Rhizospheres can promote self-transmissible plasmid transfer,however,the corresponding mechanism has not received much attention.Plant-microbe remediation is an effective way to promote pollutant biodegradation;howeve...Rhizospheres can promote self-transmissible plasmid transfer,however,the corresponding mechanism has not received much attention.Plant-microbe remediation is an effective way to promote pollutant biodegradation;however,some pollutants,such as naphthalene,are harmful to plants and result in inefficient plant-microbe remediation.In this study,trans-fer of a TOL-like plasmid,a self-transmissible plasmid loaded with genetic determinants for pollutant degradation,among different bacteria was examined in bulk and rhizosphere soils as well as addition of maize root exudate and its artificial root exudate(ARE).The results showed that the numbers of transconjugants and recipients as well as bacterial metabolic activities,such as xylE mRNA expression levels and catechol 2,3-dioxygenase(C23O)activ-ities of bacteria,remained high in rhizosphere soils,when compared with bulk soils.The number of transconjugants and bacterial metabolic activities increased with the increasing exudate and ARE concentrations,whereas the populations of donor and recipient bacteria were substantially unaltered at all concentrations.All the experiments consistently showed that a certain number of bacteria is required for self-transmissible plasmid transfer,and that the increased plasmid transfer might predominantly be owing to bacterial metabolic activ-ity stimulated by root exudates and ARE.Furthermore,ARE addition increased naphthalene degradation by transconjugants in both culture medium and soil.Thus,the combined action of a wide variety of components in ARE might contribute to the increased plasmid transfer and naphthalene degradation.These findings suggest that ARE could be an effectively al-ternative for plant-microbe remediation of pollutants in environments where plants cannot survive.展开更多
Objective The gene expression of skeletal muscle under ischemic condition by direct gene injectionwas observed in order to find a new gene delivery method to treat chronic arterial occlusion disease. Methods Weestabli...Objective The gene expression of skeletal muscle under ischemic condition by direct gene injectionwas observed in order to find a new gene delivery method to treat chronic arterial occlusion disease. Methods Weestablished the rabbit hindlimb ischemic model and used plasmid PSV-β-gal as a reporter gene. We transferedgene intramuscularly and detected the activity of β-galactosidase by histochemistry method. Results Weobserved that the gene expression of skeletal muscle under ischemic condition was higher than normalmuscle. Conclusion The result demonstrated that the direct gene injection was suitable for the chronic peripheralarterial occlusion disease, and might be a novel gene delivery method for this disease.展开更多
Summary: To construct basic fibroblast growth factor (bFGF) eukaryotic expression vector and to evaluate the possibility of bFGF gene therapy in orthopedic disease, the pCD-rbFGF recombinant plasmid was constructed by...Summary: To construct basic fibroblast growth factor (bFGF) eukaryotic expression vector and to evaluate the possibility of bFGF gene therapy in orthopedic disease, the pCD-rbFGF recombinant plasmid was constructed by cloning rat basic fibroblast growth factor (bFGF) cDNA into an eukaryotic expression vector, pcDNA 3. Rat osteoblasts were transfected with pCD-rbFGF plasmid by lopofectin mediated gene transfer, the transient expression was detected by streptavidin-biotin-enzyme complex (SABC) method. It was observed that the expression of rat bFGF gene was detected 72 h after transfected distinctly. Basic fibroblast growth factor gene therapy is a method of potential for a wide array of orthopedic diseases.展开更多
The method of gene transfer into corneal endothelium was investigated to provide a foundation for the study of TGF-β1 gene transfer to inhibit corneal graft rejection.Two days after direct injection of p MAM TGF-β...The method of gene transfer into corneal endothelium was investigated to provide a foundation for the study of TGF-β1 gene transfer to inhibit corneal graft rejection.Two days after direct injection of p MAM TGF-β1 mediated by liposom e into the anterior cham ber of rabbits,one half of corneas were made into paraffin slides and the endothelial layer was carefully torn from the other half to make a single layer slide of endothelia.By means of im munohistochemical technique, the plasmid p MAM TGF- β1 expression product TGF- β1 in the endothelia was detected.Specific TGF- β1 expression was positive in the endothelia on both the paraffin slide and the single layer slide.The results showed that by direct injection into the anterior cham ber,foreign plasmid DNA could be transferred into the endothelia and its expression was obtained.This may provide a foun- dation for further study on TGF-β1 participating in local induction of corneal imm une tolerance.展开更多
