Objective： To construct a recombinant adenovirus vector-carrying human growth and differentiation factor-5 （GDF-5） gene, investigate the biological effects of adenovirus-mediated GDF-5 （Ad-GDF-5） on extracellular...Objective： To construct a recombinant adenovirus vector-carrying human growth and differentiation factor-5 （GDF-5） gene, investigate the biological effects of adenovirus-mediated GDF-5 （Ad-GDF-5） on extracellular matrix （ECM） expression in human degenerative disc nucleus pulposus （NP） cells, and explore a candidate gene therapy method for intervertebral disc degeneration （IDD）. Methods： Human NP cells of a degenerative disc were isolated, cultured, and infected with Ad-GDF-5 using the AdEasy-1 adenovirus vector system. On Days 3, 7, 14, and 21, the contents of the sulfated glycosaminoglycan （sGAG）, deoxyribonucleic acid （DNA） and hydroxyproline （Hyp）, synthesis of proteoglycan and collagen II, gene expression of collagen II and aggrecan, and NP cell proliferation were assessed. Results： The adenovirus was an effective vehicle for gene delivery with prolonged expression of GDF-5. Biochemical analysis revealed increased sGAG and Hyp contents in human NP cells infected by Ad-GDF-5 whereas there was no conspicuous change in basal medium （BM） or Ad-green fluorescent protein （GFP） groups. Only cells in the Ad-GDF-5 group promoted the production of ECM, as demonstrated by the secretion of proteoglycan and up-regulation of collagen II and aggrecan at both protein and mRNA levels. The NP cell proliferation was significantly promoted. Conclusions： The data suggest that Ad-GDF-5 gene therapy is a potential treatment for IDD, which restores the functions of degenerative intervertebral disc through enhancing the ECM production of human NP ceils.展开更多
Objective To observe the effect of growth differentiation factor-5 (GDF-5) on the growth and anabolic metabolism of articular chondrocytes. Methods The articular chondrocytes isolated from rats were treated with vario...Objective To observe the effect of growth differentiation factor-5 (GDF-5) on the growth and anabolic metabolism of articular chondrocytes. Methods The articular chondrocytes isolated from rats were treated with various concentrations of rmGDF-5, and the growth of chondrocytes measured by MTT assay, the cellular cartilage matrices formation detected sulfated glycosaminoglycan by Alcian blue staining and type Ⅱcollagen by RT-PCR. Results After 7 days culture, MTT assay showed that GDF-5 enhanced the growth of chondrocytes in a dose-dependent manner, RT-PCR showed that GDF-5 clearly induced the synthesis of type Ⅱ collagen because of the col2a1 mRNA band more and more strong in a dose-dependent. Chondrocytes were cultured with GDF-5 for 14 days, the intensity of Alcian blue staining was greatly enhanced, especially, at a high concentration of 1000ng/mL, and GDF-5 enhanced the accumulation of the Alcian blue-stainable material in a concentration-dependent manner and in a does-dependent manner. Conclusion GDF-5 enhanced the growth of mature articular chondrocytes, and stimulated the cellular cartilage matrices formation in mono-layer culture.展开更多
Summary: The integral mature peptide gene of human growth differentiation factor-5 (GDF-5) was cloned to provide the essential foundation for study on the biological characteristics of GDF-5 at gene and protein levels...Summary: The integral mature peptide gene of human growth differentiation factor-5 (GDF-5) was cloned to provide the essential foundation for study on the biological characteristics of GDF-5 at gene and protein levels. Two primers were chemosynthesized according to the hGDF-5 sequence reported in Genbank. The hGDF-5 gene was gained by RT-PCR methods from the total RNA extracted from human fetus cartilage tissue, and was cloned into vector pMD18-T. The sequence of recombinant plasmid pMD18-T-hGDF-5 was analyzed by sequence analysis. DNA agarose gel electrophoresis showed that the product of RT-PCR was about 380bp, and double enzyme digestion of the recombinant plasmid corresponded with it. The result of sequence assay was in agreement with the reported hGDF-5 sequence in Genbank. Our results showed that the integral mature peptide gene of human GDF-5 was cloned successfully from human fetal cartilage tissue, and totally identified with the sequence of human GDF-5 in Genbank.展开更多
