Objective To explore the change of endogenic nerve growth factor （NGF） expression in human glioma cells infected with human cytomegalovirus （HCMV）. Methods U251 cells were cultured in RPMI 1640 culture medium and ...Objective To explore the change of endogenic nerve growth factor （NGF） expression in human glioma cells infected with human cytomegalovirus （HCMV）. Methods U251 cells were cultured in RPMI 1640 culture medium and infected with HCMV AD 169 strain in vitro to establish a cell model of viral infection. Morphologic changes of U251 cells were observed under inverted microscope before and after infection with HCMV. Expression of NGF gene and protein of cells was detected by RT-PCR and Western blotting before and after infection with HCMV. Results The cytopathic effects of HCMV-infected cells appeared on day 5 after infection. However, differential NGF expression was evident on day 7. NGF expression was decreased significantly in U251 cells on day 7 after infection in comparison with control group （P〈0.05）. Conclusion HCMV can down-regulate endogenous NGF levels in human glioma cell line U251.展开更多
The effect and mechanism of carmustine（BCNU） combined with all-trans retinoic acid（ATRA） on the apoptosis of human glioblastoma U251 cells were investigated by means of 3-（4,5-dimethylthiazol-2-yl）-2,5-diphe- ny...The effect and mechanism of carmustine（BCNU） combined with all-trans retinoic acid（ATRA） on the apoptosis of human glioblastoma U251 cells were investigated by means of 3-（4,5-dimethylthiazol-2-yl）-2,5-diphe- nyltetrazolium bromide（MTT） assay, flow cytometry, reverse transcription-polymerase chain reaction（RT-PCR） and Western blot analysis. The results show that BCNU or ATRA shows time- and dose-dependent inhibition effects on human glioblastoma U251 cells and the combination of BCNU with ATRA shows an synergistic inhibition effect on human glioblastoma U251 cells, and the combined BCNU and ATRA can significantly inhibit the proliferation of human glioblastoma U251 cells, and induce the apoptosis of them, making the cells arrest in the stage of G1 phase, the stage of S and G2 phases decline, the rate of the apoptosis of human glioblastoma U251 cells increase, the corresponding mRNA expression of cyclin E and cyclin-dependent kinase 2（CDK2） downregulated and the correspon- ding mRNA expression of p27kip 1 unregulated. In addition, the combined BCNU and ATRA reduced the protein expression of nuclear factor kappa B（NF-κB）. Taken together, these results suggest that the treatment of human glioblastoma U251 cells with a combination application of ATRA and BCNU can exert synergistic effect, the course of this kind of combination chemotherapy may likely be associated with multiple molecular mechanisms for apoptosis, furthermore, the cyclin E and p27kip 1 should be considered as novel targets for controlling the growth of glioblastoma cells.展开更多
The proteomics of the differential protein expressions in human glioma cell line U251 cells infected with human cytomegalovirus （HCMV） was investigated at the protein level by using the surface enhanced laser desorp...The proteomics of the differential protein expressions in human glioma cell line U251 cells infected with human cytomegalovirus （HCMV） was investigated at the protein level by using the surface enhanced laser desorption/ionization （SELDI） protein chip system in order to develop a method of study for the pathogenesis of HCMV infection. In this study, the cultured U251 cells were infected with HCMV in good condition and the supernatants of lysates and the extracellular fluids of the cultivated infected cells were quantitatively defined for the expressed proteins. The proteomics of the differential protein expression in cells before and after infection was analyzed by WCX2 arrays on the protein chip reader. It was demonstrated that the eytopathic effects of infected cells appeared on the 5th day after infection, however, the differential protein expression was evident at 6 h after infection as revealed by RT-PCR and mass spectrometry. The protein peaks captured from different batches of samples, from the same sample detected with different arrays or for the different times were all equivalent. With the molecular weight range from 2000 Da to 3000 Da, chip captured 82 peaks from the intracellular fluids and 11 protein peak from the cellular fluid in which compared with the control group, the protein peaks with molecular weight of 13 536.3 Da, 10 046.1 Da and 17 106.2 Da were close to those of β-amyloid protein, caspase-1 precursor and LPS-induced TNF-α factor respectively, which showed brief up-regulation 4 h after infection, and continued to raise 48 h later. These results infer that these proteins may be related to the apoptosis induced by HCMV infection, thus suggesting that the apoptosis induced by HCMV infection may play a role in the pathogenesis of HCMV infection.展开更多
