MFN2提升iPSCs向MSCs分化和牙周再生能力的作用及其机制研究

Mesenchymal stem cells are considered as most suitable cells for periodontal tissue regeneration. However, due to the shortcoming of hard to obtain, easily senescent, restricts its clinical application. The emergence of iPSCs not only provides a sufficient source for MSCs, but also has the advantages of easy to modify. Yet, the differentiation efficiency of iPSCs into MSCs process is low and the time of differentiation is consuming.Meanwhile, elevating the regeneration ability raise the issue of concern. Our group has confirmed in previous work, MFN2 can promote the differentiation of iPSCs into MSCs. Meanwhile, MFN2 has been reported to be widely involved in mitochondrial biogenesis. Mitochondial biogenesis play an important role in maintaining the pluripotent activity and promoting differentiation in iPSCs. Therefore, we put foward the following hypothesis: MFN2 can promote mitochondrial biogenesis to increase the differentiation efficiency of iPSCs into MSCs, and enhance its ability to differentiate into target cells. The project intends to establish MFN2 gene knock out and knock in iPSCs, then induce its differentiation into MSCs, define the role of MFN2 in differentiation. The RNA-seq technique was used to analyze the transcriptome at different time point, exploring the role of MFN2 in mitochondrial biogenesis in the differentiation process. Last, after MFN2 overexpression, iPSCs are induced into MSCs to evaluate the ability of periodontal tissue regeneration in vivo/in vitro to provide a new idea for periodontal tissue regeneration.

MSCs被认为是最适宜于牙周再生的细胞，但因其存在难获取、易衰老等问题，限制了其临床应用。iPSCs不仅为MSCs提供了充足的来源，也更易于修饰，但iPSCs分化为MSCs效率低，时间长，如何提升MSCs的再生能力也值得关注。本课题组前期工作证实，MFN2可促进iPSCs分化为MSCs。且MFN2被报道能够增强线粒体生物学功能，而后者在iPSCs多能性和分化方面发挥了重要作用。因此，我们提出以下假说：MFN2通过促进线粒体生物功能来提高iPSCs向MSCs转化率，并增强其向目标细胞分化的能力。本项目拟建立MFN2基因修饰的iPSCs细胞模型，明确MFN2在分化过程中的具体作用；并在不同时间点利用RNA-seq技术对转录组进行分析，探究MFN2在分化过程中对线粒体生物学功能的调控作用。最后，将iPSCs基因修饰后分化为MSCs,在体内、外评价其牙周组织再生能力，为牙周组织再生提供新思路。

牙周病是细菌感染引起的慢性炎症性疾病，不仅会导致牙齿脱落，还会威胁全身健康。牙周病治疗的最终目标是再生牙周组织，但传统治疗手段往往难以达到牙周再生的效果。以干细胞为基础的组织工程是牙周组织再生的潜在可行方法。MSCs被认为是最适宜的牙周再生的细胞，iPSCs来源的MSCs具有来源丰富、均质性好，且易于基因修饰的优点，如何提升iPSCs分化为MSCs的效率是突破iPSCs来源MSCs应用的瓶颈问题。MFN2被报道能够增强线粒体生物学功能，且课题组前期研究发现MFN2可以促进iPSCs向MSCs分化。本研究采用慢病毒转染的方法构建MFN2敲低及过表达的iPSCs，并通过体外研究探讨MFN2基因对iPSCs的多能性和MSCs分化的影响，进一步体外研究探讨MFN2对iPSC-MSs成骨向、牙周向分化的影响，再结合支架材料将MFN2修饰的iPSC-MSCs植入小鼠体内，评价其在动物体内牙周向分化的效果。研究结果显示，iPSCs分化为MSCs的过程中多能性降低，敲低MFN2促进iPSCs向中胚层分化，且能促进iPSCs向MSCs分化，MFN2敲低还能促进iPSC-MSCs成骨分化；敲低MFN2后促进iPSC-MSCs在体外和小鼠体内的牙周膜向、成牙骨质和成骨向分化。本研究内容明确了MFN2对iPSCs分化及iPSC-MSCs分化的影响和机制，且初步验证了MFN2敲低后的iPSC-MSCs具有牙周组织再生的应用前景，为进一步开展iPSC-MSCs在牙周再生治疗中的研究及临床转化提供理论基础。