Tripchlorolide (TC) is a potent antitumor reagent purified from a Chinese herb Tripterygium Wilfordii Hook. f.. However, its cellular effects and mechanism of action are unknown. We showed here that TC induced apoptos...Tripchlorolide (TC) is a potent antitumor reagent purified from a Chinese herb Tripterygium Wilfordii Hook. f.. However, its cellular effects and mechanism of action are unknown. We showed here that TC induced apoptosis of Chinese Hamster Ovary (CHO) cells in time- and dose-dependent manners. TC resulted in the degradation of Bcl-2, the translocation of Bax from the cytosol to mitochondria, and the release of cytochrome c from mitochondria. Stable overexpression of human Bcl-2 could reduce the apoptosis of TCtreated cells by blocking the translocation of Bax and the release of cytochrome c. These results indicate that TC induces apoptosis of CHO cell by activating the mitochondrion-mediated apoptotic pathway involving the proteins of Bcl-2 family and cytochrome c.展开更多
Glutathione peroxidase (GPX1) was the first identified selenium-dependent enzyme, and this enzyme has been most useful as a biochemical indicator of selenium (Se) status and the parameter of choice for determining Se ...Glutathione peroxidase (GPX1) was the first identified selenium-dependent enzyme, and this enzyme has been most useful as a biochemical indicator of selenium (Se) status and the parameter of choice for determining Se requirements. We have continued to study Se regulation of GPX1 to better understand the underlying mechanism and to gain insight into how cells themselves regulate nutrient status. In progressive Se deficiency in rats, GPX1 activity,protein and mRNA all decrease in a dramatic, coordinated and exponential fashion such that Se-deficient GPX1 mRNA levels are 6-15% of Sexadequate levels. mRNA levels for other Sedependent proteins are far less decreased in the same animals. The mRNA levels for a second Se-dependent peroxidase, phospholipid hydroperoxide glutathione peroxidase (GPX4 ), are little affected by Se deficiency, demonstrating that Se regulation of GPX1 is unique. Se regulation of GPX1 activity in growing male and female rats shows that the Se requirernent is 100 ng/g diet, based on liver GPX1 activity; use of GPX1 mRNA as the parameter indicates that the Se requirement is nearer to 50 ng Se/g diet in both male and female rats. This approach will readily detect an altered dietary Se requirement, as shown by the incremental increases in dietary Se requirement by 150, 100 or 50 ng Se/g diet in Seudeficient rat pups repleted with Se for 3, 7 or 14 d, respectively. Studies with CHO cells stably transfected with recombinant GPX1 also show that overexpression of GPX1 does not alter the minimum level of media Se necessary for Se-adequate levels of GPX1 activity or mRNA. We hypothesize that classical GPX1 has an integral biological role in the mechanism used by cells to regulate Se status,making GPX1 an especially useful and effective parameter for determining Se requirements in animals展开更多
Objective To express the recombinant human bone morphogenetic protein-7 (rhBMP-7) in Chinese hamster ovary (CHO) cells and to establish the in vitro biological activity assay of rhBMP-7. Methods Human BMP-7 cDNA was s...Objective To express the recombinant human bone morphogenetic protein-7 (rhBMP-7) in Chinese hamster ovary (CHO) cells and to establish the in vitro biological activity assay of rhBMP-7. Methods Human BMP-7 cDNA was subcloned into pcDNA3.1 mammalian expression vector and transfected to CHO cells by using the lipofectin transfection method. BMP-7 expression cell culture supernatants were harvested and purified for target protein. To analyze the bioactivity of the secreted rhBMP-7, a novel in vitro assay was established by measuring its alkaline phosphatase (ALP) stimulating of osteoblast cell line, W-20-17. Results BMP-7 stably expressing cell clone was selected, which secreted mature disulfide-linked homodimer form of hBMP-7 and had an apparent molecular weight of 36kDa. rhBMP-7 with >95% purity was obtained using 3 step chromatography method. Bioactivity assay showed that the purified protein specifically stimulated W-20-17 cell producing ALP, with a 4-fold increase of ALP activity at 100ng/ml or more, and the EC50 of 15.6ng/ml. Conclusion Purified rhBMP-7 from this CHO expression system has significant biological activity in induction of osteoblast phenotype, which demonstrates potential bone regeneration activity.展开更多
