基于单细胞技术筛选具有成骨分化优势的颅面骨来源BMSCs亚类的研究

Mesenchymal stem cells (BMSCs) has broad applications in the treatment of diseases and tissue regeneration. Although BMSCs have demonstrated great therapeutic potential, the heterogeneity of BMSCs may be responsible for the incongruent data obtained in BMSC-based preclinical studies and clinical trials. To clarify the molecular basis of this heterogeneity, especially the subtype which has the high potential of osteogenesis, we will try to employed single cell RNA sequencing to compare the gene expression profiles of the each single craniofacial BMSCs ,to divide them into different subtypes, revealing a relationship between gene expression and variability in subtype function. To illustrate the characteristic of the subtypes which has high or low potential of osteogenesis, we will establish animal models which BMSCs osteogenesis ability is impaired. Single cell analysis used to assess the transcriptional diversity of murine mesenchymal stem cells derived from bone marrow could help us not only clarify the subtypes which has the high potential of osteogenesis of BMSCs, but also estimate the gene function of this subtype in different phases in bone regeneration. This study will provide new ideas and theoretical basis for biological markers of craniofacial BMSCs osteogenesis ability, and developing new prevention and treatment strategies for craniofacial bone regeneration.

骨髓间充质干细胞（BMSCs）在医学中有着广泛应用。BMSCs具有多向分化能力，其分化差异是由群体细胞的异质性决定的，不同亚类的BMSCs功能各异。在颌骨组织再生研究领域，只有选择具有高成骨分化潜能的BMSCs亚类，才能提高成骨效率。因此，筛选具有成骨分化优势的亚类成为本领域研究新的关键问题。而单细胞测序技术可以分析群体细胞的异质性，精确评价其中单个细胞的功能。本研究拟在BMSCs成骨异质性研究中引入单细胞测序技术，在单细胞水平探索骨BMSCs成骨能力与细胞转录组表达谱的相关性；并通过建立衰老、骨质疏松等成骨能力降低的动物模型，系统研究不同生理病理状态下BMSCs成骨能力异质性，探索高成骨分化潜能BMSCs的标志物；在动物模型体内验证高成骨能力BMSCs亚类在颅面骨修复再生中的优势。本研究从新的视角探讨颅面骨BMSCs的成骨分化机制和分选方法，有助于提升相关骨病治疗效果。

颌骨组织缺损的再生一直是困扰临床的重大问题，颌骨来源骨髓间充质干细胞（BMSCs）作为良好的组织工程种子细胞，已被证实具有多向分化潜力和显著异质性。因此，利用单细胞测序明确并筛选具有成骨分化优势的BMSCs亚类，成为推进骨再生领域研究的关键问题。本项目基于大鼠、小鼠等实验动物建立一系列颅颌面部骨相关疾病模型，通过流式分选合并单细胞测序，利用WGCNA、GO富集、KEGG富集等方式分析不同模型来源的细胞表达谱差异，筛选出具有强成骨优势的BMSCs亚类，为后续动物验证及生物工程治疗提供分子生物学依据。进一步将对比该成骨优势亚类与其他颅颌面部骨代谢疾病微环境中提取的BMSCs，通过micro-ct，组织学，透射电镜及外泌体示踪，外泌体质谱及miRNA测序，证明BMSCs的异质性在外泌体中也有体现，为后期应用提供理论基础。此外，本项目也探讨了在机械应力对颌骨及牙槽骨吸收的影响，通过转录组测序和染色质免疫共沉淀测序，从组织水平解析了该病理过程的分子水平变化，也为进一步认识颅颌面部病理状态下的骨代谢奠定了基础，以期为利用组织工程技术治疗颅颌面部骨缺损等疾病提供理论依据。