OBJECTIVE: To study whether human umbilical cord blood CD34+ cells transduced with human aldehyde dehydrogenase class-1 (ALDH-1) and multidrug resistance gene (MDR1) have increases resistance to 4-Hydroperoxycyclo-pho...OBJECTIVE: To study whether human umbilical cord blood CD34+ cells transduced with human aldehyde dehydrogenase class-1 (ALDH-1) and multidrug resistance gene (MDR1) have increases resistance to 4-Hydroperoxycyclo-phosphamide (4-HC) and P-glycoprotein effluxed drugs. METHODS: A bicistronic retroviral vector G1Na-ALDH1-IRES-MDR1 was constructed and used to transfect the packaging cell lines GP + E86 and PA317 by LipofectAMINE method, using the medium containing VCR and 4-HC agents for cloning selection and ping-ponging supernatant infection between the ecotropic producer clone and the amphotropic producer clone, we obtained high titer amphotropic PA317 producing cells with high titers up to 5.6 x 10(5) CFU/ml. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human ALDH-1 and MDR1cDNA under the stimulation of hemopoietic growth factors. RESULTS: Bicistronic retroviral vector construction was verified by restriction endonuclease analysis. Polymerase chain reaction (PCR), reverse transcription (RT)-PCR, Southern blot, Northern blot, fluorescenceactivated cell sorting (FACS) method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analyses showed that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgenes recipient cells confered 4-fold stronger resistance to 4-HC and 5.5 to 7.2-fold P-glycoprotein effluxed drug than untransduced cells. CONCLUSION: The bicistronic retroviral vector-mediated transfer of two different types of drug resistance genes into human cord blood CD34+ cells and co-expression provided an experimental foundation for improving combination chemotherapy tolerance in tumor clinical trial.展开更多
Background Transforming growth factor-β1 (TGF-β1) exerts strong fibrogenic potential in culture-activated HSCs Smad 4 is a key intracellular mediator for the transforming growth factor-β (TGF-β) superfamily of...Background Transforming growth factor-β1 (TGF-β1) exerts strong fibrogenic potential in culture-activated HSCs Smad 4 is a key intracellular mediator for the transforming growth factor-β (TGF-β) superfamily of growth factors The aim of this study was to assess the effects of the antisense Smad 4 gene on Ito cell line, LI90 Methods The recombinant retroviral vector pLXSN-Smad 4 was constructed by cloning the rat antisense Smad 4 cDNA into the retroviral vector pLXSN Retroviruses with or without the antisense gene were obtained by transfecting pLXSN-Smad 4 and pLXSN vectors into PA317 cells Human hepatic stellate cells (HSCs) LI90 were infected with these retroviruses followed by selection with G418 The expression of Smad 4 was detected by Northern and Western blots Cell biological characteristics, including cell growth curve, 3H-TdR and 3H-proline uptake by HSCs and the production of extracellular matrix were assessed Results mRNA and protein expressions of Smad 4 in LI90 cells transfected with retrovirus containing the antisense Smad 4 gene were much lower than those in LI90 cells transfected with empty vector or parental LI90 cells Cells hypoexpressing the Smad 4 gene exhibited a slower rate of growth, a lower uptake of 3H-TdR and 3H-proline ( P <0 01), and smaller production of th extracellular matrix, compared with parental LI90 cells and cells transfected with empty retrovirus Conclusions The antisense Smad 4 gene can suppress the expression of the Smad 4 gene, reduce endogenous production of Smad 4 mRNA and protein, block TGF-β1 signaling pathway, inhibit activation of Ito cells, obstruct the growth of Ito cells, decrease the production of the extracellular matrix (ECM) Our results may provide a basis for the development of antifibrotic gene therapy展开更多
Retromer and sorting nexins(SNXs)transport cargoes from endosomes to the trans-Golgi network or plasma membrane.Recent studies have unveiled the emerging roles for retromer and SNXs in the life cycle of viruses,includ...Retromer and sorting nexins(SNXs)transport cargoes from endosomes to the trans-Golgi network or plasma membrane.Recent studies have unveiled the emerging roles for retromer and SNXs in the life cycle of viruses,including members of Coronaviridae,Flaviviridae and Retroviridae.Key components of retromer/SNXs,such as Vps35,Vps26,SNX5 and SNX27,can affect multiple steps of the viral life cycle,including facilitating the entry of viruses into cells,participating in viral replication,and promoting the assembly of virions.Here we present a comprehensive updated review on the interplay between retromer/SNXs and virus,which will shed mechanistic insights into controlling virus infection.展开更多
The aim of this paper is to explore the effects of transfection of Foxp3 gene on the phenotype and function of naive CD4^(+)T cells.The pMSCV-Foxp3 retroviral vec-tor encoding Foxp3 gene was transduced into the PT67 p...The aim of this paper is to explore the effects of transfection of Foxp3 gene on the phenotype and function of naive CD4^(+)T cells.The pMSCV-Foxp3 retroviral vec-tor encoding Foxp3 gene was transduced into the PT67 packaging cell line.Virus-containing supernatant was applied to differentiate CD4^(+)CD25^(-) T cells.The resulting cells were sorted with flow cytometry.The expressions of CD25,CD127,CTLA-4 and the proliferation of trans-fected T cells were examined.The effect of transfected CD4^(+)T cells on the proliferation and cytokine production of CD4^(+)CD25^(-) T cells was examined.Foxp3-gene trans-fected CD4^(+)T cells could express Foxp3 and transfection of Foxp3 gene up-regulated the expressions of CD25 and CTLA-4,but down-regulated CD127 expression.After transfection,the proliferation of CD4^(+)T cells was elimi-nated.Transfected T cells inhibited the proliferation of CD4^(+)CD25^(-) T cells.CD4^(+)CD25^(-) T cells acquired a reg-ulatory phenotype and function after it was transduced with the Foxp3 gene.This suggested a key role of Foxp3 in the generation of CD4^(+)CD25^(+) regulatory T cells.展开更多
