AIM: To investigate the role of TIP30 in apoptotic signal pathway in hepatoblastoma cells and to provide a basis for TIP30 as a gene therapy candidate in the regression of hepatoblastoma cells.METHODS: Apoptosis of hu...AIM: To investigate the role of TIP30 in apoptotic signal pathway in hepatoblastoma cells and to provide a basis for TIP30 as a gene therapy candidate in the regression of hepatoblastoma cells.METHODS: Apoptosis of human hepatoblastoma cell lines HepG2 (p53 wild), Hep3B (p53 null) and PLC/RPF/5 (p53mutant) infected with Ad-TIP30 (bearing a wild type human Tip30 gene) were analyzed and p53, Bax and Bclxl expression levels were compared among these cells.MlT assay, DNA fragmentation, in situ 3' end labeling of DNA, annexin-Ⅴ FITC staining were used to detect cell death and apoptosis in cells at various time intervals subsequent to infection, and to determine whether TIP30 had an effect on the expression levels of some apoptosis-related gene products such as Bax, p53 and Bcl-xl. A similar time course experiment was performed by Western blotting.RESULTS: In MTT assay, the viability of HepG2 cells decreased significantly from 99.7% to 10% and displayed more massive cell death within 5-8 d than Hep3B and PLC/RPF/5 cells, with their viability decreased from 97.8% to 44.3% and 98.1% to 50.4%, respectively. In annexin-ⅤFITC assay, the percentage of apoptosis cells in HepG2cells was two to three-fold higher than that in control cells (infected with Ad-GFP), two-fold higher than that in Hep3B cells and 1.4-fold higher than that in PLC/RPF/5 cells 36 h after infection, respectively. Moreover, in HepG2 cells, the p53 began to increase 6-8 h after infection, reaching a maximum level between 8 and 12 h after infection and then dropped. Bax showed a similar increase in the cells as p53 reached the maximum at 8-12 h and subsequently decreased. Interestingly, Bcl-xl protein levels were down regulated during 24 to 36 h after Ad-TIP30 infection. In contrast, ectopic expression of TIP30 in Hep3B and PLC/RPF/5 cells had no effect on the regulation of Bax expression, but had an effect on Bcl-xl levels. In comparison with HepG2 cells, these data suggested that up-regulation of p53 levels by TIP30 might be a pre-requisite for Bax and Bax/Bcl-xl ratio increase. We hypothesized that TIP30 might regulate Bax gene partly through p53, which sensitizes cells to apoptosis by involving a p53 apoptosis signal transduction pathway.CONCLUSION: TIP30 plays an important role in predisposing hepatoblastoma cells to apoptosis through regulating expression levels of these genes. Ad-TIP30carrying exogenous TIP30-anti-tumor genes may be regarded as a potential candidate for the treatment of hepatocellular carcinoma.展开更多
AIM: To detect the expression of CK20mRNA in peripheral blood of Pancreatic cancer and evaluate its clinical significance.METHODS: Expression of CK20mRNA in peripheral blood was detected by fluorogenic qualitative rev...AIM: To detect the expression of CK20mRNA in peripheral blood of Pancreatic cancer and evaluate its clinical significance.METHODS: Expression of CK20mRNA in peripheral blood was detected by fluorogenic qualitative reverse transcription-polymerase chain reaction (RT-PCR) in 40cases of pancreatic cancer at the night before operation,in 5 cases of benign pancreatic diseases, in 5 cases of healthy individuals. The relationships were investigated between CK20mRNA expression and the clinicopathological variables, and clinical follow-up outcome in those patients with pancreatic cancer having undergone radical resection.RESULTS: Of the 40 patients with pancreatic cancer, 23(57.5%) cases were positive for CK20mRNA expression.CK20mRNA expression was significantly correlated with lymphatic metastasis (P = 0.008), histopathological grading (P = 0.009), and pathological stage (P = 0.021);there was no significant correlation between CK20mRNA expression and age, gender, tumor diameter, and depth of invasion. The cumulative metastasis rates of patients with CK20mRNA expression were higher than those of patients with no CK20mRNA expression within 6 mo (34.7%vs5.9%, P= 0.043) or 12 mo (73.9% vs35.3%, P= 0.02)after operation. CK20mRNA expression in peripheral blood of pancreatic cancer indicated poorer prognosis. The survival rate of patients with CK20mRNA expression was lower than that of patients with negative CK20mRNA expression (Log-Rank = 13.31, P = 0.0003).CONCLUSION: CK20mRNA is a sensitive and specific molecular marker for the detection of micrometastasis in peripheral blood of patients with pancreatic cancer. The CK20mRNA expression in peripheral blood is correlated with biological characteristic of pancreatic cancer. It can help to predict the prognosis of pancreatic cancer after operation, and to determine which patient will benefit from aggressive adjuvant therapies.展开更多
