Effects of tumor necrosis factor-α on osteogenic differentiation and Notch signaling pathway in human periodontal ligament stem cells

doi:10.7518/hxkq.2018.02.013

Abstract

Abstract:

Objective To evaluate the effects of tumor necrosis factor-α (TNF-α) on osteogenic differentiation and Notch signaling pathway of periodontal ligament stem cells (PDLSCs) and to investigate the regulatory role of Notch signaling path-way on the osteogenic differentiation of PDLSCs under the influence of TNF-α. Methods PDLSCs were obtained through enzyme digestion and tissue block method. The expression levels of stem cell surface markers CD105, CD90, CD146, CD45, and CD31 were detected by fluorescence activated cell sorter (FACS). PDLSCs were divided into experimental (10 ng·mL^-1TNF-α) and control groups (0 ng·mL^-1TNF-α). The proliferation ability of PDLSCs was detected using cell counting kit-8 (CCK-8). The effect of TNF-α on the osteogenic ability of PDLSCs were tested by measuring alkaline phosphatase (ALP) activity and conducting alizarin red staining and quantitative real-time polymerase chain reaction (PCR). We tested Notch signal pathway receptors Notch1, Notch2, ligand JAG1, JGA2, and downstream gene Hes-1. Changes in DLL1 expression were detected by quantitative real-time PCR. ResultsFACS profiling showed that PDLSCs were strongly positive for CD105, CD90, and CD146 but negative for CD45 and CD31. CCK-8 results showed that TNF-α could promote the proli-feration of PDLSCs (P<0.05). ALP activity in the experi-mental group was lower than that in the control group (P<0.05). Alizarin red staining showed that the experimental group had decreased mineralized nodules as compared with the control group. Quantitative real-time PCR results showed that the mRNA expression of osteogenic marker genes cementum attachment protein (CAP), osteopontin (OPN), and Runt-related transcription factor 2 (Runx2) significantly decreased in the experimental group as compared with those in the control group (P<0.05). The expression levels of Notch1, Notch2, JAG1, JGA2 and Hes-1 were significantly decreased (P<0.05), whereas those of Notch3 and DLL1 were increased in Notch signaling pathway-related molecules (P<0.05). Conclusion TNF-α can promote PDLSCs proliferation and inhibit bone differentiation and Notch signaling pathway expression, indicating that the Notch signaling pathway regulates PDLSCs osteogenic differentiation.

Key words: periodontal ligament stem cells, Notch signal pathway, tumor necrosis factor-α, osteogenic differentiation

CLC Number:

Q257

Yu Ma, Shuhui Li, Xinxin Ding, Peiling. Wu. Effects of tumor necrosis factor-α on osteogenic differentiation and Notch signaling pathway in human periodontal ligament stem cells[J]. West China Journal of Stomatology, 2018, 36(2): 184-189.

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基因	引物序列（5’—3’）
CAP	Forward：5CTGGCTCACCTTCTACGACA
	Reverse：TACCTCAAGCAAGGCAAATG
OPN	Forward：CATACAAGGCCATCCCCGTT
	Reverse：GGGTTTCAGCACTCTGGTCA
Runx-2	Forward：ATCTCCGCAGGTCACTACCA
	Reverse：TGTGCTGAAGAGGCTGTTTG
Notch1	Forward：ACCAATACAACCCTCTGCGG
	Reverse：GGCCCTGGTAGCTCATCATC
Notch2	Forward：GGGACCCTGTCATACCCTCT
	Reverse：GAGCCATGCTTACGCTTTCG
Notch3	Forward：CAACCTGGCAGGGAGTTTCA
	Reverse：TCAGGCACTCATCCACATCG
JAG1	Forward：TCACGGGAAGTGCAAGAGTC
	Reverse：GTTTCACAGTAGGCCCCCTC
JGA2	Forward：GCATCAACATCGACGAGTGC
	Reverse：GATCTCCCGAACCCGATCAC
DLL1	Forward：GCAGCTCTTCACCCTGTTCT
	Reverse：GGTGCAGGAGAAGTCGTTCA
Hes-1	Forward：ATGACAGTGAAGCACCTCCG
	Reverse：CGTTCATGCACTCGCTGAAG
GAPDH	Forward：TCAAGGCTGAGAACGGGAAG
	Reverse：TGGACTCCACGACGTACTCA