J. Mater. Sci. Technol. ›› 2016, Vol. 32 ›› Issue (9): 978-985.DOI: 10.1016/j.jmst.2016.08.001
• Orginal Article • Previous Articles
Chen Shaoming1,2,Gao Manman2,3,a,Zhou Zhiyu2,3,a,Liang Jiabi1,Gong Ming2,3,Dai Xuejun2,3,Liang Tangzhao2,Ye Jiacheng2,Wu Gang4,5,Zou Lijin6,Wang Yingjun4,5,Zou Xuenong2,3,*()
Received:
2015-10-16
Accepted:
2015-11-30
Online:
2016-09-20
Published:
2016-11-02
Contact:
Zou Xuenong
Chen Shaoming,Gao Manman,Zhou Zhiyu,Liang Jiabi,Gong Ming,Dai Xuejun,Liang Tangzhao,Ye Jiacheng,Wu Gang,Zou Lijin,Wang Yingjun,Zou Xuenong. Opposite Regulation of Chondrogenesis and Angiogenesis in Cartilage Repair ECM Materials under Hypoxia[J]. J. Mater. Sci. Technol., 2016, 32(9): 978-985.
Gene symbol | Forward primer | Reversed primer |
---|---|---|
Rn18s | CCTGGATACCGCAGCTAGGA | GCGGCGCAATACGAATGAATGCCCC |
Acan | AAGAATCAAGTGGAGCCGTGT | CAGAGTCATTGGAGCGAAGGT |
Col2α1 | TGGTGGAGCAGCAAGAGCAAG | AGTGGACAGTAGACGGAGGAAAG |
Sox9 | TGAACGAGAGCGAGAAGAGAC | AGATAGCATTAGGAGAGATGTGAGT |
VEGFα | GGACCCTGGCTTTACTGCTGTACC | TCACCGCCTTGGCTTGTCACA |
HIF1α | CCACAGGACAGTACAGGATG | TCAAGTCGTGCTGAATAATACC |
MMP13 | CCCTTCCCTATGGTGATGATGATG | AGAGTCGCAGGATGGTAGTATGAT |
ADAMTS5 | GCTAAGGGCACAGGCTACTATG | CTCCGCACACTCCACACTTG |
mmu-miR-146b | AACCGCCTGAGAACTGAAT | - |
mmu-miR-140-5p | CAGTGGTTTTACCCTATGGTAG | - |
U6 | CTCGCTTCGGCAGCACA | AACGCTTCACGAATTTGCGT |
Table 1 Primer sequences of genes and miRNAs
Gene symbol | Forward primer | Reversed primer |
---|---|---|
Rn18s | CCTGGATACCGCAGCTAGGA | GCGGCGCAATACGAATGAATGCCCC |
Acan | AAGAATCAAGTGGAGCCGTGT | CAGAGTCATTGGAGCGAAGGT |
Col2α1 | TGGTGGAGCAGCAAGAGCAAG | AGTGGACAGTAGACGGAGGAAAG |
Sox9 | TGAACGAGAGCGAGAAGAGAC | AGATAGCATTAGGAGAGATGTGAGT |
VEGFα | GGACCCTGGCTTTACTGCTGTACC | TCACCGCCTTGGCTTGTCACA |
HIF1α | CCACAGGACAGTACAGGATG | TCAAGTCGTGCTGAATAATACC |
MMP13 | CCCTTCCCTATGGTGATGATGATG | AGAGTCGCAGGATGGTAGTATGAT |
ADAMTS5 | GCTAAGGGCACAGGCTACTATG | CTCCGCACACTCCACACTTG |
mmu-miR-146b | AACCGCCTGAGAACTGAAT | - |
mmu-miR-140-5p | CAGTGGTTTTACCCTATGGTAG | - |
U6 | CTCGCTTCGGCAGCACA | AACGCTTCACGAATTTGCGT |
Fig. 1. Relative expression of genes related with chondrogenesis of C3H10T1/2 cell line in pellet culture, showing that Sox9, COL2α1 and Acan are up-regulated, while MMP13 and ADAMTS5 are down-regulated. *P?<?0.05.
Fig. 2. Relative expression of genes related with chondrogenesis and angiogenesis of C3H10T1/2 cells in pellet culture under hypoxic (2% O2) environment. HIF1α was up-regulated, while VEGFα showed down-expression. *P?<?0.05.
Fig. 3. Relative expression of related miRNAs. miR-140-5p is up-regulated while miR-146b is down-regulated during pellet culture of C3H10T1/2 cell line under hypoxic (2% O2) environment. *P?<?0.05.
Fig. 4. 2?×?106 C3H10T1/2 cells seeded in the ECM materials (A); Hoechst 33258 fluorescent staining validated the good cytocompatibility with no dead cells (B); Immunohistochemical staining showed less expression of type II Collagen on day 7 (C) than day 21 (D).
Fig. 5. Relative expression of genes and miRNAs related with chondrogenesis of C3H10T1/2 cells in ECM materials culture. ECM materials coordinated with chondrogenic medium promoted chondrogenesis. *P?<?0.05.
Fig. 6. Relative expression of genes and miRNAs related with chondrogenesis and angiogenesis in ECM materials under hypoxic (2% O2) environment. Genes regulating chondrogenesis exhibited up expression while those regulating angiogenesis exhibited down expression. At the same time, miR-140-5p was up-regulated and miR-146b was down-regulated. *P?<?0.05.
Fig. 7. Dual-luciferase reporter assays of miR-140-5p regulating VEGFα. miR-140-5p down regulated VEGFα expression by directly targeting the 3′-UTR sequence. *P?<?0.05.
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