基金theNationalNaturalScienceFoundationofChina (No 39770 331)
文摘OBJECTIVE: To study whether human umbilical cord blood CD34+ cells transduced with human aldehyde dehydrogenase class-1 (ALDH-1) and multidrug resistance gene (MDR1) have increases resistance to 4-Hydroperoxycyclo-phosphamide (4-HC) and P-glycoprotein effluxed drugs. METHODS: A bicistronic retroviral vector G1Na-ALDH1-IRES-MDR1 was constructed and used to transfect the packaging cell lines GP + E86 and PA317 by LipofectAMINE method, using the medium containing VCR and 4-HC agents for cloning selection and ping-ponging supernatant infection between the ecotropic producer clone and the amphotropic producer clone, we obtained high titer amphotropic PA317 producing cells with high titers up to 5.6 x 10(5) CFU/ml. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human ALDH-1 and MDR1cDNA under the stimulation of hemopoietic growth factors. RESULTS: Bicistronic retroviral vector construction was verified by restriction endonuclease analysis. Polymerase chain reaction (PCR), reverse transcription (RT)-PCR, Southern blot, Northern blot, fluorescenceactivated cell sorting (FACS) method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analyses showed that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgenes recipient cells confered 4-fold stronger resistance to 4-HC and 5.5 to 7.2-fold P-glycoprotein effluxed drug than untransduced cells. CONCLUSION: The bicistronic retroviral vector-mediated transfer of two different types of drug resistance genes into human cord blood CD34+ cells and co-expression provided an experimental foundation for improving combination chemotherapy tolerance in tumor clinical trial.
文摘Background Transforming growth factor-β1 (TGF-β1) exerts strong fibrogenic potential in culture-activated HSCs Smad 4 is a key intracellular mediator for the transforming growth factor-β (TGF-β) superfamily of growth factors The aim of this study was to assess the effects of the antisense Smad 4 gene on Ito cell line, LI90 Methods The recombinant retroviral vector pLXSN-Smad 4 was constructed by cloning the rat antisense Smad 4 cDNA into the retroviral vector pLXSN Retroviruses with or without the antisense gene were obtained by transfecting pLXSN-Smad 4 and pLXSN vectors into PA317 cells Human hepatic stellate cells (HSCs) LI90 were infected with these retroviruses followed by selection with G418 The expression of Smad 4 was detected by Northern and Western blots Cell biological characteristics, including cell growth curve, 3H-TdR and 3H-proline uptake by HSCs and the production of extracellular matrix were assessed Results mRNA and protein expressions of Smad 4 in LI90 cells transfected with retrovirus containing the antisense Smad 4 gene were much lower than those in LI90 cells transfected with empty vector or parental LI90 cells Cells hypoexpressing the Smad 4 gene exhibited a slower rate of growth, a lower uptake of 3H-TdR and 3H-proline ( P <0 01), and smaller production of th extracellular matrix, compared with parental LI90 cells and cells transfected with empty retrovirus Conclusions The antisense Smad 4 gene can suppress the expression of the Smad 4 gene, reduce endogenous production of Smad 4 mRNA and protein, block TGF-β1 signaling pathway, inhibit activation of Ito cells, obstruct the growth of Ito cells, decrease the production of the extracellular matrix (ECM) Our results may provide a basis for the development of antifibrotic gene therapy
基金supported by grants from National Natural Science Foundation of China(81871663 and 82072270)Undergraduate Science and Technology Innovation Plan of International Class of Clinical Medicine in Shandong First Medical University(ZYKC2019-016)Academic promotion programme of Shandong First Medical University(2019LJ001)。
文摘Retromer and sorting nexins(SNXs)transport cargoes from endosomes to the trans-Golgi network or plasma membrane.Recent studies have unveiled the emerging roles for retromer and SNXs in the life cycle of viruses,including members of Coronaviridae,Flaviviridae and Retroviridae.Key components of retromer/SNXs,such as Vps35,Vps26,SNX5 and SNX27,can affect multiple steps of the viral life cycle,including facilitating the entry of viruses into cells,participating in viral replication,and promoting the assembly of virions.Here we present a comprehensive updated review on the interplay between retromer/SNXs and virus,which will shed mechanistic insights into controlling virus infection.
基金supported by the National Natural Science Foundation of China(Grant No.30470772).
文摘The aim of this paper is to explore the effects of transfection of Foxp3 gene on the phenotype and function of naive CD4^(+)T cells.The pMSCV-Foxp3 retroviral vec-tor encoding Foxp3 gene was transduced into the PT67 packaging cell line.Virus-containing supernatant was applied to differentiate CD4^(+)CD25^(-) T cells.The resulting cells were sorted with flow cytometry.The expressions of CD25,CD127,CTLA-4 and the proliferation of trans-fected T cells were examined.The effect of transfected CD4^(+)T cells on the proliferation and cytokine production of CD4^(+)CD25^(-) T cells was examined.Foxp3-gene trans-fected CD4^(+)T cells could express Foxp3 and transfection of Foxp3 gene up-regulated the expressions of CD25 and CTLA-4,but down-regulated CD127 expression.After transfection,the proliferation of CD4^(+)T cells was elimi-nated.Transfected T cells inhibited the proliferation of CD4^(+)CD25^(-) T cells.CD4^(+)CD25^(-) T cells acquired a reg-ulatory phenotype and function after it was transduced with the Foxp3 gene.This suggested a key role of Foxp3 in the generation of CD4^(+)CD25^(+) regulatory T cells.
