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中华口腔医学研究杂志(电子版) ›› 2020, Vol. 14 ›› Issue (04) : 214 -220. doi: 10.3877/cma.j.issn.1674-1366.2020.04.003

所属专题: 文献

基础研究

低氧环境下Notch信号通路对人牙髓干细胞成牙本质向分化的影响
关丽娜1, 杨帆2, 尹东锋1, 杨自更1, 魏敦宏1, 雒可夫1, 王瑞1,()   
  1. 1. 新疆军区总医院,乌鲁木齐 830000
    2. 军事口腔医学国家重点实验室,口腔疾病国家临床医学研究中心,陕西省口腔医学重点实验室,空军军医大学口腔医院,西安 710032
  • 收稿日期:2020-03-26 出版日期:2020-08-01
  • 通信作者: 王瑞

Effects of Notch signaling pathway on the dentin differentiation of human dental pulp stem cells in hypoxic environment

Lina Guan1, Fan Yang2, Dongfeng Yin1, Zigeng Yang1, Dunhong Wei1, Kefu Luo1, Rui Wang1,()   

  1. 1. General Hospital of Xinjiang Military Command, PLA, Urumqi 830000, China
    2. State Key Laboratory of Military Stomatology and National Clinical Research Center for Oral Diseases and Shaanxi Key Laboratory of Stomatology, Department of Rehabilitation Medicine, School of Stomatology, Air Force Medical University, PLA, Xi′an 710032, China
  • Received:2020-03-26 Published:2020-08-01
  • Corresponding author: Rui Wang
  • About author:
    Corresponding author: Wang Rui, Email:
  • Supported by:
    Natural Science Foundation of Xinjiang Uygur Autonomous Region(2016D01C391)
引用本文:

关丽娜, 杨帆, 尹东锋, 杨自更, 魏敦宏, 雒可夫, 王瑞. 低氧环境下Notch信号通路对人牙髓干细胞成牙本质向分化的影响[J/OL]. 中华口腔医学研究杂志(电子版), 2020, 14(04): 214-220.

Lina Guan, Fan Yang, Dongfeng Yin, Zigeng Yang, Dunhong Wei, Kefu Luo, Rui Wang. Effects of Notch signaling pathway on the dentin differentiation of human dental pulp stem cells in hypoxic environment[J/OL]. Chinese Journal of Stomatological Research(Electronic Edition), 2020, 14(04): 214-220.

目的

探讨低氧环境下通过低氧诱导因子-1α(HIF-1α)调控Notch信号通路对人牙髓干细胞(HDPSC)成牙本质向分化能力的影响。

方法

组织块酶消化法培养HDPSC,给予氯化钴(CoCl2)诱导的化学性低氧环境,Western blot检测HIF-1α蛋白表达,茜素红染色检测细胞矿化结节形成能力,反转录聚合酶链反应(RT-PCR)检测Notch下游靶基因Hes1与成牙本质相关基因的表达;进一步加入Notch信号通路特异性阻断剂γ分泌酶抑制剂(GSI),观察以上指标的变化。报告基因方法检测HIF-1α对Notch信号通路的调控作用。采用R version 3.5.3软件进行统计分析,计量资料进行正态检验,多组间比较采用单因素方差分析(方差齐)或Kruskal-Wallis H检验(方差不齐),两两比较采用LSD-t检验(方差齐)或Mann-Whitney U检验(方差不齐)。

结果

(1)CoCl2诱导的低氧条件下HDPSC中的HIF-1α蛋白水平上调,Notch下游靶基因Hes1 mRNA相对表达量为1.46 ± 0.12,相比常氧组(1.06 ± 0.09)显著增高(t = -4.64,P = 0.012);GSI处理阻断Notch信号通路后,低氧条件下HDPSC中HIF-1α蛋白水平下调,Hes1 mRNA相对表达量降低至0.82 ± 0.14,与处理前相比差异有统计学意义(t = 5.98,P = 0.004);常氧条件下GSI处理后的HDPSC中HIF-1α蛋白水平也明显下调,Hes1 mRNA相对表达量(0.30 ± 0.09)相比处理前也显著降低(t = 10.08,P = 0.001);(2)矿化诱导后的茜素红染色结果显示:低氧处理后,HDPSC细胞成牙本质分化能力下降;GSI处理后,成牙本质向分化能力增强。成骨/牙本质相关基因mRNA表达水平检测结果显示:低氧处理后,BSPOCNDSPP的mRNA相对表达量分别为0.53 ± 0.14、0.43 ± 0.20、0.48 ± 0.11,相比常氧组(1.21 ± 0.12、1.08 ± 0.19、1.03 ± 0.13)显著降低(tBSP = 6.30,PBSP = 0.003;tOCN = 4.07,POCN = 0.015;tDSPP = 5.67,PDSPP = 0.005);GSI处理后,低氧条件下BSPOCNDSPP mRNA相对表达量分别为0.99 ± 0.13、1.09 ± 0.13、0.95 ± 0.16,与处理前相比显著增高(tBSP = -4.17,PBSP = 0.014;tOCN = -4.83,POCN = 0.012;tDSPP = -4.30,PDSPP = 0.017),常氧条件下BSPOCNDSPP mRNA相对表达量分别为1.73 ± 0.20、1.55 ± 0.08、1.52 ± 0.14,与处理前相比显著增高(tBSP = -3.84,PBSP = 0.027;tOCN = -3.99,POCN = 0.035;tDSPP = -4.43,PDSPP = 0.011)。(3)荧光素酶报告基因结果显示,HIF-1α可调控NICD启动子,使双荧光素酶相对表达活性为5.37 ± 0.12,与对照组(2.09 ± 0.15)相比显著增强(t = -28.92,P<0.001),提示HIF-1α可能使Notch信号通路激活。

结论

低氧引起HDPSC中HIF-1α升高并可能通过激活Notch信号通路抑制其成牙本质向分化能力。

Objective

To investigate whether and how hypoxia affects the differentiation capacity of human dental pulp stem cells (HDPSCs) via Notch signaling.