The characteristics of a cultured cell line do not always remain stable and may change upon continuous passage. Most continuous cell lines, even after cloning, possess several genotypes that are constantly changing. T...The characteristics of a cultured cell line do not always remain stable and may change upon continuous passage. Most continuous cell lines, even after cloning, possess several genotypes that are constantly changing. There are numerous selective and adaptive culture processes, in addition to genetic instability, that may improve phenotypic change in cell growth, virus susceptibility, gene expression, and production of virus. Similar detrimental effects of long term passaging of insect cells have also been reported for continuous cell lines, for example, Tn5B 1-4 cells, which are the most widely used for the baculovirus expression vector system (BEVS), provide superior production of recombinant proteins,however, this high productivity may be more evident in low passage cells. In this paper, we describe the isolation of a cell clone, Tn5B-40, from low passage Tn5B 1-4 cells. The growth characteristics,productions of virus, and high level of recombinant protein productions were determined. The results showed the susceptibility of both clone and Tn5B 1-4 cells to wild-type AcNPV was approximately the same rate with over 95% of infection; when the cloned cells were infected with recombinant baculoviruses expressing β-galactosidase and secreted alkaline phosphatase (SEAP), expression of the recombinant proteins from the cloned cells exceeded that from the parental Tn5B 1-4 cells.展开更多
文摘AIM: To investigate the role of TIP30 in apoptotic signal pathway in hepatoblastoma cells and to provide a basis for TIP30 as a gene therapy candidate in the regression of hepatoblastoma cells.METHODS: Apoptosis of human hepatoblastoma cell lines HepG2 (p53 wild), Hep3B (p53 null) and PLC/RPF/5 (p53mutant) infected with Ad-TIP30 (bearing a wild type human Tip30 gene) were analyzed and p53, Bax and Bclxl expression levels were compared among these cells.MlT assay, DNA fragmentation, in situ 3' end labeling of DNA, annexin-Ⅴ FITC staining were used to detect cell death and apoptosis in cells at various time intervals subsequent to infection, and to determine whether TIP30 had an effect on the expression levels of some apoptosis-related gene products such as Bax, p53 and Bcl-xl. A similar time course experiment was performed by Western blotting.RESULTS: In MTT assay, the viability of HepG2 cells decreased significantly from 99.7% to 10% and displayed more massive cell death within 5-8 d than Hep3B and PLC/RPF/5 cells, with their viability decreased from 97.8% to 44.3% and 98.1% to 50.4%, respectively. In annexin-ⅤFITC assay, the percentage of apoptosis cells in HepG2cells was two to three-fold higher than that in control cells (infected with Ad-GFP), two-fold higher than that in Hep3B cells and 1.4-fold higher than that in PLC/RPF/5 cells 36 h after infection, respectively. Moreover, in HepG2 cells, the p53 began to increase 6-8 h after infection, reaching a maximum level between 8 and 12 h after infection and then dropped. Bax showed a similar increase in the cells as p53 reached the maximum at 8-12 h and subsequently decreased. Interestingly, Bcl-xl protein levels were down regulated during 24 to 36 h after Ad-TIP30 infection. In contrast, ectopic expression of TIP30 in Hep3B and PLC/RPF/5 cells had no effect on the regulation of Bax expression, but had an effect on Bcl-xl levels. In comparison with HepG2 cells, these data suggested that up-regulation of p53 levels by TIP30 might be a pre-requisite for Bax and Bax/Bcl-xl ratio increase. We hypothesized that TIP30 might regulate Bax gene partly through p53, which sensitizes cells to apoptosis by involving a p53 apoptosis signal transduction pathway.CONCLUSION: TIP30 plays an important role in predisposing hepatoblastoma cells to apoptosis through regulating expression levels of these genes. Ad-TIP30carrying exogenous TIP30-anti-tumor genes may be regarded as a potential candidate for the treatment of hepatocellular carcinoma.
