成釉细胞无血清条件培养液微环境下BMP信号通路对诱导多能干细胞牙向分化的作用机制的研究

Traditional dental therapies for the replacement of missing teeth largely utilize partial or complete dentures and titanium implants capped with prosthetic crowns. Although these prostheses serve a purpose, they are not equivalent, neither in function nor aesthetics, to natural teeth. Recent progress in dental tissue engineering has generated functional bioengineered teeth using stem cells may soon be available to replace missing teeth.The study of induced pluripotent stem (iPS) cells that is one of the key problems in the field of biological engineering is essential in areas of stem cell and regenerative medicine. iPS cells behave similarly to ESCs, exhibiting the capacity to differentiate into virtually any cell type, while avoiding ethical issues surrounding the use of human embryos and immune rejection. Thus, the creation of iPS cells presents potentially tremendous opportunities as candidate cells for teeth regeneration. The recent use of iPS cells for tooth regeneration under specific microenvironment is becoming increasingly concerned. But the mechanism of specific microenvironment on iPS directional odontogenic differentiation is unclear. Our present studies explore the possibility of mouse iPS cells differentiation into odontogenic differentiation induced by ameloblasts serum-free conditioned medium (ASF-CM) supplemented with bone morphogenetic protein4 (BMP4). The studies suggested ASF-BMP4 induced mouse iPS cells can differentiate into ameloblast-like and odontoblast-like cells. We assume that BMP signaling pathway plays an important role in the odontogenic differentiation from iPS cells induced by ASF-CM. Therefor，it is necessary to clarify the mechanism of BMP singaling pathyway in regulating the odontogenic differentiation from iPS cells induced by ASF-CM. Our aim is establishing a method for improving iPS directional differentiation potency by regulating BMP signal pathway key molecules, and observation of the ability of formation tooth structures of iPS cells in vivo based on in vitro regulation. These researches provide a theoretical basis for the study of tissue engineering tooth regeneration.

传统牙齿缺失的替代修复治疗不能与天然牙齿相媲美，利用干细胞构建有生物活性的组织工程化牙齿打破了以往牙科治疗的框架。诱导多能干细胞（iPS）具有和胚胎干细胞（ES）类似的功能，并且避免了ES研究面临的伦理学和免疫排斥方面的限制，成为组织工程牙齿再生研究最佳候选种子细胞。利用特定微环境诱导iPS分化实现牙齿再生日益受到关注。但是微环境对iPS牙向分化的诱导机制不清。我们前期研究证实成釉细胞无血清条件培养液（ASF-CM）中加入骨形成蛋白-4（BMP4）能促进小鼠iPS向成釉细胞谱系和成牙本质细胞谱系分化。据此提出假设ASF-CM中的BMP信号通路对iPS牙向分化有重要的调控作用。本项目拟在前期研究基础上从BMP信号通路分子水平探索ASF-CM微环境对iPS牙向分化的作用机制，通过正性和负性调控BMP信号通路关键分子，建立提高iPS牙向分化能力的方法，为组织工程牙齿再生研究提供理论依据。

由在成体细胞中高表达四种特殊转录因子（Oct3 /4、Sox2、Klf4和c-Myc）而生成的诱导多能干细胞（iPS，induced pluripotent stem cell）可以用来产生间充质干细胞(MSLCs)，并在特定的微环境下，定向诱导成所需要的细胞进行组织修复。这些细胞由于有助于牙及牙周组织的再生与修复因而在牙科领域中具有广阔应用的前景。骨形成蛋白- 4(BMP4，bone morphogenetic protein- 4)作为TGF 家族重要的一员，在脊椎动物的牙齿发育过程中广泛表达及参与并发挥了重要的作用。我们构建过表达BMP4基因的慢病毒载体,探讨其过表达后对小鼠诱导性多能干细胞(iPS)生物学活性的影响。采用慢病毒转染建立稳定过表达BMP4的iPS 细胞株。CCK8检测细胞增殖的活性。ALP活性检测细胞分化程度。Q-PCR检测AMBN 、CK14、DSPP、BSP、Runx2 mRNA表达的变化。CCK8结果显示，过表达BMP4 组细胞与空载体组相比能显著提高细胞的增殖活性（P＜0.001）。ALP活性测定结果显示，过表达BMP4组的ALP活性相对于空载体组和空白组增强，提示过表达BMP4能够促进iPS细胞的分化能力。RT-PCR的结果显示，过表达BMP4组Runx2的mRNA表达水平显著增高（P＜0.001）。此外，过表达BMP4组的AMBNmRNA相对表达水平为4.473±0.456，是空载体组的1.435倍（P＜0.001）。过表达BMP4组的CK14mRNA相对表达量是空载体组1.79倍（P＜0.001）；DSPPmRNA的相对表达量在过表达BMP4组中为53.196±11.225，是空载体组的14.942倍（P＜0.001）；过表达BMP4组BSPmRNA的相对表达量27.013±4.235,是空载体组的5.581倍（P＜0.001）。BMP4基因过表达能够显著促进iPS细胞的牙向分化能力，显著促进了AMBN 、CK14、DSPP、BSP、Runx2 mRNA的表达。