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张凡柯, 杜雅晶, 周陈晨, 等. 小鼠成牙关键调控基因的筛选与功能验证实验研究[J]. 四川大学学报(医学版), 2020, 51(4): 472-479. DOI: 10.12182/20200760605
引用本文: 张凡柯, 杜雅晶, 周陈晨, 等. 小鼠成牙关键调控基因的筛选与功能验证实验研究[J]. 四川大学学报(医学版), 2020, 51(4): 472-479. DOI: 10.12182/20200760605
ZHANG Fan-ke, DU Ya-jing, ZHOU Chen-chen, et al. Screen and Function Study of the Key Regulative Odontogenic Genes in Mice[J]. Journal of Sichuan University (Medical Sciences), 2020, 51(4): 472-479. DOI: 10.12182/20200760605
Citation: ZHANG Fan-ke, DU Ya-jing, ZHOU Chen-chen, et al. Screen and Function Study of the Key Regulative Odontogenic Genes in Mice[J]. Journal of Sichuan University (Medical Sciences), 2020, 51(4): 472-479. DOI: 10.12182/20200760605

小鼠成牙关键调控基因的筛选与功能验证实验研究

Screen and Function Study of the Key Regulative Odontogenic Genes in Mice

  • 摘要:
      目的  筛选小鼠成牙关键基因,并验证关键基因对羊膜上皮细胞(WISH)成牙诱导分化的能力。
      方法  通过免疫组化染色、实时荧光定量PCR(RT-qPCR)检测骨形成蛋白4(BMP4)、成纤维细胞生长因子8(FGF8)、音猬因子(SHH)、淋巴增强因子(LEF1)4种分子蛋白和基因在小鼠早期牙胚发育过程〔小鼠胚胎发育第10.5天(E10.5)、E11.5、E14.5〕中的时空差异化表达,筛选可能的成牙关键基因;非牙源性上皮WISH成骨诱导培养3周,采用茜素红(ALZ)染色与RT-qPCR检测碱性磷酸酶(ALP)观察骨向分化能力;通过胚层重组实验进行组织重组,将BMP4蛋白加入重组组织块,并移植于小鼠肾被膜下,观察验证关键基因的蛋白能否诱导非牙源性上皮成牙分化。
      结果  免疫组化染色和RT-qPCR结果证实E10.5胎鼠BMP4蛋白和基因在第一、第二腮弓上皮存在差异性表达,且从E10.5~E14.5其表达与成牙能力从上皮向间充质的转移一致;FGF8蛋白和基因虽在第一、第二腮弓上皮存在差异性表达,但其表达与成牙能力的转移不一致;LEF1、SHH蛋白和基因在第一、二腮弓上皮不存在差异表达,且与成牙能力的转移不一致,因此,筛选出BMP4基因为可能的成牙关键基因。WISH成骨诱导3周,ALZ染色阳性,并形成钙结节,RT-qPCR检测ALP mRNA表达升高;胚层重组实验结果显示,加入外源性的BMP4蛋白可使第二腮弓间充质分别与第二腮弓上皮及WISH均有牙齿样结构形成。
      结论  BMP4、FGF8、SHH、LEF1的蛋白和基因在小鼠牙胚早期发育过程中呈时空特异性表达;BMP4的蛋白和基因在第一、二腮弓上皮差异表达,且其蛋白可使与第二腮弓间充质重组的非牙源性上皮获得成牙能力。BMP4为可能的成牙关键基因。

     

    Abstract:
      Objective  To screen the key odontogenic genes in mice and verify the odontogenic inducing effect on amniotic epithelial cells (WISH).
      Methods  The spatially and temporally different expression of bone morphogenetic proteins 4 (BMP4), fibroblast growth factor 8 (FGF8), sonic hedgehog (SHH), lymphoid enhancer factor 1 (LEF1) proteins and their genes expression in the early odontogenesis stage (embryo day 10.5 (E10.5)、E11.5、E14.5) in fetal mice were detected by immunohistochemistry staining and quantitative real-time PCR (RT-qPCR). According to the results, we screened the probable key odontogenic genes. Then adding osteogenic inducing solution to induce non-odontogenic epithelium cells, WISH. After 3 weeks culture of non-odontogenic epithelial WISH for osteogenic induction, the epithelial-mesenchymal transformation cap ability was evaluated by using Alizarin (ALZ) red staining and RT-qPCR on the alkaline phosphatase (ALP) mRNA expression level. Using germ layer recombination experiment to observe and verify whether the screened genes can induce non-odontogenic epithelium cells acquire odontogenesis ability. The recombined tissue grafts containing key genes were transplanted beneath the renal capsule of mice.
      Results  The results of immunohistochemistry staining and RT-qPCR showed that on E10.5 BMP4 protein and gene were differently expressed in the first and second branchial arch epithelium, which synchronized the odontogenic capability transferring from epithelium to mesenchyme from E10.5-E14.5. Though the expression of FGF8 protein and gene existed such difference in the first and second branchial arch epithelium, there was no synchronization in transfer. The expression of LEF1 and SHH proteins and genes had neither difference nor synchronization. So far, we considered the BMP4 was the probable key odontogenic gene. Through 3 weeks’ osteogenic induction, ALZ red stained positively and calcium nodules were observed in WISH, and the expression level of ALP mRNA increased. In the germ layer recombination experiment, exogenous BMP4 protein enabled the second branchial arch mesenchyme forming tooth-like structures after recombined with the second branchial arch epithelium or WISH.
      Conclusions  The proteins and genes of BMP4, FGF8, SHH and LEF1 are spatially and temporally differently expressed in the early tooth development stage in mice. The protein and gene of BMP4 are differently expressed between the first and second branchial arch epithelium and enables the non-odontogenic epithelium acquiring odontogenic ability. BMP4 is the possible key odontogenic gene.

     

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