The surface of polypropylene (PP) membrane was modified by low temperature plasma with ammonia. The effect of exposure time was investigated by means of contact angle measurement. The results show that low temperature...The surface of polypropylene (PP) membrane was modified by low temperature plasma with ammonia. The effect of exposure time was investigated by means of contact angle measurement. The results show that low temperature ammonia plasma treatment can enhance its hydrophilicity. Chinese hamster ovary (CHO) cells attachment on the modified membrane was enhanced and the growth rate on the membrane was faster than unmodified one.展开更多
To clone the murine α fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1 6 cells, and then the murine α fetoprotein gene was a...To clone the murine α fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1 6 cells, and then the murine α fetoprotein gene was amplified by RT PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. After transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8kb murine α fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.展开更多
Objective To characterize a possible retention function of unique sequence in the 5＇end of rat testis GABAA receptor β3t splicing variant Methods Rat testis GABAA receptor β3t splicing variant cDNA was cloned and t...Objective To characterize a possible retention function of unique sequence in the 5＇end of rat testis GABAA receptor β3t splicing variant Methods Rat testis GABAA receptor β3t splicing variant cDNA was cloned and two eukaryotic expression recombinant plasmids of pEGFP-N1 and pEGFP-C1 were constructed respectively by fusing green fluorescent protein to the N or C-terminus of β3t isoform. The recombinant plasmids were transfected into CHO cells by calcium phosphate co-precipitation method Fluorescence microscope and laser confocal microscope were used to analyze localization of β3t in the transfected cells. ConA-Texas-Red was used to label cell ER and the localization of rat testis β3t splicing variant in CHO cells was determined. Results When rat testis β3t splicing variant was expressed in CHO cells, two expression patterns were delineated, the distributions of uniform and mainly discrete intracellular compartments respectively, The chimera product failed to be translocated into the cell surface when expressed in ClIO cells; whereas the β3 subunit of rat brain was incorporated into the plasma membrane. Conclusion The inability of β3t to target into the ER may be a consequence of the unique 25 specific amino acid segments in the N terminus.展开更多
Human c-myc cDNA was fused with the hormonebinding domain (HBD) cDNA of murine estrogen receptorgene and the chimeric gene was introduced into the CHOcells. The fusion protein, c-MycER, becomes activatedwhen the synth...Human c-myc cDNA was fused with the hormonebinding domain (HBD) cDNA of murine estrogen receptorgene and the chimeric gene was introduced into the CHOcells. The fusion protein, c-MycER, becomes activatedwhen the synthetic steroid, 4-hydroxy-tamoxifen (OHT),binds HBD. Activated c-MycER, likely c-Myc, can inducequiescent CHO cells reentry into S phase and subsequentcell death under serum-free condition. In addition, theexpression of some proposed c-myc target genes such asODC, MrDb, cad, rcc1 and rc1 were found to increase uponOHT induction before S, phase entry and apoptosis, indicating that these target genes are involved in cell cycleregulation and/or apoptosis control. However, the mutantD106-143c-MycER protein does not have above activities.展开更多
Genetically-engineered CHO cell lines, rβ- 13 and CLF-8B2, were cultivated with the MC- 1 microcarrier culture system. The cell density could be enhanced by increasing the concentration of microcarrier. At a microcar...Genetically-engineered CHO cell lines, rβ- 13 and CLF-8B2, were cultivated with the MC- 1 microcarrier culture system. The cell density could be enhanced by increasing the concentration of microcarrier. At a microcarrier concentration of 10 mg/ml. the cell density could reach 4 to 5 × 106 cells/ml. It was shown that these cell lines would spontaneously release from the microcarrier to attach to and proliferate on fresh microcarriers. We were thus able to scale up cultivation using a simple method. i. e. by adding fresh microcarriers and medium directly into the culture system to about 2, 4 or 8 times the original volume. Using a perfusion culture system. we have successfully cultivated CLF-8B2 cells in a 2 L bioreactor for several weeks at medium perfusion rates of 0. 5 to 3working volumes. Prourokinase was stably secreted.展开更多
基金supported by Grant 39825115 of the National Outstanding Young Scientists,a special grant of major state basic research program of China(No.G1999053901)a grant from the Chinese Academy of Sciences KSCX2-SW-203 to JR Wu.
