Journal of Prevention and Treatment for Stomatological Diseases ›› 2021, Vol. 29 ›› Issue (7): 449-455.doi: 10.12016/j.issn.2096-1456.2021.07.003

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Effects of hypoxia inducible factor-1α on osteogenic differentiation and angiogenesis related factors of bone marrow mesenchymal stem cells

ZUO Xinhui1(),LI Jun1,HAN Xiangzhen1,LIU Xiaoyuan1,HE Huiyu1,2()   

  1. 1. Department of Prosthodontics, the First Affiliated Hospital of Xinjiang Medical University (Affiliated Stomatological Hospital), Urumqi 830054, China
    2. Xinjiang Uygur Autonomous Region Institute of Stomatology, Urumqi 830011, China
  • Received:2020-08-18 Revised:2021-01-05 Online:2021-07-20 Published:2021-04-19
  • Contact: Huiyu HE E-mail:13838371011@163.com;hehuiyu01@126.com
  • Supported by:
    Xinjiang Uygur Autonomous Region Science and Technology Support Project(2018E02060);Postgraduate Innovation and Entrepreneurship Project(XJ2020G216)

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Abstract:

Objective To investigate the level of hypoxia inducible factor-1α (HIF-1α) on osteoblasts and angiogenesis-associated cytokines in bone marrow mesenchymal stem cells (BMSCs) from SD rats.Methods BMSCs were isolated and cultured and identified by flow cytometry. Plasmid vectors containing upregulated and downregulated HIF-1α gene and a control vector were constructed. The plasmids were transfected into BMSCs by Lipofectamine?LTX transfection reagent, and the cells were divided into an overexpression experimental group, an overexpression control group, a low expression experimental group and a low expression control group. All components were stained with a lizarin red 3 d and 7 d after osteogenesis induction. The mRNA expression levels of the target gene HIF-1α, osteogenic differentiation-specific markers, including Runt-related transcription factor 2 (Runx2) and angiogenic markers, including platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β (TGF-β), were detected by RT-PCR. Western blot was used to detect the protein expression of the target proteins HIF-1α, Runx2, and PDGF-BB.Results The CD29- and CD45-positivity rates of BMSC surface markers identified by flow cytometry were 98.2% and 4.2%, respectively. RT-PCR results showed that the mRNA expression of HIF-1α, Runx2, TGF-β and PDGF-BB was observably increased (P < 0.001). The mRNA expression levels of HIF-1α, Runx2, TGF-β and PDGF-BB in BMSCs from the low expression experimental group were significantly reduced (P < 0.001). Western blot results showed that the expression levels of HIF-1α, Runx2 and PDGF-BB in BMSCs from the overexpression experimental group were all increased (P < 0.001). The expression levels of HIF-1α, Runx2 and PDGF-BB in BMSCs from the low expression experimental group were reduced (P < 0.001). Alizarin red staining results showed that the area of calcium nodules in the low expression experimental group was smaller than that in low expression control group, the area of red calcium nodules in the over expression experimental group was larger than that in over expression control group, and with the increase of osteogenic induction time, the calcification area of each group also increased.Conclusion Upregulation and downregulation of HIF-1α can regulate the osteogenic differentiation and the expression of angiogenesis related factors of BMSCs.

Key words: hypoxia inducible factor-1α, bone marrow mesenchymal stem cells, osteogenic differentiation, angiogenesis, Runt-related transcription factor 2, platelet derived growth factor-BB, transforming growth factor-β, phosphatidylinositol 3-kinase, protein kinase B, bone tissue engineering

CLC Number: 

  • R78

Table 1

Synthetic primers used for RT-PCR"

Primers Sequence
GAPDH F:5’-GGCACAGTCAAGGCTGAGAATG-3’
R:5’-ATGGTGGTGAAGACGCCAGTA-3’
HIF-1α F:5’-CACCGCAACTGCCACCACTG-3’
R:5’-TGAGGCTGTCCGACTGTGAGTAC-3’
Runx2 F:5’-CCATAACGGTCTTCACAAATCCT-3’
R:5’-TCTGTCTGTGCCTTCTTGGTTC-3’
PDGF-BB F:5’-TCTCTGCTGCTACCTGCGTCTG-3’
R:5’-AAGGAGCGGATGGAGTGGTCAC-3’
TGF-β F:5’-GGCACCATCCATGACATGAACCG-3’
R:5’-GCCGTACACAGCAGTTCTTCTCTG-3’

Figure 1

Cell culture of BMSCs（× 50） a: the primary cells were round; b: P3 cells, the cells adhered to the wall and grew in long fusiform or whirlpool shape;BMSCs： bone marrow mesenchymal stem cells"

Figure 2

BMSC transfected with plasmid after 48 h （× 100） BMSCs that were successfully transfected with DNA plasmid showed green fluorescence expression, and the cell growth density was approximately 75%. The expression level of the low expression experimetal group was approximately 76%, and that of the control group was approximately 70%. The fluorescence level of the overexpression experimetal group was 73%, and that of the control group was approximately 68%. BMSCs： bone marrow mesenchymal stem cells"

Figure 3

BMSC alizarin red staining results for each group（×50） The area of calcium nodules in the low expression group was smaller than that in the control group and smaller than the area of nodules in the overexpression experimental group. The area of red calcium nodules in the overexpression experimental group was significantly larger than that in the control group, and the calcification area in each group increased significantly with increasing osteogenic induction time. BMSCs： bone marrow mesenchymal stem cells"

Table 2

mRNA expression of osteogenic differentiation and angiogenesis related factors $\bar{x}\pm s$"

Gene names Low expression
experimental group		 Low expression
control group		 t P Over expression
experimental group		 Over expression
control group		 t P
HIF-1α 0.373 ± 0.020 1.023 ± 0.231 22.522 <0.001 1.563 ± 0.128 1.013 ± 0.171 0.896 <0.001
Runx2 0.618 ± 0.012 1.025 ± 0.229 32.776 <0.001 25.462 ± 0.406 1.023 ± 0.204 6.278 <0.001
PDGF-BB 0.097 ± 0.011 1.009 ± 0.155 16.531 <0.001 3.322 ± 0.111 1.009 ± 0.147 1.705 <0.001
TGF-β 0.447 ± 0.013 1.023 ± 0.222 9.203 <0.001 1.734 ± 0.016 1.006 ± 0.112 17.389 <0.001

Figure 4

Protein expression of osteogenic differentiation and angiogenesis related factors 1: low expression experimental group; 2: low expression control group; 3: over expression experimental group; 4: over expression control group; HIF-1α: hypoxia inducible factor-1α; Runx2: runt-related transcription factor 2; PDGF-BB: platelet derived growth factor-BB; TGF-β: transforming growth factor-β; ***P<0.001"

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