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
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