Objective：To investigate the effect of the gap junction blocker 1-heptanol on the in vitro chondrogenic differentiation of mouse bone marrow mesenchymal stem cells（MSCs） following induction by GDF-5. Methods：MSCs ...Objective：To investigate the effect of the gap junction blocker 1-heptanol on the in vitro chondrogenic differentiation of mouse bone marrow mesenchymal stem cells（MSCs） following induction by GDF-5. Methods：MSCs were isolated from mouse bone marrow and cultured in vitro. After 3 passages cells were induced to undergo chondrogenic differentiation with recombinant human GDF-5（100 ng/ml）, with or without 1-heptanol（2.5 la mol/L）. The effect of 1-heptanol on MSCs proliferation was investigated using the MTT assay. Type II collagen mRNA and protein were examined by RT-PCR and immunocytochemistry respectively, and the sulfate glycosaminoglycan was assessed by Alcian blue dye staining. Connexin43（Cx43） protein was examined by western blotting. Results：GDF-5 induced proliferation and chondrogenic differentiation of MSCs. While 1-heptanol treatment had no effect on this proliferation, it inhibited the expression of both type II collagen mRNA and protein. The Alcian blue staining revealed that 1-heptanol also inhibited the deposition of the typical cartilage extracellular matrix promoted by recombinant GDF-5. Western blotting demonstrated that 1-heptanol had no effect on the expression of Cx43. Conclusion：These results suggest that mouse bone marrow MSCs can be differentiated into a chondrogenic phenotype by GDF- 5 administration in vitro. While the gap junction blocker, 1-heptanol, did not reduce gap junction Cx43, these intercellular communication pathways clearly played an important functional role in GDF-5-induced cartilage differentiation.展开更多
Objective:To construct a novel non-viral vector loaded with growth and differentiation factor-5(GDF-5) plasmid using chitosan,hyaluronic acid,and chondroitin sulfate for osteoarthritis (OA)gene therapy.Methods: Nano-m...Objective:To construct a novel non-viral vector loaded with growth and differentiation factor-5(GDF-5) plasmid using chitosan,hyaluronic acid,and chondroitin sulfate for osteoarthritis (OA)gene therapy.Methods: Nano-microspheres (NMPs)were prepared by mixing chitosan,hyaluronic acid,and chondreitin sulfate.GDF-5 plasmid was encapsulated in the NMPs through electrostatic adsorption.The basic characteristics of the NMPs were observed,and then they were co-cultured with chondrocytes to observe their effects on extracellular matrix (ECM) protein expression.Finally,NMPs loaded with GDF-5were inje.cted into the articular cavities of rabbits to observe their therapeutic effects on OA in vivo.Results:NMPs exhibited good physicochemical properties and low cytotoxicity.Their average diameter was (0.61±0.20)μm,and encapsulation efficiency was (38.19±0.36)%.According to Cell Counting Kit-8(CCK-8)assay,relative cell viability was 75%-99%when the total weight of NMPs was less than 560μg. Transfection efficiency was (62.0±2.1)% in a liposome group,and (60.0±1.8)% in the NMP group.There was no sig- nificant difference between the two groups (P>0.05).Immunohistochemical staining results suggested that NMPs can successfully transfect chondrocytes and stimulate ECM protein expression in vitro.Compared with the control groups, the NMP group significantly promoted the expression of chondrocyte ECM in vivo (P<0.05),as shown by analysis of the biochemical composition of chondrocyte ECM.When NMPs were injected into OA model rabbits,the expression of ECM proteins in chondrocytes was significantly promoted and the progression of OA was slowed down.Conclusions: Based on these data,we think that these NMPs with excellent physicochemical and biological properties could be promising non-viral vectors for OA gene therapy.展开更多