文摘Tripchlorolide (TC) is a potent antitumor reagent purified from a Chinese herb Tripterygium Wilfordii Hook. f.. However, its cellular effects and mechanism of action are unknown. We showed here that TC induced apoptosis of Chinese Hamster Ovary (CHO) cells in time- and dose-dependent manners. TC resulted in the degradation of Bcl-2, the translocation of Bax from the cytosol to mitochondria, and the release of cytochrome c from mitochondria. Stable overexpression of human Bcl-2 could reduce the apoptosis of TCtreated cells by blocking the translocation of Bax and the release of cytochrome c. These results indicate that TC induces apoptosis of CHO cell by activating the mitochondrion-mediated apoptotic pathway involving the proteins of Bcl-2 family and cytochrome c.
文摘Glutathione peroxidase (GPX1) was the first identified selenium-dependent enzyme, and this enzyme has been most useful as a biochemical indicator of selenium (Se) status and the parameter of choice for determining Se requirements. We have continued to study Se regulation of GPX1 to better understand the underlying mechanism and to gain insight into how cells themselves regulate nutrient status. In progressive Se deficiency in rats, GPX1 activity,protein and mRNA all decrease in a dramatic, coordinated and exponential fashion such that Se-deficient GPX1 mRNA levels are 6-15% of Sexadequate levels. mRNA levels for other Sedependent proteins are far less decreased in the same animals. The mRNA levels for a second Se-dependent peroxidase, phospholipid hydroperoxide glutathione peroxidase (GPX4 ), are little affected by Se deficiency, demonstrating that Se regulation of GPX1 is unique. Se regulation of GPX1 activity in growing male and female rats shows that the Se requirernent is 100 ng/g diet, based on liver GPX1 activity; use of GPX1 mRNA as the parameter indicates that the Se requirement is nearer to 50 ng Se/g diet in both male and female rats. This approach will readily detect an altered dietary Se requirement, as shown by the incremental increases in dietary Se requirement by 150, 100 or 50 ng Se/g diet in Seudeficient rat pups repleted with Se for 3, 7 or 14 d, respectively. Studies with CHO cells stably transfected with recombinant GPX1 also show that overexpression of GPX1 does not alter the minimum level of media Se necessary for Se-adequate levels of GPX1 activity or mRNA. We hypothesize that classical GPX1 has an integral biological role in the mechanism used by cells to regulate Se status,making GPX1 an especially useful and effective parameter for determining Se requirements in animals
文摘Objective To express the recombinant human bone morphogenetic protein-7 (rhBMP-7) in Chinese hamster ovary (CHO) cells and to establish the in vitro biological activity assay of rhBMP-7. Methods Human BMP-7 cDNA was subcloned into pcDNA3.1 mammalian expression vector and transfected to CHO cells by using the lipofectin transfection method. BMP-7 expression cell culture supernatants were harvested and purified for target protein. To analyze the bioactivity of the secreted rhBMP-7, a novel in vitro assay was established by measuring its alkaline phosphatase (ALP) stimulating of osteoblast cell line, W-20-17. Results BMP-7 stably expressing cell clone was selected, which secreted mature disulfide-linked homodimer form of hBMP-7 and had an apparent molecular weight of 36kDa. rhBMP-7 with >95% purity was obtained using 3 step chromatography method. Bioactivity assay showed that the purified protein specifically stimulated W-20-17 cell producing ALP, with a 4-fold increase of ALP activity at 100ng/ml or more, and the EC50 of 15.6ng/ml. Conclusion Purified rhBMP-7 from this CHO expression system has significant biological activity in induction of osteoblast phenotype, which demonstrates potential bone regeneration activity.
