尼古丁对BMSCs修复关节软骨缺损的干预作用及机制研究

Bone marrow-derived mesenchymal stem cells (BMSCs) are believed to possess extensive clinical application prospects in regeneration and repair of articular cartilage. The current researches mainly focus on the therapeutic effects of BMSCs' chondrogenic differentiation on cartilage repair, however, the concerns about unfavorable factors affecting the potential of BMSCs' chondrogenic differentiation are still existed and deficient to be comprehensively explored. Considering a relatively high worldwide smoking rate，our previous work have demonstrated that nicotine suppresses potential of BMSCs' chondrogenic differentiation by decreasing synthesis of extracellular matrix and the expression of articular cartilage phenotypic gene. Here, we hypothesize that nicotine could bind to nAChRs located on cell membrane of BMSCs, and the subsequent increased concentration of intracellular calcium as well as the elevated activity of calcineurin would activate NFATc2, which might further interfere the chondrogenic differentiation of BMSCs via suppressing Sox9 expression. Therefore, this project aims to firstly elucidate the potential epigenetic regulatory mechanism of nicotine on BMSCs' chondrogenic differentiation in vitro. Then, the in vivo test would be employed to further validate the adverse effects and mechanism of nicotine on BMSCs' chondrogenic differentiation and its application on cartilage repair. The project will contribute to the risk assessment of clinical autologous BMSCs grafting to the patient with smoking history, and provide experimental basis for the clinical application of cartilage tissue engineering.

骨髓间充质干细胞(BMSCs)回植诱导分化修复关节软骨缺损具有良好应用前景，但目前研究多集中于BMSCs向软骨分化的修复效果，对影响其自身分化潜能的不利因素尚缺乏研究。基于社会的高吸烟率，本课题组前期发现，尼古丁可抑制BMSCs软骨分化中的基质蛋白合成和表型功能基因表达。综合文献,我们推测：尼古丁通过与BMSCs膜上尼古丁受体α7-nAChR结合后，提高胞内钙离子浓度和磷酸酯酶活性来激活NFATc2，后者通过抑制Sox9 表达干预BMSCs的软骨分化。本课题拟在前期研究发现的基础上，进一步阐明尼古丁抑制BMSCs向软骨分化的表观遗传转录调控机制，并在整体水平证实尼古丁对BMSCs软骨分化和软骨缺损修复的干预作用。本项目将有助于评估具有吸烟背景的软骨缺损患者采用自体BMSCs进行缺损修复治疗的风险，为指导软骨组织工程在临床中的应用提供实验依据。上述工作及研究设想在国内外尚未见报道。

关节软骨组织因缺乏足够的血管支配，自我修复能力极差，软骨损伤常导致关节功能障碍及退变，目前研究多集中如何有效修复关节软骨损伤，对其修复过程中不利因素影响研究甚少。尼古丁可影响组织损伤修复，明确尼古丁对组织工程干细胞修复软骨缺损的影响对于软骨缺损研究具有重要指导意义。本项目建立大鼠BMSCs体外软骨分化模型，在其分化过程中给予不同浓度尼古丁处理，检测软骨细胞表型、分子信号通路及关键基因表遗传变化，从细胞水平证实尼古丁抑制 BMSCs 软骨定向分化作用，并阐明其表观遗传转录调控机制，明确其作用靶标；进一步建立大鼠软骨缺损模型，应用BMSCs移植修复，在修复过程中给于尼古丁干预，干预同时给予尼古丁作用靶标抑制剂进行反证，从整体水平证实尼古丁对 BMSCs 软骨分化和软骨缺损修复的干预作用，并验证其作用靶标。研究结果表明，尼古丁可剂量依赖性抑制BMSCs的软骨定向分化，其机制为尼古丁通过上的BMSCs的α7-nAChR激活Ca2+/CaN/NFATc2信号通路，招募HDAC1降低软骨分化核心转录因子Sox9启动子区组蛋白乙酰化水平，从而抑制Sox9的表达，减少软骨表型基因Col2A1的表达，进而抑制BMSCs的软骨分化。同时使用si-RNA敲低BMSCs中NFATc2的表达，发现其可逆转尼古丁对BMSCs软骨分化的抑制作用，证实NFATc2为尼古丁抑制作用的靶标。在整体水平，低于日常尼古丁依赖人群剂量的尼古丁暴露可对BMSCs移植修复软骨缺损产生不良干预作用，使用NFATc2抑制剂时可有效降低尼古丁对BMSCs修复软骨缺损的不利影响，同时也从整体水平证实NFATc2是尼古丁抑制BMSCs修复软骨缺损的分子靶标。本研究有助于评估具有吸烟背景的软骨缺损患者采用自体BMSCs进行软骨诱导分化和缺损修复治疗的风险，提供干预治疗的靶标；最大程度上避免尼古丁对BMSCs移植修复软骨缺损的不利影响，实现高质量的关节软骨组织修复，为指导软骨组织工程在临床中的应用提供实验依据。