基金Project supported by the National Natural Science Foundation of China(Nos.81171472 and 81201407)the Innovation Team Project of Sichuan Provincial Education Department(No.13TD0030)+1 种基金the Major Transformation Cultivation Project of Sichuan Provincial Education Department(No.15CZ0021)the Science and Technology Project of Nanchong City(No.14A0021),China
文摘Objective： To construct a recombinant adenovirus vector-carrying human growth and differentiation factor-5 （GDF-5） gene, investigate the biological effects of adenovirus-mediated GDF-5 （Ad-GDF-5） on extracellular matrix （ECM） expression in human degenerative disc nucleus pulposus （NP） cells, and explore a candidate gene therapy method for intervertebral disc degeneration （IDD）. Methods： Human NP cells of a degenerative disc were isolated, cultured, and infected with Ad-GDF-5 using the AdEasy-1 adenovirus vector system. On Days 3, 7, 14, and 21, the contents of the sulfated glycosaminoglycan （sGAG）, deoxyribonucleic acid （DNA） and hydroxyproline （Hyp）, synthesis of proteoglycan and collagen II, gene expression of collagen II and aggrecan, and NP cell proliferation were assessed. Results： The adenovirus was an effective vehicle for gene delivery with prolonged expression of GDF-5. Biochemical analysis revealed increased sGAG and Hyp contents in human NP cells infected by Ad-GDF-5 whereas there was no conspicuous change in basal medium （BM） or Ad-green fluorescent protein （GFP） groups. Only cells in the Ad-GDF-5 group promoted the production of ECM, as demonstrated by the secretion of proteoglycan and up-regulation of collagen II and aggrecan at both protein and mRNA levels. The NP cell proliferation was significantly promoted. Conclusions： The data suggest that Ad-GDF-5 gene therapy is a potential treatment for IDD, which restores the functions of degenerative intervertebral disc through enhancing the ECM production of human NP ceils.
文摘Objective To observe the effect of growth differentiation factor-5 (GDF-5) on the growth and anabolic metabolism of articular chondrocytes. Methods The articular chondrocytes isolated from rats were treated with various concentrations of rmGDF-5, and the growth of chondrocytes measured by MTT assay, the cellular cartilage matrices formation detected sulfated glycosaminoglycan by Alcian blue staining and type Ⅱcollagen by RT-PCR. Results After 7 days culture, MTT assay showed that GDF-5 enhanced the growth of chondrocytes in a dose-dependent manner, RT-PCR showed that GDF-5 clearly induced the synthesis of type Ⅱ collagen because of the col2a1 mRNA band more and more strong in a dose-dependent. Chondrocytes were cultured with GDF-5 for 14 days, the intensity of Alcian blue staining was greatly enhanced, especially, at a high concentration of 1000ng/mL, and GDF-5 enhanced the accumulation of the Alcian blue-stainable material in a concentration-dependent manner and in a does-dependent manner. Conclusion GDF-5 enhanced the growth of mature articular chondrocytes, and stimulated the cellular cartilage matrices formation in mono-layer culture.
文摘Summary: The integral mature peptide gene of human growth differentiation factor-5 (GDF-5) was cloned to provide the essential foundation for study on the biological characteristics of GDF-5 at gene and protein levels. Two primers were chemosynthesized according to the hGDF-5 sequence reported in Genbank. The hGDF-5 gene was gained by RT-PCR methods from the total RNA extracted from human fetus cartilage tissue, and was cloned into vector pMD18-T. The sequence of recombinant plasmid pMD18-T-hGDF-5 was analyzed by sequence analysis. DNA agarose gel electrophoresis showed that the product of RT-PCR was about 380bp, and double enzyme digestion of the recombinant plasmid corresponded with it. The result of sequence assay was in agreement with the reported hGDF-5 sequence in Genbank. Our results showed that the integral mature peptide gene of human GDF-5 was cloned successfully from human fetal cartilage tissue, and totally identified with the sequence of human GDF-5 in Genbank.
