目的研究生长分化因子-5(GDF-5)在大鼠肢体早期发育过程中的表达规律,探讨GDF-5调控肢体发育的相关机制。方法采用半定量RT-PCR和W estern b lotting分别检测GDF-5在大鼠胚胎早期发育过程中肢芽组织mRNA和蛋白表达的变化。结果大鼠胚胎...目的研究生长分化因子-5(GDF-5)在大鼠肢体早期发育过程中的表达规律,探讨GDF-5调控肢体发育的相关机制。方法采用半定量RT-PCR和W estern b lotting分别检测GDF-5在大鼠胚胎早期发育过程中肢芽组织mRNA和蛋白表达的变化。结果大鼠胚胎肢体发育的早期阶段GDF-5 mRNA呈持续表达,其中在胚胎13、14d(E 13、E 14)呈高表达,继而表达减弱,GDF-5蛋白表达规律与mRNA变化趋势一致。结论GDF-5在大鼠胚胎早期肢体发育中对调控软骨发育和关节形成有很重要的作用。展开更多
背景:移植间充质干细胞预防和治疗椎间盘退变是一种可行的方法,将SOX-9和GDF-5共同转染骨髓间充质干细胞,使其向髓核细胞转化,以期获得更大的髓核诱导和促增殖效应。目的:探讨SOX-9和GDF-5基因共同诱导兔骨髓间充质干细胞向类髓核细胞...背景:移植间充质干细胞预防和治疗椎间盘退变是一种可行的方法,将SOX-9和GDF-5共同转染骨髓间充质干细胞,使其向髓核细胞转化,以期获得更大的髓核诱导和促增殖效应。目的:探讨SOX-9和GDF-5基因共同诱导兔骨髓间充质干细胞向类髓核细胞分化的效果。方法:提取、分离、纯化4周龄新西兰大白兔骨髓间充质干细胞,取第3代骨髓间充质干细胞分为5组体外诱导其向类髓核细胞分化,分别为未转染组、空载体转染组、SOX-9转染组、GDF-5转染组、共转染组。转染后第14天采用RT-PCR检测SOX-9,GDF-5和Ⅱ型胶原的m RNA表达,免疫组化染色法检测髓核细胞标记物KRT19表达。结果与结论:共转染组SOX-9 m RNA表达高于转染SOX-9组,差异有显著性意义(P<0.05);共转染组GDF-5m RNA表达高于转染GDF-5组,差异有显著性意义(P<0.05)。共转染组Ⅱ型胶原表达高于转染SOX-9组、转染GDF-5组,差异有显著性意义(P<0.05)。SOX-9转染组及GDF-5转染组KRT19呈阳性表达,共转染组呈强阳性表达,可见被转染的骨髓间充质干细胞向类髓核细胞分化,且双基因转染诱导骨髓间充质干细胞向类髓核细胞分化的能力和分泌细胞外基质的能力明显高于单基因转染。展开更多
Objective： To investigate the expression of growth differentiation factor 5（GDF-5） during limb skeletal development of mice and the effect of GDF-5 on bone marrow mesenchymal stem cells in vitro. Methods ： The exp...Objective： To investigate the expression of growth differentiation factor 5（GDF-5） during limb skeletal development of mice and the effect of GDF-5 on bone marrow mesenchymal stem cells in vitro. Methods ： The expression of GDF- 5 mRNA and protein in mouse fetal limb buds were detected in embryonic day 11.5-15.5 （E11.5-15.5） by RT-PCR and Western blotting respectively. Type Ⅱ collagen protein was examined with immunocytochemistry and the sulfate glycosaminoglycan was measured by Alcian blue. Results： During early stage of developmental skeletogenesis, the expression of GDF-5mRNA was constant and began with embryos E11.5, highlighted at embryos E12.5 and E13.5, subsequently dropped at embryos E14.5 and E15.5. There was very significant difference （P 〈 0.01） in average light density ratio of GDF-5/β-actin between E12.5-13.5 and the other three days. The expression of GDF-5 protein had a similar change with mRNA during limb skeletogenesis. Immunocytochemistry showed that GDF-5 could promote expression of Type Ⅱ collagen protein and histological staining of proteoglycan with Alcian blue revealed the deposition of typical cartilage extracellular matrix components. Conclusion： GDF-5 can enhance chondrogenic differentiation of mouse bone marrow mesenchymal stem cells in vitro, which plays an important role in limb skeletal development and joint formation.展开更多
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
文摘背景:移植间充质干细胞预防和治疗椎间盘退变是一种可行的方法,将SOX-9和GDF-5共同转染骨髓间充质干细胞,使其向髓核细胞转化,以期获得更大的髓核诱导和促增殖效应。目的:探讨SOX-9和GDF-5基因共同诱导兔骨髓间充质干细胞向类髓核细胞分化的效果。方法:提取、分离、纯化4周龄新西兰大白兔骨髓间充质干细胞,取第3代骨髓间充质干细胞分为5组体外诱导其向类髓核细胞分化,分别为未转染组、空载体转染组、SOX-9转染组、GDF-5转染组、共转染组。转染后第14天采用RT-PCR检测SOX-9,GDF-5和Ⅱ型胶原的m RNA表达,免疫组化染色法检测髓核细胞标记物KRT19表达。结果与结论:共转染组SOX-9 m RNA表达高于转染SOX-9组,差异有显著性意义(P<0.05);共转染组GDF-5m RNA表达高于转染GDF-5组,差异有显著性意义(P<0.05)。共转染组Ⅱ型胶原表达高于转染SOX-9组、转染GDF-5组,差异有显著性意义(P<0.05)。SOX-9转染组及GDF-5转染组KRT19呈阳性表达,共转染组呈强阳性表达,可见被转染的骨髓间充质干细胞向类髓核细胞分化,且双基因转染诱导骨髓间充质干细胞向类髓核细胞分化的能力和分泌细胞外基质的能力明显高于单基因转染。
基金National Natural Science Foundation of China(No. 30471753)
文摘Objective： To investigate the expression of growth differentiation factor 5（GDF-5） during limb skeletal development of mice and the effect of GDF-5 on bone marrow mesenchymal stem cells in vitro. Methods ： The expression of GDF- 5 mRNA and protein in mouse fetal limb buds were detected in embryonic day 11.5-15.5 （E11.5-15.5） by RT-PCR and Western blotting respectively. Type Ⅱ collagen protein was examined with immunocytochemistry and the sulfate glycosaminoglycan was measured by Alcian blue. Results： During early stage of developmental skeletogenesis, the expression of GDF-5mRNA was constant and began with embryos E11.5, highlighted at embryos E12.5 and E13.5, subsequently dropped at embryos E14.5 and E15.5. There was very significant difference （P 〈 0.01） in average light density ratio of GDF-5/β-actin between E12.5-13.5 and the other three days. The expression of GDF-5 protein had a similar change with mRNA during limb skeletogenesis. Immunocytochemistry showed that GDF-5 could promote expression of Type Ⅱ collagen protein and histological staining of proteoglycan with Alcian blue revealed the deposition of typical cartilage extracellular matrix components. Conclusion： GDF-5 can enhance chondrogenic differentiation of mouse bone marrow mesenchymal stem cells in vitro, which plays an important role in limb skeletal development and joint formation.
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.
