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中华口腔医学研究杂志(电子版)

中华口腔医学研究杂志(电子版) ›› 2019, Vol. 13 ›› Issue (02) : 71 -76. doi: 10.3877/cma.j.issn.1674-1366.2019.02.002

所属专题： 文献；

基础研究

小分子混合物在神经诱导液中对根尖牙乳头干细胞成神经分化的影响
陈琪欣1, 袁长永2, 刘宗响3, 朱绍跃4, 胡刚刚1, 王鹏来2,†()   
  1. 1. 徐州医科大学口腔医学院　221004
    2. 徐州医科大学口腔医学院　221004；徐州医科大学附属口腔医院种植科　221002
    3. 徐州医科大学附属口腔医院科教科　221002
    4. 徐州医科大学附属口腔医院正畸科　221002
  • 收稿日期:2018-12-21 出版日期:2019-04-01
  • 通信作者: 王鹏来

Small molecules supplemented in neuronal induction medium enhance neurogenic differentiation of stem cells from apical papilla

Qixin Chen1, Changyong Yuan2, Zongxiang Liu3, Shaoyue Zhu4, Ganggang Hu1, Penglai Wang2,†()   

  1. 1. School of Stomatology, Xuzhou Medical University, Xuzhou 221004, China
    2. School of Stomatology, Xuzhou Medical University, Xuzhou 221004, China; Depaetment of Implant Dentistry, The Affiliated Stomatology Hospital of Xuzhou Medical University, Xuzhou 221002, China
    3. Science and Education Section, The Affiliated Stomatology Hospital of Xuzhou Medical University, Xuzhou 221002, China
    4. Depaetment of Orthodontics, The Affiliated Stomatology Hospital of Xuzhou Medical University, Xuzhou 221002, China
  • Received:2018-12-21 Published:2019-04-01
  • Corresponding author: Penglai Wang
  • About author:
    Corresponding author: Wang Penglai, Email:
  • Supported by:
    Young Scientists Fund of the National Natural Science Foundation of China(81700954)
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引用本文：

陈琪欣, 袁长永, 刘宗响, 朱绍跃, 胡刚刚, 王鹏来. 小分子混合物在神经诱导液中对根尖牙乳头干细胞成神经分化的影响[J]. 中华口腔医学研究杂志(电子版), 2019, 13(02): 71-76.

Qixin Chen, Changyong Yuan, Zongxiang Liu, Shaoyue Zhu, Ganggang Hu, Penglai Wang. Small molecules supplemented in neuronal induction medium enhance neurogenic differentiation of stem cells from apical papilla[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2019, 13(02): 71-76.

目的

评估小分子混合物在神经诱导液中对根尖牙乳头干细胞（SCAP）向神经细胞分化能力的影响。

方法

分别使用神经诱导液（对照组）和添加小分子混合物的神经诱导液（实验组）培养SCAP，8 d后在倒置显微镜下进行形态学观察，使用实时荧光定量聚合酶链反应（PCR）和免疫蛋白印迹法检测神经丝（NFM）、微管相关蛋白2（MAP2）和神经元特异性烯醇化酶（NSE）的表达情况，使用免疫荧光反应实验检测β3微管蛋白（TUBB3）的表达情况。采用SPSS 16.0软件包对数据进行统计学分析。

结果

形态学观察结果显示，实验组SCAP形成了更多神经样突触。实时荧光定量PCR结果显示，实验组NFM、MAP2和NSE的mRNA相对表达量分别为6.608 ± 2.836、29.40 ± 6.645和6.428 ± 1.025，较对照组（1.074 ± 0.202、1.092 ± 0.115和1.140 ± 0.281）显著提高，差异均有统计学意义（tNFM = 3.387，PNFM = 0.0276；tMAP2 = 7.490，PMAP2 = 0.0017；tNSE = 8.615，PNSE = 0.0010）。免疫蛋白印迹实验结果显示，实验组NFM、MAP2和NSE印迹较对照组更为明显。免疫荧光反应实验结果显示，实验组SCAP中TUBB3表达明显强于对照组。

结论

小分子混合物添加入神经诱导液中能够促进SCAP向神经细胞分化。

关键词: 干细胞，牙乳头, 神经发生, 小分子混合物, 神经诱导液
Objective

To observe the effect of small molecules supplemented in neuronal induction medium on the neurogenic differentiation of stem cells from apical papilla (SCAPs) .