文摘AIM: To detect the expression of CK20mRNA in peripheral blood of Pancreatic cancer and evaluate its clinical significance.METHODS: Expression of CK20mRNA in peripheral blood was detected by fluorogenic qualitative reverse transcription-polymerase chain reaction (RT-PCR) in 40cases of pancreatic cancer at the night before operation,in 5 cases of benign pancreatic diseases, in 5 cases of healthy individuals. The relationships were investigated between CK20mRNA expression and the clinicopathological variables, and clinical follow-up outcome in those patients with pancreatic cancer having undergone radical resection.RESULTS: Of the 40 patients with pancreatic cancer, 23(57.5%) cases were positive for CK20mRNA expression.CK20mRNA expression was significantly correlated with lymphatic metastasis (P = 0.008), histopathological grading (P = 0.009), and pathological stage (P = 0.021);there was no significant correlation between CK20mRNA expression and age, gender, tumor diameter, and depth of invasion. The cumulative metastasis rates of patients with CK20mRNA expression were higher than those of patients with no CK20mRNA expression within 6 mo (34.7%vs5.9%, P= 0.043) or 12 mo (73.9% vs35.3%, P= 0.02)after operation. CK20mRNA expression in peripheral blood of pancreatic cancer indicated poorer prognosis. The survival rate of patients with CK20mRNA expression was lower than that of patients with negative CK20mRNA expression (Log-Rank = 13.31, P = 0.0003).CONCLUSION: CK20mRNA is a sensitive and specific molecular marker for the detection of micrometastasis in peripheral blood of patients with pancreatic cancer. The CK20mRNA expression in peripheral blood is correlated with biological characteristic of pancreatic cancer. It can help to predict the prognosis of pancreatic cancer after operation, and to determine which patient will benefit from aggressive adjuvant therapies.
文摘The characteristics of a cultured cell line do not always remain stable and may change upon continuous passage. Most continuous cell lines, even after cloning, possess several genotypes that are constantly changing. There are numerous selective and adaptive culture processes, in addition to genetic instability, that may improve phenotypic change in cell growth, virus susceptibility, gene expression, and production of virus. Similar detrimental effects of long term passaging of insect cells have also been reported for continuous cell lines, for example, Tn5B 1-4 cells, which are the most widely used for the baculovirus expression vector system (BEVS), provide superior production of recombinant proteins,however, this high productivity may be more evident in low passage cells. In this paper, we describe the isolation of a cell clone, Tn5B-40, from low passage Tn5B 1-4 cells. The growth characteristics,productions of virus, and high level of recombinant protein productions were determined. The results showed the susceptibility of both clone and Tn5B 1-4 cells to wild-type AcNPV was approximately the same rate with over 95% of infection; when the cloned cells were infected with recombinant baculoviruses expressing β-galactosidase and secreted alkaline phosphatase (SEAP), expression of the recombinant proteins from the cloned cells exceeded that from the parental Tn5B 1-4 cells.
