抗坏血酸碳点通过p38信号通路促进骨髓间充质干细胞黏附作用的机理

Cell adhesion plays an important role in histogenesis and tissue regeneration. Some research has showed that p38 signaling pathway is associated with cell adhesion. We previously found that ascorbic acid-based carbon dots (AACDs) can promote adhesion of bone marrow mesenchymal stem cells（BMMSCs）, with the change of p38 phosphorylation level. In order to explore if AACDs’ role on adhesion of BMMSCs is mediated by p38 signaling pathway, we will study the effect of AACDs on cell adhesion ability, expression of adhesion-associated molecules ，p38 and osteogenic-related molecules in BMMSCs by trypsinization method, real-time PCR, western blot and injection of BMMSCs in vivo, and identify the role of AACDs on adhesion of BMMSCs; then we will study the relationship between p38 signaling activity and expression of cell adhesion associated molecules under the influence of AACDs by gain- and loss- of function, and illuminate the role and mechanism of AACDs on adhesion of BMMSCs mediated by p38 signaling pathway; lastly, we will observe the effect of AACDs on cell sheet formation ability of BMMSCs and the effect of the cell sheet on repair of rat cranial defect by histological staining, immunohistochemistry and microCT, which will lay a foundation for application of AACDs in bone defect.

细胞黏附在组织发生和损伤再生中发挥重要作用。研究表明，p38信号通路与细胞黏附密切相关，我们发现抗坏血酸碳点(AACDs)可以促进骨髓间充质干细胞（BMMSCs）的黏附，并伴有p38磷酸化水平的改变。为探索AACDs促进BMMSCs黏附是否由p38信号通路介导，采用胰酶消化法、Real-Time PCR、Western Blot等生物学技术和体内注射方法研究AACDs处理后BMMSCs黏附能力以及黏附相关分子、p38信号分子和成骨相关分子表达的变化，明确AACDs对BMMSCs黏附的作用；通过基因功能激活和失活方法验证AACDs作用下p38信号通路同黏附相关分子表达的关系，阐明AACDs通过p38信号通路对BMMSCs黏附作用机理；利用组织学、免疫组化和micro CT等技术，观察AACDs处理后BMMSCs膜片形成能力及其对大鼠骨缺损模型修复作用，为AACDs治疗骨破坏性疾病奠定基础。

细胞黏附在组织发生和损伤再生中发挥重要作用。近年来，利用纳米材料表面特性调控细胞黏附已取得了一定的成绩，但是其具体作用的生物学机理并不明确。本项目通过微波法制备了抗坏血酸碳点并对其理化性质进行了表征，利用酶消化法证实了抗坏血酸碳点促进细胞黏附的作用。以此为基础，进一步通过real-time PCR、western blot、ALP染色等技术手段说明抗坏血酸碳点促进成骨细胞分化的作用，并采用HE染色和免疫组化染色证实抗坏血酸碳点可促进细胞膜片形成。接着，为阐明抗坏血酸碳点促进细胞黏附的分子机制，利用si-RNA、小分子抑制剂以及RNA-seq等方法，明确TGF-β/p38/Snail信号通路和PI3K/Akt/mTOR介导的自噬在抗坏血酸碳点促进细胞黏附中发挥重要作用，最后，利用组织学、免疫组化和micro CT等技术，在体内证实抗坏血酸碳点诱导的细胞膜片具有更强的促组织修复作用，为碳点的临床应用奠定了基础。