摘要
Although cartilage tissue engineering has been developed for decades, it is still unclear whether angio- genesis was the accompaniment of chondrogenesis in cartilage regeneration. This study aimed to explore the process of anti-angiogenesis during cartilage regenerative progress in cartilage repair extracellular matrix （ECM） materials under Hypoxia. C3H10T1/2 cell line, seeded as pellet or in ECM materials, was added with chondrogenic medium or DMEM medium for 21 days under hypoxia or normoxia environment. Genes and miRNAs related with chondrogenesis and angiogenesis were detected by RT-qPCR technique on Days 7, 14, and 21. Dual-luciferase report system was used to explore the regulating roles of miRNAs on angiogenesis. Results showed that the chondrogenic medium promotes chondrogenesis both in pellet and ECM materials culture. HIF1α was up-regulated under hypoxia compared with normoxia （P 〈 0.05）. Meanwhile, hypoxia enhanced chondrogenesis, miR-140-Sp exhibited higher expression while miR-146b exhibited lower expression. The chondrogenic phenotype was more stabilized in the ECM materials in chondrogenic medium than DMEM medium, with lower VEGFα expression even under hypoxia. Dual-luciferase report assays demonstrated that miR-140-5p directly targets VEGFct by binding its 3＇- UTR. Taken together, chondrogenic cytokines, ECM materials and hypoxia synergistically promoted chondrogenesis and inhibited angiogenesis, miR-140-5p olaved an imnortant role in this process.
Although cartilage tissue engineering has been developed for decades, it is still unclear whether angio- genesis was the accompaniment of chondrogenesis in cartilage regeneration. This study aimed to explore the process of anti-angiogenesis during cartilage regenerative progress in cartilage repair extracellular matrix （ECM） materials under Hypoxia. C3H10T1/2 cell line, seeded as pellet or in ECM materials, was added with chondrogenic medium or DMEM medium for 21 days under hypoxia or normoxia environment. Genes and miRNAs related with chondrogenesis and angiogenesis were detected by RT-qPCR technique on Days 7, 14, and 21. Dual-luciferase report system was used to explore the regulating roles of miRNAs on angiogenesis. Results showed that the chondrogenic medium promotes chondrogenesis both in pellet and ECM materials culture. HIF1α was up-regulated under hypoxia compared with normoxia （P 〈 0.05）. Meanwhile, hypoxia enhanced chondrogenesis, miR-140-Sp exhibited higher expression while miR-146b exhibited lower expression. The chondrogenic phenotype was more stabilized in the ECM materials in chondrogenic medium than DMEM medium, with lower VEGFα expression even under hypoxia. Dual-luciferase report assays demonstrated that miR-140-5p directly targets VEGFct by binding its 3＇- UTR. Taken together, chondrogenic cytokines, ECM materials and hypoxia synergistically promoted chondrogenesis and inhibited angiogenesis, miR-140-5p olaved an imnortant role in this process.
基金
supported by the National Basic Research Program of China(973 Program,No.2012CB619100)
the National Natural Science Foundation of China(Nos.31430030,0731001,and 81071512)
the Natural Science Foundation of Guangdong Province(No.2014A030310466)
the China Scholarship Council