Methods

SCAPs were cultured in neuronal induction medium with or without small molecules. After 8 days of culture, the cellular morphology was observed with invert microscope. qRT-PCR and western blot were used to analyze the expression of neurofilament (NFM) , microtubule-associated protein 2 (MAP2) and neuron specific enolase (NSE) . Immunofluorescence was used to detect tubulin beta 3 (TUBB3) . The data was analyzed using SPSS 16.0 software package.

Results

It was observed that SCAPs treated with small molecules exhibited marked difference in cellular morphology compared with the untreated control, which displayed more neurite outgrowth. The mRNA relative expression of NFM, MAP2 and NSE with small molecules (6.608 ± 2.836, 29.40 ± 6.645 and 6.428 ± 1.025) was higher than that of the control (1.074 ± 0.202, 1.092 ± 0.115 and 1.140 ± 0.281) , which was statistically significant (tNFM= 3.387, PNFM = 0.0276; tMAP2 = 7.490, PMAP2 = 0.0017; tNSE = 8.615, PNSE = 0.0010) . In Western blot assay, the expression of NFM, MAP2 and NSE with small molecules was higher than that of the control, too. The immunofluorescence expression of TUBB3 in the experimental group was also higher than that of the control.

Conclusion

Neuronal induction medium with small molecules can enhance neurogenic differentiation of SCAPs.

Key words: Stem cells, apical papilla, Neurogenesis, Small molecules, Neuronal induction medium
表1 根尖牙乳头干细胞不同培养基的神经诱导液成分
组别 基础培养基 其他添加物 生长因子 小分子混合物
神经诱导液 Advance DMEM/F12： 0.5%（v/v）N2+1%（v/v） 20 ng/mL bFGF 无
（对照组） Neurobasal A（1∶1） B27+100 mmol/L cAMP ? ?
神经诱导液添加 Advance DMEM/F12： 0.5%（v/v）N2+1%（v/v） 20 ng/mL bFGF 0.5 mmol/L VPA，+3 μmol/L CHIR99021+1 μmol/L
小分子混合物 Neurobasal A（1∶1） B27+100 mmol/L cAMP ? Repsox，+10 μmol/L Forskolin+10 μmol/L SP600125,+5 μmol/L GO6983,+5 μmol/L Y-27632
（实验组） ? ? ?
表2 实时荧光定量聚合酶链反应引物序列
基因 引物序列
NSE F：5′-GTCCCACGTGTCTTCCACTT-3′
? R：5′-TGGGATCTACAGCCACATGA-3′
MAP2 F：5′-TTGGTGCCGAGTGAGAAGAA-3′
? R：5′-GGTCTGGCAGTGGTTGGTTAA-3′
NFM F：5′-GTCAAGATGGCTCTGGATATAGAAATC-3′
? R：5′-TACAGTGGCCCAGTGATGCTT-3′
图1 根尖牙乳头干细胞（SCAP）在添加小分子的神经诱导液培养8 d后与对照组的形态变化（低倍放大）A：对照组，可见较多神经突触样伸展；B：实验组
图2 实时荧光定量聚合酶链反应实验检测诱导8 d后根尖牙乳头干细胞（SCAP）神经标志物的表达情况　A：神经丝（NFM）；B：微管相关蛋白2（MAP2）；C：神经元特异性烯醇化酶（NSE）；实验组较对照组相对表达量差异有统计学意义（aP<0.05）
图3 免疫荧光实验检测诱导8 d后根尖牙乳头干细胞（SCAP）中β3微管蛋白（TUBB3）的表达情况（低倍放大）A：对照组DAPI定位细胞核；B：对照组细胞对TUBB3的荧光表达情况；C：对照组DAPI与TUBB3荧光表达合并；D：实验组DAPI定位细胞核；E：实验组细胞对TUBB3的荧光表达情况；F：实验组DAPI与TUBB3荧光表达合并
图4 免疫印记实验检测诱导8 d后根尖牙乳头干细胞（SCAP）蛋白水平的表达情况
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