Methods

Primary cultured HDPSCs were stimulated by CoCl2, which could induce a chemical hypoxic environment. HIF-1α protein level was detected by Western blot. The mRNA expression levels of Notch and dentin related genes were detected by RT-PCR. Alizarin red staining was used to detect the ability of HDPSCs to form mineralized nodules. Notch signaling pathway specific blocker GSI was further added to observe the changes of the indicators above. Reporter gene method was then used to detect the regulation of HIF-1α gene on Notch signaling pathway. R software (version 3.5.3) was used for statistical analysis. All measurement data were done by normality test. One-Way ANOVA (homogeneity of variance) or Kruskal-Wallis H test (heterogeneity of variance) was used for the multi-group comparison, and LSD-t test (homogeneity of variance) or Mann-Whitney U test (heterogeneity of variance) was used for the pair-wise comparison.

Results

(1) HIF-1α protein level was up-regulated by the chemical hypoxic environment induced by CoCl2, and then reduced when GSI was added. The mRNA expression level of Notch target gene Hes1 was also significantly increased from 1.06 ± 0.09 to 1.46 ± 0.12 (t = -4.64, P = 0.012) under the hypoxic condition, and then significantly decreased to 0.82 ± 0.14 when GSI was added (t = 5.98, P = 0.004) . Moreover, the same resultsthat the mRNA expression level of Hes1 was significantly decreased from 1.06 ± 0.09 to 0.30 ± 0.09 when GSI was added (t = 10.08, P = 0.001) , were observed under a normoxic condition. (2) The results of alizarin red staining showed that the dentin differentiative potential of HDPSCs was decreased under the hypoxic environment, and then enhanced when GSI was added. The mRNA expression levels of osteogenic/dentin related genes BSP, OCN and DSPP were 1.21 ± 0.12, 1.08 ± 0.19 and 1.03 ± 0.13 under the normoxic environment, and 0.53 ± 0.14, 0.43 ± 0.20 and 0.48 ± 0.11 under the hypoxic environment, where there was a statistically significant difference between the two conditions (tBSP = 6.30, PBSP = 0.003; tOCN = 4.07, POCN = 0.015; tDSPP = 5.67, PDSPP = 0.005) . When GSI was added, the mRNA expression levels of BSP, OCN and DSPP under the hypoxic environment were then significantly increased to 0.99 ± 0.13, 1.09 ± 0.13 and 1.09 ± 0.13 (tBSP = -4.17, PBSP = 0.014; tOCN = -4.83, POCN = 0.012; tDSPP = -4.30, PDSPP = 0.017) . Moreover, the same results that the mRNA expression level of BSP, OCN and DSPP were significantly increased to 1.73 ± 0.20, 1.55 ± 0.08 and 1.52 ± 0.14 respectively after GSI was added (tBSP = -3.84, PBSP = 0.027; tOCN = -3.99, POCN = 0.035; tDSPP = -4.43, PDSPP = 0.011) , were observed under the normoxic condition. (3) The results of luciferase reporter gene experiment showed that the activity of Notch signaling pathway was significantly increased from 2.09 ± 0.15 to 5.37 ± 0.12 after the addition of HIF-1α (t = -28.92, P<0.001) .

Conclusion

Hypoxia caused an increase of HIF-1α in HDPSCs and HIF-1α may inhibit the dentin differentiative potential of HDPSCs by activating the Notch signaling pathway.

表1 人牙髓干细胞(HDPSC)的分组方法与给药处理
表2 实时荧光定量聚合酶链反应(PCR)引物序列
图1 低氧和Notch信号通路对HIF-1α蛋白表达的影响 GSI为γ分泌酶抑制剂
图2 低氧和Notch信号通路对Hes1基因表达的影响 GSI为γ分泌酶抑制剂;组间相比,差异有统计学意义(aP<0.05)
图3 人牙髓干细胞(HDPSC)成牙本质向分化能力检测结果(茜素红染色) GSI为γ分泌酶抑制剂
图4 低氧和Notch信号通路对牙本质分化相关基因表达的影响 A:BSP;B:OCN;C:DSPP;GSI为γ分泌酶抑制剂;组间相比,差异有统计学意义(aP<0.05)
表3 低氧与Notch信号通路对人牙髓干细胞(HDPSC)中成牙本质相关基因mRNA表达水平的影响( ± s
图5 荧光素酶活性检测HIF-1α对Notch活性的调控作用 GSI为γ分泌酶抑制剂;组间相比,差异有统计学意义(aP<0.05)
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