基金The National Natural Science Foundation of China (No. 29776027)
文摘The surface of polypropylene (PP) membrane was modified by low temperature plasma with ammonia. The effect of exposure time was investigated by means of contact angle measurement. The results show that low temperature ammonia plasma treatment can enhance its hydrophilicity. Chinese hamster ovary (CHO) cells attachment on the modified membrane was enhanced and the growth rate on the membrane was faster than unmodified one.
文摘To clone the murine α fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1 6 cells, and then the murine α fetoprotein gene was amplified by RT PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. After transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8kb murine α fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.
基金The study was supported by National Basic Science Key Infrastructure Development Program (973 program), National Ministry of Science and Technology (Grant No: G19990559) and in part by E-institutes fund of Shanghai Municipal Education Commission. (Grant No:E03003)
文摘Objective To characterize a possible retention function of unique sequence in the 5＇end of rat testis GABAA receptor β3t splicing variant Methods Rat testis GABAA receptor β3t splicing variant cDNA was cloned and two eukaryotic expression recombinant plasmids of pEGFP-N1 and pEGFP-C1 were constructed respectively by fusing green fluorescent protein to the N or C-terminus of β3t isoform. The recombinant plasmids were transfected into CHO cells by calcium phosphate co-precipitation method Fluorescence microscope and laser confocal microscope were used to analyze localization of β3t in the transfected cells. ConA-Texas-Red was used to label cell ER and the localization of rat testis β3t splicing variant in CHO cells was determined. Results When rat testis β3t splicing variant was expressed in CHO cells, two expression patterns were delineated, the distributions of uniform and mainly discrete intracellular compartments respectively, The chimera product failed to be translocated into the cell surface when expressed in ClIO cells; whereas the β3 subunit of rat brain was incorporated into the plasma membrane. Conclusion The inability of β3t to target into the ER may be a consequence of the unique 25 specific amino acid segments in the N terminus.
文摘Human c-myc cDNA was fused with the hormonebinding domain (HBD) cDNA of murine estrogen receptorgene and the chimeric gene was introduced into the CHOcells. The fusion protein, c-MycER, becomes activatedwhen the synthetic steroid, 4-hydroxy-tamoxifen (OHT),binds HBD. Activated c-MycER, likely c-Myc, can inducequiescent CHO cells reentry into S phase and subsequentcell death under serum-free condition. In addition, theexpression of some proposed c-myc target genes such asODC, MrDb, cad, rcc1 and rc1 were found to increase uponOHT induction before S, phase entry and apoptosis, indicating that these target genes are involved in cell cycleregulation and/or apoptosis control. However, the mutantD106-143c-MycER protein does not have above activities.
文摘Genetically-engineered CHO cell lines, rβ- 13 and CLF-8B2, were cultivated with the MC- 1 microcarrier culture system. The cell density could be enhanced by increasing the concentration of microcarrier. At a microcarrier concentration of 10 mg/ml. the cell density could reach 4 to 5 × 106 cells/ml. It was shown that these cell lines would spontaneously release from the microcarrier to attach to and proliferate on fresh microcarriers. We were thus able to scale up cultivation using a simple method. i. e. by adding fresh microcarriers and medium directly into the culture system to about 2, 4 or 8 times the original volume. Using a perfusion culture system. we have successfully cultivated CLF-8B2 cells in a 2 L bioreactor for several weeks at medium perfusion rates of 0. 5 to 3working volumes. Prourokinase was stably secreted.
