Objective To assess the effects of both TrkA and p75NTR on nerve growth factor (NGF)-induced differentiation of neuroblastoma cells.Methods Retroviral vectors were constructed to express the high affinity NGF receptor...Objective To assess the effects of both TrkA and p75NTR on nerve growth factor (NGF)-induced differentiation of neuroblastoma cells.Methods Retroviral vectors were constructed to express the high affinity NGF receptor (TrkA) and low affinity NGF receptor (p75NTR). Neuroblastoma cell line IMR-32 was transfected by the vectors expressing either TrkA or p75NTR or both by using lipofectmine?reagent separately or cotransfected at the same time. Southern blot, Northern blot, RT-PCR and flow cytometry were used to determine the success of the transfection. MTT technique was to monitor the cell proliferation. Colony formation in soft agar and tumor forming assay in nude mice were used to test the biological characteristics of the tumor cells. Terminal-deoxynucleotidytransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay was used to test the apoptosis of the tumor cells.Results Stable transformant cell lines expressing TrkA, p75NTR or both genes were established. Studies on these transformant cell lines have shown different NGF responses. The pTSNTR transfection only resulted in the mild differentiation response, and transfection of TrkA gene caused remarkable neurite extension, up-regulation of neurofilament and decreased expression of N-myc oncogene after NGF treatment. The cotransfection of the two genes into this cell line resulted in the more rapid and more apparent morphological changes than single TrkA transfected cells after NGF treatment. The cotransfected cells underwent apoptosis after withdrawal of NGF.Conclusions The results indicate that coexpression of both low- and high-affinity NGF receptors are not only more efficient in restoration of NGF-induced differentiation pathway, but also be able to activate the pro-apoptotic activity of low-affinity NGF receptor and make the tumor cells become NGF-dependent and irreversibly differentiated.展开更多
Objective： We investigated the influence of CDK1 and CDK2 expression inhibited by cotransfection of CDK1 and CDK2 siRNA on cell cycle and apoptosis, explored the exact role of cell cycle master regulator in tumor cel...Objective： We investigated the influence of CDK1 and CDK2 expression inhibited by cotransfection of CDK1 and CDK2 siRNA on cell cycle and apoptosis, explored the exact role of cell cycle master regulator in tumor cell apoptosis process. Methods： The siRNA targeting the CDK1 and CDK2 genes were synthesized and simultaneously cotransfected into Hela cells by lipofectamine 2000.48 or 60 h after the cotransfection, CDK1 and CDK2 protein expressions were examined by Western blot. Cell cycle distribution was analyzed by flow cytometry. Cell apoptosis was detected by the Annexin V/PI method. The changes of the transfected cell morphological under a microscope after Wright-Giemsa Staining were studied. Results： CDK1 and CDK2 protein expression was decreased at 48 or 60 h after cotransfection. The accumulation of the G2/M and S phase population in cell cycle of the cotransfected cells at 48 or 60 h after transfection was enhanced obviously compared with control. The ratio of apoptotic cell of cotransfected cells at 48 or 60 h after transfection was increased significantly compared with control. More binucleate or multinucleate ceJls among cotransfected cells were observed under the microscope. Conclusion： The decreased expression of CDK1 and CDK2 by cotransfection of CDK1 and CDK2 siRNA not only leads to tumor cell cycle arrest in S phase and G2/M phase, but also induces tumor cell apoptosis.展开更多
The virus-like particle（VLPs） vaccine is an ideal HIV-1 vaccine, which can simultaneously induce a neutralizing antibody reaction and cell-mediated immunity effectively. In this study, two kinds of plasmids have bee...The virus-like particle（VLPs） vaccine is an ideal HIV-1 vaccine, which can simultaneously induce a neutralizing antibody reaction and cell-mediated immunity effectively. In this study, two kinds of plasmids have been used, one can express the HIV-1 main structure proteins, Gagpol and Env, and the other contains an antibiotic gene. The two kinds of plasmids have been cotransfected into 293 cells. A stable cell line that can express Gagpol and Env proteins efficiently and lastingly has been screened. It has been confirmed that Gagpol and Env proteins in the cell culture supernatant can be self-assembled into virus-like particles. The authors have detected the secretion of VLPs in the cell medium, defined the peak of the secretion, and followed and monitored the stability of expression.展开更多
The rapid growth of the global HIV/AIDS epidemic makes it a high priority to develop an effective vaccine.Since a live attenuated or inactivated HIV vaccine is not likely to be approved for clinical application due to...The rapid growth of the global HIV/AIDS epidemic makes it a high priority to develop an effective vaccine.Since a live attenuated or inactivated HIV vaccine is not likely to be approved for clinical application due to safety concerns,HIV virus like particles(VLPs) offer an attractive alternative because they are considered safer since they lack viral genome.We got a stable eukaryotic cell line by G418 resistance selection,engineered to express the HIV-1 structure protein Gag and Env efficiently and stably.We confirmed the presence of Gag and Env proteins in the cell culture supernatant and that they could self-assemble into VLPs.These VLPs were found to be able to elicit specific humoral and cellular immune response after immunization without any adjuvant.展开更多
Insect baculovirus(IBV) is a biological insecticide in agriculture and forestry, butits slow speed of killing pests and narrow range of host make its application quitelimited. Hence, polyhedrin (ocu) gene of IBV was c...Insect baculovirus(IBV) is a biological insecticide in agriculture and forestry, butits slow speed of killing pests and narrow range of host make its application quitelimited. Hence, polyhedrin (ocu) gene of IBV was chosen for construction of展开更多
基金This work was supported by the National Natural Science Foundation for outstanding young scientist(No.39625012)and the National Education Committee for Transcentury Outstanding Young People.
