肿瘤坏死因子-α对牙周膜干细胞骨向分化及Notch信号通路的影响

doi:10.7518/hxkq.2018.02.013

摘要/Abstract

摘要：

目的研究肿瘤坏死因子-α（TNF-α）对牙周膜干细胞（PDLSCs）骨向分化及Notch信号通路的影响,初步探讨在TNF-α影响下Notch信号通路对PDLSCs成骨分化的调控作用。方法采用酶消化结合组织块法获得PDLSCs,通过流式细胞仪检测间充质干细胞（MSCs）表面标记物CD105、CD90、CD146、CD45、CD31的表达,将PDLSCs分为实验组（10 ng·mL^-1TNF-α）和对照组（不含TNF-α）,使用CCK-8法检测PDLSCs增殖能力;通过碱性磷酸酶（ALP）活性、茜素红染色、实时定量聚合酶链反应（PCR）检测TNF-α对PDLSCs成骨能力的影响;实时定量PCR 检测Notch信号通路受体Notch1、Notch2、Notch3,配体JAG1、JGA2、DLL1,细胞内效应分子Hes-1的表达。结果流式细胞仪检测结果显示CD105、CD90及CD146表达阳性,CD45、CD31表达阴性;CCK-8法结果显示TNF-α能够促进PDLSCs增殖能力（P<0.05）;ALP活性检测结果显示,实验组与对照组相比,ALP活性降低（P<0.05）;茜素红染色结果显示,与对照组相比,实验组矿化结节减少;实时定量PCR结果显示,与对照组相比,成骨标志基因牙骨质附着蛋白（CAP）、骨桥蛋白（OPN）、Runt相关转录因子2（Runx2）表达量明显降低（P<0.05）;Notch信号通路相关分子中Notch1、Notch2、JAG1、JGA2、Hes-1的表达水平显著降低（P<0.05）,而Notch3、DLL1的表达水平增高,差异有统计学意义（P<0.05）。结论 TNF-α能够促进PDLSCs增殖而抑制其骨向分化,同时抑制Notch信号通路的表达,说明Notch信号通路对PDLSCs骨向分化具有一定的调控作用。

关键词: 牙周膜干细胞, Notch信号通路, 肿瘤坏死因子-α, 成骨分化

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

中图分类号:

Q257

马玉, 李淑慧, 丁欣欣, 吴佩玲. 肿瘤坏死因子-α对牙周膜干细胞骨向分化及Notch信号通路的影响[J]. 华西口腔医学杂志, 2018, 36(2): 184-189.

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