基金supported by the National Natural Science Foundation of China(30471753)
文摘Objective：To investigate the effect of the gap junction blocker 1-heptanol on the in vitro chondrogenic differentiation of mouse bone marrow mesenchymal stem cells（MSCs） following induction by GDF-5. Methods：MSCs were isolated from mouse bone marrow and cultured in vitro. After 3 passages cells were induced to undergo chondrogenic differentiation with recombinant human GDF-5（100 ng/ml）, with or without 1-heptanol（2.5 la mol/L）. The effect of 1-heptanol on MSCs proliferation was investigated using the MTT assay. Type II collagen mRNA and protein were examined by RT-PCR and immunocytochemistry respectively, and the sulfate glycosaminoglycan was assessed by Alcian blue dye staining. Connexin43（Cx43） protein was examined by western blotting. Results：GDF-5 induced proliferation and chondrogenic differentiation of MSCs. While 1-heptanol treatment had no effect on this proliferation, it inhibited the expression of both type II collagen mRNA and protein. The Alcian blue staining revealed that 1-heptanol also inhibited the deposition of the typical cartilage extracellular matrix promoted by recombinant GDF-5. Western blotting demonstrated that 1-heptanol had no effect on the expression of Cx43. Conclusion：These results suggest that mouse bone marrow MSCs can be differentiated into a chondrogenic phenotype by GDF- 5 administration in vitro. While the gap junction blocker, 1-heptanol, did not reduce gap junction Cx43, these intercellular communication pathways clearly played an important functional role in GDF-5-induced cartilage differentiation.
基金Project supported by the National Natural Science Foundation of China(No.81201407)the Bureau of Science&Technology and Intellectual Property Nanchong City(Nos.NSMC20170203 and NSMC20170310)+1 种基金the Health and Family Planning Commission of Sichuan Province(No.17JP496)the Research Projects of North Sichuan Medical College(No.CBY13-A-ZD09),China
文摘Objective:To construct a novel non-viral vector loaded with growth and differentiation factor-5(GDF-5) plasmid using chitosan,hyaluronic acid,and chondroitin sulfate for osteoarthritis (OA)gene therapy.Methods: Nano-microspheres (NMPs)were prepared by mixing chitosan,hyaluronic acid,and chondreitin sulfate.GDF-5 plasmid was encapsulated in the NMPs through electrostatic adsorption.The basic characteristics of the NMPs were observed,and then they were co-cultured with chondrocytes to observe their effects on extracellular matrix (ECM) protein expression.Finally,NMPs loaded with GDF-5were inje.cted into the articular cavities of rabbits to observe their therapeutic effects on OA in vivo.Results:NMPs exhibited good physicochemical properties and low cytotoxicity.Their average diameter was (0.61±0.20)μm,and encapsulation efficiency was (38.19±0.36)%.According to Cell Counting Kit-8(CCK-8)assay,relative cell viability was 75%-99%when the total weight of NMPs was less than 560μg. Transfection efficiency was (62.0±2.1)% in a liposome group,and (60.0±1.8)% in the NMP group.There was no sig- nificant difference between the two groups (P>0.05).Immunohistochemical staining results suggested that NMPs can successfully transfect chondrocytes and stimulate ECM protein expression in vitro.Compared with the control groups, the NMP group significantly promoted the expression of chondrocyte ECM in vivo (P<0.05),as shown by analysis of the biochemical composition of chondrocyte ECM.When NMPs were injected into OA model rabbits,the expression of ECM proteins in chondrocytes was significantly promoted and the progression of OA was slowed down.Conclusions: Based on these data,we think that these NMPs with excellent physicochemical and biological properties could be promising non-viral vectors for OA gene therapy.