Obiective TO improve the plasmid vectors in gene therapy, adeno - associated virus (AAV) basedplasmid expressing vectors containing hIL - 2 gene or mIFN-γ gene were constructed and its expression intransfected cells ...Obiective TO improve the plasmid vectors in gene therapy, adeno - associated virus (AAV) basedplasmid expressing vectors containing hIL - 2 gene or mIFN-γ gene were constructed and its expression intransfected cells was studied. Methods By means of step to step cloning, promoter CMVp was placed at thedownstream of 5’ inverted terminal repeat from AAV (AAV - ITR) of pAP, hIL - 2 gene or mIFN -γ gene insertedinto pAC between CMVp and polyA. Then intron A was inserted into pAC - hIL - 2 or pAC- mIFN-γ betweenCMVp and IL - 2 gene or IFNγ gene to construct pAI- hIL - 2 or pAI- mIFN -γ. Liposome - plasmid complexeswere formed by mixing Dosper with these AAV- based plasmids containing hIL - 2 gene or mIFN- γgene. Results High biotogical activities of IL - 2 or IFN- γ could be detected in the supernatants of NIH3T3 andMM45T Li cells after transfection. Insertion of intron A into pAC- hIL - 2 or pAC- mIFN - γ improved theexpression of IL - 2 or IFN- γ. Conclusion These data demonstrated that the constructed AAV-based plasmidexpressing vectors could ejlciently express therapeutic genes in cultured cells and could be used as a nonviral genetransfer system in human gene therapy.展开更多
After organ transplantation, rapid repair of injured vascular endothelial cell (VEC) is a key to prevent graft chronic dysfunction besides control of immunological rejection. Many studies have confirmed that vascular ...After organ transplantation, rapid repair of injured vascular endothelial cell (VEC) is a key to prevent graft chronic dysfunction besides control of immunological rejection. Many studies have confirmed that vascular endothelial growth factor 165 (VEGF165) could accelerate the repair of VEC injury, decrease thrombosis and thrombotic occlusion, and inhibit hyperplasia of the intima. This study was designed to construct eukaryotic expression plasmid pBudCE4.1/VEGF165, and observe its effect on the prolife ration of VEC. METHODS:The VEGF165 gene cloned from human heart tissue by RT-PCR was cloned into eukaryotic expression plasmid pBudCE4.1. The recombinant expression plasmid pBudCE4.1/VEGF165 was identified by restriction enzyme (Hind III and BamH I) digestion analysis, and was sequenced. The pBudCE4.1/VEGF165 was introduced into VEC through lipofection transfection. The VEGF165 mRNA expression by Northern blot and VEGF165 protein expression was detected by immunocytochemical staining. The effect of expression protein on VEC proliferation was detected by flow cytometry. RESULTS:The RT-PCR product of the VEGF165 gene was about 576bp. Sequencing analysis revealed that the sequence of the amplified VEGF165 gene was identical with that in GenBank. Restrictive enzyme digestion analysis showed that recombinant expression plasmid pBudCE4.1/ tVEGF165 had been constructed successfully. The expression of VEGF165 at mRNA and protein levels in the transformed VSMCs had been demonstrated by Northern blot and immunocytochemical staining respectively. The expressed product of VEGF165 could notably accelerate the proliferation of VECs. CONCLUSIONS:pBudCE4.1/VEGF165 is successfully cons- tructed and is expressed in VECs. Expressed VEGF165 can accelerate the VEC proliferation. The present study has laid a foundation for potential use of VEGF165 gene transfection to prevent and treat vascular stenosis in the transplanted organ.展开更多
BACKGROUND: The highly specific vascular endothelialgrowth factor (VEGF) induces the growth of vascular en-dothelial cell. This study was to construct the eukaryoticexpression plasmid of vascular endothelial growth fa...BACKGROUND: The highly specific vascular endothelialgrowth factor (VEGF) induces the growth of vascular en-dothelial cell. This study was to construct the eukaryoticexpression plasmid of vascular endothelial growth factorl65(VEGF165) and observe its expression in vascular smoothmuscles (VSMCs).METHODS: The primers were designed and synthesizedaccording to the gene sequences of human VEGF165. TheVEGF165 gene was obtained from umbilic artery tissue bythe method of RT-PCR, then it was cloned to eukaryoticexpression plasmid pBudCE4.1 by recombination strategy.The eukaryotic expression plasmid named pBudCE4.1/VEGF165 was identified by restriction enzyme digestion,and was sequenced. The pBudCE4.1/VEGF165 was trans-fected into VSMCs by using lipofection. The VEGF165 ex-pression of mRNA and protein was detected by RT-PCRand Western blot respectively.RESULTS: VEGF165 was shown about 576bp by RT-PCR.Sequencing revealed the amplified VEGF165 gene was iden-tical with that in the GeneBank. Restrictive enzyme (HindBam HI) digestion analysis showed that recombinantexpression plasmid pBudCE4. l/tVEGF165 had been con-structed successfully. The expression of VEGF165 at mRNAand protein levels in the transformed VSMCs had beendemonstrated by RT-PCR and Western blot.CONCLUSIONS: The recombinant eukaryotic expressionplasmid pBudCE4.1/VEGF165 has been successfully con-structed and expressed in transformed VSMCs. The presentstudy has laid a foundation for VEGF165 gene therapy ofvascular stenosis in the transplant organ.展开更多
基金This work was supported by the Shanghai Municipal Science and Technology Commission(No.16391902100)the Shanghai Construction Group(No.19JCSF-12)the Ministry of Agriculture,P.R.China and Shanghai Engineering Research Center of Plant Germplasm Resources(No.17DZ2252700).