Previous studies indicated that B7-H4,the youngest B7 family,negatively regulates T cell-mediated immunity and is significantly overexpressed in many human tumors.Tumor stem cells are purported to play a role in tumor...Previous studies indicated that B7-H4,the youngest B7 family,negatively regulates T cell-mediated immunity and is significantly overexpressed in many human tumors.Tumor stem cells are purported to play a role in tumor renewal and resistance to radiation and chemotherapy.However,the link between B7-H4and tumor stem cells is unclear.In this study,we investigated B7-H4 expression in the medium of human glioma U251 cell cultures.Immunofluorescence results showed that U251 cells cultured in serum-free medium(supplemented with 2%B27,20 ng/mL epidermal growth factor,20 ng/mL basic fibroblast growth factor)maintained stem-like cell characteristics,including expression of stem cell marker CD133 and the neural progenitor cell markers nestin and SOX2.In contrast,U251 cells cultured in serum-containing medium highly expressed differentiation marker glial fibrillary acidic protein.Flow cytometry analysis showed serum-free medium-cultured U251 cells expressed higher intracellular B7-H4 than serumcontaining medium-cultured U251 cells(24%-35%vs.8%-11%,P<0.001).Immunofluorescence in purified monocytes from normal human peripheral blood mononuclear cells revealed moderate expression of B7-H4 after stimulation with conditioned medium from U251 cells cultured in serum-containing medium.Moreover,conditioned medium from U251 stem-like cells had a significant stimulation effect on B7-H4expression compared with serum-containing conditioned medium(P<0.01).Negative costimulatory molecule B7-H4 was preferentially expressed in U251 stem-like cells,and conditioned medium from these cells more effectively induced monocytes to express B7-H4 than conditioned medium from U251 cells cultured in the presence of serum.Our results show that U251 stem-like cells may play a more crucial role in tumor immunoloregulation with high expression of B7-H4.展开更多
基金supported by the National Natural Science Foundation of China, (No. 30770105)Mt. Tai Scholar Construction Engineering Special Foundation of Shandong Province
文摘Objective To explore the change of endogenic nerve growth factor （NGF） expression in human glioma cells infected with human cytomegalovirus （HCMV）. Methods U251 cells were cultured in RPMI 1640 culture medium and infected with HCMV AD 169 strain in vitro to establish a cell model of viral infection. Morphologic changes of U251 cells were observed under inverted microscope before and after infection with HCMV. Expression of NGF gene and protein of cells was detected by RT-PCR and Western blotting before and after infection with HCMV. Results The cytopathic effects of HCMV-infected cells appeared on day 5 after infection. However, differential NGF expression was evident on day 7. NGF expression was decreased significantly in U251 cells on day 7 after infection in comparison with control group （P〈0.05）. Conclusion HCMV can down-regulate endogenous NGF levels in human glioma cell line U251.
基金Supported by the National Natural Science Foundation of China(No.30672159)the Fund of Jilin Provincial Science & Technology Department, China(No.200905173)
文摘The effect and mechanism of carmustine（BCNU） combined with all-trans retinoic acid（ATRA） on the apoptosis of human glioblastoma U251 cells were investigated by means of 3-（4,5-dimethylthiazol-2-yl）-2,5-diphe- nyltetrazolium bromide（MTT） assay, flow cytometry, reverse transcription-polymerase chain reaction（RT-PCR） and Western blot analysis. The results show that BCNU or ATRA shows time- and dose-dependent inhibition effects on human glioblastoma U251 cells and the combination of BCNU with ATRA shows an synergistic inhibition effect on human glioblastoma U251 cells, and the combined BCNU and ATRA can significantly inhibit the proliferation of human glioblastoma U251 cells, and induce the apoptosis of them, making the cells arrest in the stage of G1 phase, the stage of S and G2 phases decline, the rate of the apoptosis of human glioblastoma U251 cells increase, the corresponding mRNA expression of cyclin E and cyclin-dependent kinase 2（CDK2） downregulated and the correspon- ding mRNA expression of p27kip 1 unregulated. In addition, the combined BCNU and ATRA reduced the protein expression of nuclear factor kappa B（NF-κB）. Taken together, these results suggest that the treatment of human glioblastoma U251 cells with a combination application of ATRA and BCNU can exert synergistic effect, the course of this kind of combination chemotherapy may likely be associated with multiple molecular mechanisms for apoptosis, furthermore, the cyclin E and p27kip 1 should be considered as novel targets for controlling the growth of glioblastoma cells.
