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中华口腔医学研究杂志(电子版) ›› 2014, Vol. 8 ›› Issue (06) : 464 -470. doi: 10.3877/cma.j.issn.1674-1366.2014.06.005

基础研究

组蛋白去甲基化酶KDM5A 在人牙髓细胞及成牙本质细胞分化中的表达
李金铃1, 廖章松1, 饶利佳1, 徐琼1,()   
  1. 1.510055 广州,中山大学光华口腔医学院·附属口腔医院,广东省口腔医学重点实验室
  • 收稿日期:2014-10-16 出版日期:2014-12-01
  • 通信作者: 徐琼

Expression of histone demethylase KDM5A in human dental pulp cells during odontogenic differentiation process

Jinling Li1, Zhangsong Liao1, Lijia Rao1, Qiong Xu1,()   

  1. 1.Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China
  • Received:2014-10-16 Published:2014-12-01
  • Corresponding author: Qiong Xu
引用本文:

李金铃, 廖章松, 饶利佳, 徐琼. 组蛋白去甲基化酶KDM5A 在人牙髓细胞及成牙本质细胞分化中的表达[J/OL]. 中华口腔医学研究杂志(电子版), 2014, 8(06): 464-470.

Jinling Li, Zhangsong Liao, Lijia Rao, Qiong Xu. Expression of histone demethylase KDM5A in human dental pulp cells during odontogenic differentiation process[J/OL]. Chinese Journal of Stomatological Research(Electronic Edition), 2014, 8(06): 464-470.

目的

探讨组蛋白赖氨酸去甲基化酶KDM5A 在人牙髓细胞(hDPC)中的表达模式及成牙本质分化诱导对其表达量的影响。

方法

体外培养hDPC,实时荧光定量聚合酶链反应和Western blot 检测第1 代至第8 代(P1 ~P8 代)hDPC 中KDM5A mRNA 和蛋白的表达量;免疫荧光检测KDM5A在hDPC 中的分布;对P3 代细胞进行成牙本质分化诱导,于第7 天和第14 天分别检测KDM5A mRNA 和蛋白的表达水平。

结果

体外传代培养hDPC 中可检测到KDM5A 的表达,KDM5A mRNA 和蛋白量均呈先增加后减少的趋势;hDPC 细胞质及细胞核中均表达KDM5A;成牙本质向分化诱导7 和14 d,KDM5A mRNA 和蛋白量高于未诱导组细胞,诱导14 d 表达量高于诱导7 d(P <0.05)。

结论

hDPC表达KDM5A,矿化诱导可提高KDM5A 的表达。

Objective

To investigate the expression of KDM5A in human dental pulp cells during in vitro odontogenic differentiation.

Methods

Human dental pulp cells (hDPCs) were cultured from human normal tooth pulp. KDM5A mRNA and protein in hDPCs from passage 1 to passage 8 (P1-P8) were detected using real-time quantitative PCR and western blot. Celluar KDM5A localization in hDPCs was determined by immunofluorescence. HDPCs at passage 3 were cultured in the odontoblastic differentiation induction media for 7 d and 14 d, and the mRNA and protein level of KDM5A were confirmed by real-time quantitative PCR and western blot.

Results

Real-time quantitative PCR and western blot showed that mRNA and protein expression of KDM5A increased at passage 1-2 and decreased at passage 7-8 in hDPCs. KDM5A existed in both the cytoplasm and the nucleus of the hDPCs.KDM5A mRNA and protein expression gradually increased during the odontogenic differentiation of the hDPCs (P <0.05).

Conclusion

KDM5A present in hDPCs. Odontogenic differentiation induction enhance the expression level of KDM5A.

表1 实时荧光定量PCR 引物序列
图1 实时定量荧光PCR 检测P1 ~P8 代人牙髓细胞中KDM5A mRNA 表达水平 注:与P1 代比较,P2 ~P7 代升高(aP <0.05),P8 代与P1 代相比差异无统计学意义(bP >0.05)
图2 Western blot 检测P1 ~P8 代人牙髓细胞中KDM5A 蛋白水平 注:与P1 代比较,P2 ~P6 代升高(aP <0.05),P7、P8 代后与P1 代相比差异无统计学意义(bP >0.05)
图3 免疫荧光检测KDM5A 在人牙髓细胞中的分布 注: 图3A、图3B 为实验组,KDM5A 在细胞浆和细胞核内均有表达,红色荧光为KDM5A 抗体特异性染色,蓝色荧光为细胞核特异性染料DAPI(图3A ×200、图3B ×400);图3C、图3D 为阴性对照组,未加入特异性抗体时,荧光二抗对细胞无非特异性染色(图3C ×200、图3D ×400)
图4 实时定量荧光PCR 检测人牙髓细胞成牙本质分化诱导前后的KDM5A mRNA 表达 注:与对照组组比较,KDM5A mRNA 升高,矿化诱导14 d 高于诱导7 d(aP <0.05)
图5 Western blot 检测人牙髓细胞成牙本质分化诱导前后的KDM5A 蛋白表达 注:灰度值分析显示与对照组比较,KDM5A 蛋白升高,矿化诱导14 d 高于诱导7 d(aP <0.05)
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