Objective To determine whether the synthesis of proteoglycan, collagen and associated ultrastructure are related to the adenovirus-mediated gene transferred to adult degenerative cells.Methods Adenovirus/cytomegalovir...Objective To determine whether the synthesis of proteoglycan, collagen and associated ultrastructure are related to the adenovirus-mediated gene transferred to adult degenerative cells.Methods Adenovirus/cytomegalovirus human transforming growth fector-β1 (Ad/CMV-hTGF-β1 ) was used to transfect degenerative cells. Antonopulos method, Miamine method and transmission electrion microscopy were conducted to study the synthesis of proteoglycan, collagen, and ultrastructure, respectively. Cell cultures were established from the nucleus pulpous and annulus fibrosus tissues, which were taken from surgery.Results Nucleus pulpous and annulus fibrosus cells were efficiently transduced by the adenoviral vector carrying hTGF-β1 gene. The synthesis of proteoglycan and collagen increased compared with the control group ( P < 0. 05). The metabolism of cells was slightly improved. No significant toxic effects were found.Conclusions Expression of hTGF-β1 gene is efficient to accelerates proteoglycan synthesis and thus accelerates the improvement of collagen. The function and structure of degenerative cells are improved. Ad/CMV-hTGF-β1 may be suitable for treating disc degeneration.展开更多
To provide a highly efficient adenov iral vector Ad CMV hTGFβ1 for the study of gene therapy for reversion of the intervertebral disc degeneration.Methods: A newly developed recombinant adenoviral vector constr uctio...To provide a highly efficient adenov iral vector Ad CMV hTGFβ1 for the study of gene therapy for reversion of the intervertebral disc degeneration.Methods: A newly developed recombinant adenoviral vector constr uction system was used in the study. The cDNA of hTGFβ1 was first subcloned into a shuttle plasmid pShuttle CMV. The resultant plasmid was linearized by d ig esting with restriction endonuclease PmeI, and subsequently transformed into E .coli. BJ5183 cells with an adenoviral backbone plasmid pAdEasy 1. Recombinants were selected by kanamycin resistance and confirmed by restriction endonuclease analysis. Finally, the recombinant plasmid linearized by PmeI was transfected in to 293 cells. Recombinant adenoviruses were generated within 2 weeks. Results: The recombinant adenoviral plasmids were cut by BamHI and PacI respectively, and the diagnostic fragments appeared in 0.8 % agarose electrophoresis. The infected 293 cells showed evident cytopathic effect (CPE). The productions of PCR confirmed the presence of recombinant adenovirus. The exp ression of hTGFβ1 was verified by immunohistochemical staining. Conclusions: The successful generation of the adenoviral vector Ad CMV hTGFβ1 and the confirmation of the interest gene expression make it p ossible for the experimental study of the reversion of the intervertebral disc d egeneration by gene therapy.展开更多
文摘Objective To determine whether the synthesis of proteoglycan, collagen and associated ultrastructure are related to the adenovirus-mediated gene transferred to adult degenerative cells.Methods Adenovirus/cytomegalovirus human transforming growth fector-β1 (Ad/CMV-hTGF-β1 ) was used to transfect degenerative cells. Antonopulos method, Miamine method and transmission electrion microscopy were conducted to study the synthesis of proteoglycan, collagen, and ultrastructure, respectively. Cell cultures were established from the nucleus pulpous and annulus fibrosus tissues, which were taken from surgery.Results Nucleus pulpous and annulus fibrosus cells were efficiently transduced by the adenoviral vector carrying hTGF-β1 gene. The synthesis of proteoglycan and collagen increased compared with the control group ( P < 0. 05). The metabolism of cells was slightly improved. No significant toxic effects were found.Conclusions Expression of hTGF-β1 gene is efficient to accelerates proteoglycan synthesis and thus accelerates the improvement of collagen. The function and structure of degenerative cells are improved. Ad/CMV-hTGF-β1 may be suitable for treating disc degeneration.
文摘To provide a highly efficient adenov iral vector Ad CMV hTGFβ1 for the study of gene therapy for reversion of the intervertebral disc degeneration.Methods: A newly developed recombinant adenoviral vector constr uction system was used in the study. The cDNA of hTGFβ1 was first subcloned into a shuttle plasmid pShuttle CMV. The resultant plasmid was linearized by d ig esting with restriction endonuclease PmeI, and subsequently transformed into E .coli. BJ5183 cells with an adenoviral backbone plasmid pAdEasy 1. Recombinants were selected by kanamycin resistance and confirmed by restriction endonuclease analysis. Finally, the recombinant plasmid linearized by PmeI was transfected in to 293 cells. Recombinant adenoviruses were generated within 2 weeks. Results: The recombinant adenoviral plasmids were cut by BamHI and PacI respectively, and the diagnostic fragments appeared in 0.8 % agarose electrophoresis. The infected 293 cells showed evident cytopathic effect (CPE). The productions of PCR confirmed the presence of recombinant adenovirus. The exp ression of hTGFβ1 was verified by immunohistochemical staining. Conclusions: The successful generation of the adenoviral vector Ad CMV hTGFβ1 and the confirmation of the interest gene expression make it p ossible for the experimental study of the reversion of the intervertebral disc d egeneration by gene therapy.