文摘Objective To assess the effects of both TrkA and p75NTR on nerve growth factor (NGF)-induced differentiation of neuroblastoma cells.Methods Retroviral vectors were constructed to express the high affinity NGF receptor (TrkA) and low affinity NGF receptor (p75NTR). Neuroblastoma cell line IMR-32 was transfected by the vectors expressing either TrkA or p75NTR or both by using lipofectmine?reagent separately or cotransfected at the same time. Southern blot, Northern blot, RT-PCR and flow cytometry were used to determine the success of the transfection. MTT technique was to monitor the cell proliferation. Colony formation in soft agar and tumor forming assay in nude mice were used to test the biological characteristics of the tumor cells. Terminal-deoxynucleotidytransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay was used to test the apoptosis of the tumor cells.Results Stable transformant cell lines expressing TrkA, p75NTR or both genes were established. Studies on these transformant cell lines have shown different NGF responses. The pTSNTR transfection only resulted in the mild differentiation response, and transfection of TrkA gene caused remarkable neurite extension, up-regulation of neurofilament and decreased expression of N-myc oncogene after NGF treatment. The cotransfection of the two genes into this cell line resulted in the more rapid and more apparent morphological changes than single TrkA transfected cells after NGF treatment. The cotransfected cells underwent apoptosis after withdrawal of NGF.Conclusions The results indicate that coexpression of both low- and high-affinity NGF receptors are not only more efficient in restoration of NGF-induced differentiation pathway, but also be able to activate the pro-apoptotic activity of low-affinity NGF receptor and make the tumor cells become NGF-dependent and irreversibly differentiated.
基金Supported by grants from the National Basic Research Program of China (No. 2004CB518705)National Natural Sciences Foundation of China (No.30570908)
文摘Objective： We investigated the influence of CDK1 and CDK2 expression inhibited by cotransfection of CDK1 and CDK2 siRNA on cell cycle and apoptosis, explored the exact role of cell cycle master regulator in tumor cell apoptosis process. Methods： The siRNA targeting the CDK1 and CDK2 genes were synthesized and simultaneously cotransfected into Hela cells by lipofectamine 2000.48 or 60 h after the cotransfection, CDK1 and CDK2 protein expressions were examined by Western blot. Cell cycle distribution was analyzed by flow cytometry. Cell apoptosis was detected by the Annexin V/PI method. The changes of the transfected cell morphological under a microscope after Wright-Giemsa Staining were studied. Results： CDK1 and CDK2 protein expression was decreased at 48 or 60 h after cotransfection. The accumulation of the G2/M and S phase population in cell cycle of the cotransfected cells at 48 or 60 h after transfection was enhanced obviously compared with control. The ratio of apoptotic cell of cotransfected cells at 48 or 60 h after transfection was increased significantly compared with control. More binucleate or multinucleate ceJls among cotransfected cells were observed under the microscope. Conclusion： The decreased expression of CDK1 and CDK2 by cotransfection of CDK1 and CDK2 siRNA not only leads to tumor cell cycle arrest in S phase and G2/M phase, but also induces tumor cell apoptosis.
基金the National Natural Science Foundation of China(No.30371317).
文摘The virus-like particle（VLPs） vaccine is an ideal HIV-1 vaccine, which can simultaneously induce a neutralizing antibody reaction and cell-mediated immunity effectively. In this study, two kinds of plasmids have been used, one can express the HIV-1 main structure proteins, Gagpol and Env, and the other contains an antibiotic gene. The two kinds of plasmids have been cotransfected into 293 cells. A stable cell line that can express Gagpol and Env proteins efficiently and lastingly has been screened. It has been confirmed that Gagpol and Env proteins in the cell culture supernatant can be self-assembled into virus-like particles. The authors have detected the secretion of VLPs in the cell medium, defined the peak of the secretion, and followed and monitored the stability of expression.
基金supported by the Na-tional Natural Science Foundation of China (Grant No. 30371317) to Kong Wei
文摘The rapid growth of the global HIV/AIDS epidemic makes it a high priority to develop an effective vaccine.Since a live attenuated or inactivated HIV vaccine is not likely to be approved for clinical application due to safety concerns,HIV virus like particles(VLPs) offer an attractive alternative because they are considered safer since they lack viral genome.We got a stable eukaryotic cell line by G418 resistance selection,engineered to express the HIV-1 structure protein Gag and Env efficiently and stably.We confirmed the presence of Gag and Env proteins in the cell culture supernatant and that they could self-assemble into VLPs.These VLPs were found to be able to elicit specific humoral and cellular immune response after immunization without any adjuvant.
文摘Insect baculovirus(IBV) is a biological insecticide in agriculture and forestry, butits slow speed of killing pests and narrow range of host make its application quitelimited. Hence, polyhedrin (ocu) gene of IBV was chosen for construction of