基金This work was supported by National Natural Science Foundation of China(No.30471527 and No.30540075)partly supported by Mr.Tai Scholar Construction Engineering Foundation.
文摘The proteomics of the differential protein expressions in human glioma cell line U251 cells infected with human cytomegalovirus （HCMV） was investigated at the protein level by using the surface enhanced laser desorption/ionization （SELDI） protein chip system in order to develop a method of study for the pathogenesis of HCMV infection. In this study, the cultured U251 cells were infected with HCMV in good condition and the supernatants of lysates and the extracellular fluids of the cultivated infected cells were quantitatively defined for the expressed proteins. The proteomics of the differential protein expression in cells before and after infection was analyzed by WCX2 arrays on the protein chip reader. It was demonstrated that the eytopathic effects of infected cells appeared on the 5th day after infection, however, the differential protein expression was evident at 6 h after infection as revealed by RT-PCR and mass spectrometry. The protein peaks captured from different batches of samples, from the same sample detected with different arrays or for the different times were all equivalent. With the molecular weight range from 2000 Da to 3000 Da, chip captured 82 peaks from the intracellular fluids and 11 protein peak from the cellular fluid in which compared with the control group, the protein peaks with molecular weight of 13 536.3 Da, 10 046.1 Da and 17 106.2 Da were close to those of β-amyloid protein, caspase-1 precursor and LPS-induced TNF-α factor respectively, which showed brief up-regulation 4 h after infection, and continued to raise 48 h later. These results infer that these proteins may be related to the apoptosis induced by HCMV infection, thus suggesting that the apoptosis induced by HCMV infection may play a role in the pathogenesis of HCMV infection.
基金supported by grants from the National Natural Science Foundation of China (No.81272797 to Y.Y.)nnovation Program of Shanghai Municipal Education Commission (No.13ZZ010 to Y.Y.)Shanghai Talents Development Funds (No.2011063 to Y.Y.)
文摘Previous studies indicated that B7-H4,the youngest B7 family,negatively regulates T cell-mediated immunity and is significantly overexpressed in many human tumors.Tumor stem cells are purported to play a role in tumor renewal and resistance to radiation and chemotherapy.However,the link between B7-H4and tumor stem cells is unclear.In this study,we investigated B7-H4 expression in the medium of human glioma U251 cell cultures.Immunofluorescence results showed that U251 cells cultured in serum-free medium(supplemented with 2%B27,20 ng/mL epidermal growth factor,20 ng/mL basic fibroblast growth factor)maintained stem-like cell characteristics,including expression of stem cell marker CD133 and the neural progenitor cell markers nestin and SOX2.In contrast,U251 cells cultured in serum-containing medium highly expressed differentiation marker glial fibrillary acidic protein.Flow cytometry analysis showed serum-free medium-cultured U251 cells expressed higher intracellular B7-H4 than serumcontaining medium-cultured U251 cells(24%-35%vs.8%-11%,P<0.001).Immunofluorescence in purified monocytes from normal human peripheral blood mononuclear cells revealed moderate expression of B7-H4 after stimulation with conditioned medium from U251 cells cultured in serum-containing medium.Moreover,conditioned medium from U251 stem-like cells had a significant stimulation effect on B7-H4expression compared with serum-containing conditioned medium(P<0.01).Negative costimulatory molecule B7-H4 was preferentially expressed in U251 stem-like cells,and conditioned medium from these cells more effectively induced monocytes to express B7-H4 than conditioned medium from U251 cells cultured in the presence of serum.Our results show that U251 stem-like cells may play a more crucial role in tumor immunoloregulation with high expression of B7-H4.