文摘Rhizospheres can promote self-transmissible plasmid transfer,however,the corresponding mechanism has not received much attention.Plant-microbe remediation is an effective way to promote pollutant biodegradation;however,some pollutants,such as naphthalene,are harmful to plants and result in inefficient plant-microbe remediation.In this study,trans-fer of a TOL-like plasmid,a self-transmissible plasmid loaded with genetic determinants for pollutant degradation,among different bacteria was examined in bulk and rhizosphere soils as well as addition of maize root exudate and its artificial root exudate(ARE).The results showed that the numbers of transconjugants and recipients as well as bacterial metabolic activities,such as xylE mRNA expression levels and catechol 2,3-dioxygenase(C23O)activ-ities of bacteria,remained high in rhizosphere soils,when compared with bulk soils.The number of transconjugants and bacterial metabolic activities increased with the increasing exudate and ARE concentrations,whereas the populations of donor and recipient bacteria were substantially unaltered at all concentrations.All the experiments consistently showed that a certain number of bacteria is required for self-transmissible plasmid transfer,and that the increased plasmid transfer might predominantly be owing to bacterial metabolic activ-ity stimulated by root exudates and ARE.Furthermore,ARE addition increased naphthalene degradation by transconjugants in both culture medium and soil.Thus,the combined action of a wide variety of components in ARE might contribute to the increased plasmid transfer and naphthalene degradation.These findings suggest that ARE could be an effectively al-ternative for plant-microbe remediation of pollutants in environments where plants cannot survive.
文摘Objective The gene expression of skeletal muscle under ischemic condition by direct gene injectionwas observed in order to find a new gene delivery method to treat chronic arterial occlusion disease. Methods Weestablished the rabbit hindlimb ischemic model and used plasmid PSV-β-gal as a reporter gene. We transferedgene intramuscularly and detected the activity of β-galactosidase by histochemistry method. Results Weobserved that the gene expression of skeletal muscle under ischemic condition was higher than normalmuscle. Conclusion The result demonstrated that the direct gene injection was suitable for the chronic peripheralarterial occlusion disease, and might be a novel gene delivery method for this disease.
文摘Summary: To construct basic fibroblast growth factor (bFGF) eukaryotic expression vector and to evaluate the possibility of bFGF gene therapy in orthopedic disease, the pCD-rbFGF recombinant plasmid was constructed by cloning rat basic fibroblast growth factor (bFGF) cDNA into an eukaryotic expression vector, pcDNA 3. Rat osteoblasts were transfected with pCD-rbFGF plasmid by lopofectin mediated gene transfer, the transient expression was detected by streptavidin-biotin-enzyme complex (SABC) method. It was observed that the expression of rat bFGF gene was detected 72 h after transfected distinctly. Basic fibroblast growth factor gene therapy is a method of potential for a wide array of orthopedic diseases.
基金This project was supported by a grant from the NaturalSciences Foundation of Hubei Province(No.97J0 70 )
文摘The method of gene transfer into corneal endothelium was investigated to provide a foundation for the study of TGF-β1 gene transfer to inhibit corneal graft rejection.Two days after direct injection of p MAM TGF-β1 mediated by liposom e into the anterior cham ber of rabbits,one half of corneas were made into paraffin slides and the endothelial layer was carefully torn from the other half to make a single layer slide of endothelia.By means of im munohistochemical technique, the plasmid p MAM TGF- β1 expression product TGF- β1 in the endothelia was detected.Specific TGF- β1 expression was positive in the endothelia on both the paraffin slide and the single layer slide.The results showed that by direct injection into the anterior cham ber,foreign plasmid DNA could be transferred into the endothelia and its expression was obtained.This may provide a foun- dation for further study on TGF-β1 participating in local induction of corneal imm une tolerance.
