IGF2在低氧下对牙源性干细胞功能及在牙髓血运重建术中的作用及机制研究

Root hypoplasia and regeneration is a key problem in oral treatment for children, however, the traditional therapy couldn’t reach the satisfied therapeutic effect. Dental pulp revascularization could induce the development of tooth root and enhance its structure, but couldn’t form functional active dental pulp. Our previous studies found that IGF2 are down-regulated in stem cells from apical papilla (SCAPs) than those in the tissue of apical papilla. And in vitro experiments showed that IGF2 could significantly enhance the proliferation, odontogenic differentiation, angiogenic differentiation, and neurogenic differentiation potentials of SCAPs, suggesting that IGF2 may play an important role to regulate the stem cells function in dental pulp revascularization and tooth regeneration. In this project, we want to investigate the biological regulation function of IGF2 molecules in dental mesenchymal stem cells by using human recombinant protein under hypoxia microenvironment. And then we will make the IGF2-Chitosan hydrogel microspheres, investigate its function in dental pulp revascularization by using BEAGLE dental pulp necrosis model. Finally, we will figure out the molecular mechanism to explain how IGF2 signaling regulate the function of dental mesenchymal stem cells and dental tissue regeneration. Taken together, this study will illuminate the mechanism of the IGF2 to regulate the dental stem cells function under clinic conditions, provide the key target genes and certain theoretical basis to promote stem cells mediated dental tissue regeneration. And we also provide the theoretical basis and technique parameters for enhancing dental pulp revascularization in clinic application by using IGF2.

年轻恒牙因龋病、外伤或发育畸形等原因造成的牙髓坏死导致牙根发育不完善是儿童口腔治疗的关键问题，传统治疗效果欠佳。近来采用的牙髓血运重建术能促进牙根继续发育、增强根管壁的厚度，但不能形成具有功能活性的牙髓组织。课题组前期从牙根发育微环境出发筛选出可能的关键生长因子IGF2，对其功能研究表明IGF2可显著促进根尖牙乳头干细胞的增殖、成牙分化、成神经分化和成血管分化功能，提示若将其用于牙髓血运重建术，可能具有一定的促牙髓牙本质再生作用。本课题拟将进一步研究IGF2在低氧下对牙源性干细胞增殖、定向分化等功能的影响及调控机制；进而构建IGF2壳聚糖缓释微球作为支架，在大型动物比格犬模型上研究IGF2在年轻恒牙牙髓血运重建术中的作用。为在临床条件下IGF2促进干细胞功能提供理论依据和候选靶点，并希望明确IGF2在牙髓血运重建术中的作用。

年轻恒牙因龋病、外伤或发育畸形等原因造成的牙髓坏死导致牙根发育不完善是儿童口腔治疗的关键问题，传统治疗效果欠佳。近来采用的牙髓血运重建术能促进牙根继续发育、增强根管壁的厚度，但不能形成具有功能活性的牙髓组织。课题组前期从牙根发育微环境出发筛选出可能的关键生长因子IGF2，对其功能研究表明IGF2可显著促进根尖牙乳头干细胞的增殖分化功能，提示若将其用于牙髓血运重建术，可能具有一定的促牙髓牙本质再生作用。本课题采用成骨诱导中碱性磷酸酶活性检测法首先筛选出IGF2生长因子最佳浓度为5ng/ml；CCK-8和CFSE试验检测细胞增殖结果表明5ng/ml IGF2生长因子可以促进根尖乳头干细胞的增殖。茜素红染色及钙离子浓度测定检测细胞成骨/成牙本质能力结果表明IGF2组与对照组相比分泌钙离子浓度显著增高，差异有统计学意义（P<0.05）。Real-TimeRT-PCR 检测成骨/成牙本质相关基因表达IGF2组与对照组相比，BSP、 DSPP、DMP1、 OCN、OPN基因的表达在不同时间点显著增高，差异有统计学意义。此外成血管/成神经相关的基因VEGF、NCAM、NeuroD、TH-1的表达结果表明IGF2组与对照组相比不同时间点基因表达均显著增高。神经诱导后细胞免疫荧光染色结果表明IFG2组和对照组相比，每视野阳性染色神经球多，差异具有统计学意义。蛋白质组分析结果表明加入IGF2组根尖牙乳头干细胞与对照组相比有72种蛋白分泌上调，37种蛋白分泌下调。我们进而对上调蛋白中的IGFBP5的研究表明体外诱导环境下IGFBP5能够促进牙髓干细胞成血管成神经分化。进而构建IGF2玻璃陶瓷/氧化海藻酸钠（BG/OSA）复合水凝胶缓释系统作为缓释支架，体外观察对牙源性干细胞繁殖和分化的作用；动物模型上观察IGF2对细胞归巢及牙髓再生的作用，明确IGF2在牙髓血运重建术中的可行性。