidermal growth factor receptor （EGFR） in patients with benign and malignant hydrothorax. Methods： The contents of VEGF in the pleural effusion and serum of the patients with malignant pleural effusion （n = 35） and benign pleural effusion （n = 30） were detected by double antibody sandwich enzyme linked immunosorbent assay. The gene copy number level of EGFR in pleural effusion was detected by fluorescence in situ hybridization （FISH）. The points with the highest sensitivity and specificity were selected as the critical values to calculate the diagnostic value of the VEGF in pleural effusion and serum, and EGFR gene copy number in pleural effusion. Results： The contents of VEGF in pleural effusion and serum of patients with malignant hydrothorax were （384.91 ± 120.18）, and （129.62 ±46.35） ng/L, respectively, which were significantly higher than those of the patients with benign hydrothorax （207.97 ± 64.04）, （63.49 ± 24.58） ng/L （P 〈 0.01 ）. The sensitivity and specificity of detecting VEGF in pleural effusion were 80.0% and 96.7% （the boundary value was 297.06 ng/L）, respectively for diagnosing benign and malignant hydrothorax. The sensitivity and specificity of serum were 74.3% and 96.7%, respectively （the boundary value was 99.21 ng/L） for diagnosing benign and malignant hydrothorax. The diagnostic efficiencies of EGFR and VEGF in hydrothorax were similar. There was a significant correlation between EGFR and VEGF in hydrothorax （P 〈 0.01 ）. Conclusions： VEGF and EGFR play important roles in the formation of pleural effusion. VEGF differed significantly in benign and malignant pleural effusions, which contributed to differential diagnosis results of benign and malignant pleural effusions. It is feasible to detect the gene copy number of the pleural effusion cell mass EGFR by FISH technique. Joint detection can improve the diagnostic sensitivity.
基金supported by the National Natural Science Foundation of China (Construction of artificial nerves using hTERT-transfected Schwann cells combined with chitosan sustained-release FK506 incorporated conduits),No. 30973060
文摘The immunosuppressive agent FK506 contributes to neural regeneration via growth cone and Schwann cell processes. However, it is difficult to rapidly harvest a large number of Schwann cells, and Schwann cell senescence is obvious following culture, proliferation, and amplification. The present study investigated the effects of various concentrations of FK506 on proliferation and secretion of cultured Schwann cells. In addition, the suitability of FK506 application as tissue engineered artificial nerves was considered. Bilateral sciatic nerves of 3-5-day old, Sprague Dawley rats were obtained, and primary cultured Schwann cells were identified by S-100 protein staining. Following subculture, Schwann cells purity was 〉 90%. After 3-5 days in culture, Schwann cell proliferation was visible in each group. Results verified that FK506 promoted proliferation of cultured Schwann cells and secretion of nerve growth factor. This effect was significant at a low concentration （1 × 10.8 mol/L）.
文摘Background Measurement of human epidermal growth factor receptor 2 （HER2） protein in the serum of metastatic breast cancer patients has previously been reported, but there are no consistent data to support the clinical utility of serum HER2 extracellular domain for patients with early stage breast cancer. We aimed to evaluate the correlation between serum extracellular domain levels and tissue HER2 expression, and analyzed their relationship with clinico-pathological parameters in patients with early stage disease. Methods A prospective study was conducted on 232 breast cancer patients with stage I-III prior to treatment. Preoperative serum samples were measured by enzyme-linked immunosorbent assay. Tissue HER2 status was analyzed by immunohistochemistry and fluorescence in situ hybridization assays. Results The median serum extracellular domain concentration was 6.8 ng/ml. The best diagnostic cut-off value was 7.4 ng/ml, with 62.9% sensitivity and 85.3% specificity. High serum extracellular domain levels were reported in 89 patients （38.3%）, and HER2-positive expression was observed in 77 patients （33.2%）. Multivariate analysis showed that elevated serum extracellular domain correlated with postmenopausal status （P 〈0.001）, high histological grade （P 〈0.001）, negativity of both estrogen （P=0.012） and progesterone receptors （P 〈0.001）, and high levels of carcinoembryonic antigen 153 （P=-0.048）. Conclusions We recommend that 7.4 ng/ml should be used as the cut-off value when evaluating serum extracellular domain levels in early stage of breast cancer. Patients with high serum extracellular domain levels have a certain clinico- pathological characteristics, may provide a basis for clinical practice.