文摘Obiective TO improve the plasmid vectors in gene therapy, adeno - associated virus (AAV) basedplasmid expressing vectors containing hIL - 2 gene or mIFN-γ gene were constructed and its expression intransfected cells was studied. Methods By means of step to step cloning, promoter CMVp was placed at thedownstream of 5’ inverted terminal repeat from AAV (AAV - ITR) of pAP, hIL - 2 gene or mIFN -γ gene insertedinto pAC between CMVp and polyA. Then intron A was inserted into pAC - hIL - 2 or pAC- mIFN-γ betweenCMVp and IL - 2 gene or IFNγ gene to construct pAI- hIL - 2 or pAI- mIFN -γ. Liposome - plasmid complexeswere formed by mixing Dosper with these AAV- based plasmids containing hIL - 2 gene or mIFN- γgene. Results High biotogical activities of IL - 2 or IFN- γ could be detected in the supernatants of NIH3T3 andMM45T Li cells after transfection. Insertion of intron A into pAC- hIL - 2 or pAC- mIFN - γ improved theexpression of IL - 2 or IFN- γ. Conclusion These data demonstrated that the constructed AAV-based plasmidexpressing vectors could ejlciently express therapeutic genes in cultured cells and could be used as a nonviral genetransfer system in human gene therapy.
文摘After organ transplantation, rapid repair of injured vascular endothelial cell (VEC) is a key to prevent graft chronic dysfunction besides control of immunological rejection. Many studies have confirmed that vascular endothelial growth factor 165 (VEGF165) could accelerate the repair of VEC injury, decrease thrombosis and thrombotic occlusion, and inhibit hyperplasia of the intima. This study was designed to construct eukaryotic expression plasmid pBudCE4.1/VEGF165, and observe its effect on the prolife ration of VEC. METHODS:The VEGF165 gene cloned from human heart tissue by RT-PCR was cloned into eukaryotic expression plasmid pBudCE4.1. The recombinant expression plasmid pBudCE4.1/VEGF165 was identified by restriction enzyme (Hind III and BamH I) digestion analysis, and was sequenced. The pBudCE4.1/VEGF165 was introduced into VEC through lipofection transfection. The VEGF165 mRNA expression by Northern blot and VEGF165 protein expression was detected by immunocytochemical staining. The effect of expression protein on VEC proliferation was detected by flow cytometry. RESULTS:The RT-PCR product of the VEGF165 gene was about 576bp. Sequencing analysis revealed that the sequence of the amplified VEGF165 gene was identical with that in GenBank. Restrictive enzyme digestion analysis showed that recombinant expression plasmid pBudCE4.1/ tVEGF165 had been constructed successfully. The expression of VEGF165 at mRNA and protein levels in the transformed VSMCs had been demonstrated by Northern blot and immunocytochemical staining respectively. The expressed product of VEGF165 could notably accelerate the proliferation of VECs. CONCLUSIONS:pBudCE4.1/VEGF165 is successfully cons- tructed and is expressed in VECs. Expressed VEGF165 can accelerate the VEC proliferation. The present study has laid a foundation for potential use of VEGF165 gene transfection to prevent and treat vascular stenosis in the transplanted organ.
基金This study was supported by grants from the 973 National Basic ResearchProgram of China ( 2003CB515501 ) and the National Natural ScienceFoundation of China (No. 30270514).
文摘BACKGROUND: The highly specific vascular endothelialgrowth factor (VEGF) induces the growth of vascular en-dothelial cell. This study was to construct the eukaryoticexpression plasmid of vascular endothelial growth factorl65(VEGF165) and observe its expression in vascular smoothmuscles (VSMCs).METHODS: The primers were designed and synthesizedaccording to the gene sequences of human VEGF165. TheVEGF165 gene was obtained from umbilic artery tissue bythe method of RT-PCR, then it was cloned to eukaryoticexpression plasmid pBudCE4.1 by recombination strategy.The eukaryotic expression plasmid named pBudCE4.1/VEGF165 was identified by restriction enzyme digestion,and was sequenced. The pBudCE4.1/VEGF165 was trans-fected into VSMCs by using lipofection. The VEGF165 ex-pression of mRNA and protein was detected by RT-PCRand Western blot respectively.RESULTS: VEGF165 was shown about 576bp by RT-PCR.Sequencing revealed the amplified VEGF165 gene was iden-tical with that in the GeneBank. Restrictive enzyme (HindBam HI) digestion analysis showed that recombinantexpression plasmid pBudCE4. l/tVEGF165 had been con-structed successfully. The expression of VEGF165 at mRNAand protein levels in the transformed VSMCs had beendemonstrated by RT-PCR and Western blot.CONCLUSIONS: The recombinant eukaryotic expressionplasmid pBudCE4.1/VEGF165 has been successfully con-structed and expressed in transformed VSMCs. The presentstudy has laid a foundation for VEGF165 gene therapy ofvascular stenosis in the transplant organ.
