炎症微环境下FGF2/BMP2序贯缓释调控PI3K-AKT促进SHED移植修复牙周骨缺损的研究

The study on transplantation of stem cells for periodontal tissure regeneration is one of the most popular subjects in the field of oral regeneration at present. However, there is a sharp decline in the number of living transplanted cells after transplantation because of the poor living environment such as inflammation. This phenomenon has restricted the application of stem cells seriously. It is an effective method for improving the microenvironment by introducing factors which could inhibit apoptosis and induce differentiation while cells were tranplanted. TNFα is a major pro-apoptotic factor in the inflammatory microenvironment. FGF2 is an anti-apoptotic factor. Studies revealed that FGF2 could upregulate PI3K-AKT signaling pathway, and AKT could inhibit caspase-9, GSK-3 and FoxO1 which play important roles in TNFα induced apoptosis pathway. Thereby FGF2 could inhibite apoptosis of cells. In this study we will embed both FGF2 factors and SHED into the fibrin glue scaffolds to promote the survival of stem cells after transplantation. In the meanwhile BMP2 nanoliposomes are embeded into the gel to improve the osteogenic differentiation of stem cells. In order to tracke the cells in vivo SHED are labled with superparamagnetic iron oxide particles (SPIO) before transplantation. In all this study will investigate the mechanism of FGF2 and BMP2 sequential and sustained-release system in promoting SHED survival and osteogenic differentiation for repair of periodontal bone defects in inflammatory microenvironment.

利用干细胞移植进行牙周骨组织的再生是当前口腔再生领域的研究热点，但细胞移植后由于炎症等不利因素导致细胞存活数目急剧下降，严重制约了干细胞的应用。移植干细胞的同时引入抗凋亡、促分化的因子，改善移植部位微环境，是提高干细胞再生效率的关键。研究表明凋亡抑制因子FGF2能够上调PI3K-AKT信号途径，AKT能够抑制TNFα凋亡通路中的caspase-9、GSK-3、FoxO1，从而抑制细胞凋亡。因而，本研究将FGF2因子与乳牙牙髓干细胞（SHED）同时包埋入纤维蛋白胶(fibrin)支架，促进干细胞移植后的存活，同时埋入BMP2脂质体纳米球，促进干细胞存活后的成骨分化，并利用超顺磁性氧化铁纳米颗粒（SPIO）标记SHED进行干细胞移植后的MRI活体追踪，探讨FGF2/BMP2纳米序贯缓释系统在炎症微环境下促进SHED修复牙周骨缺损的作用机制。

慢性牙周炎引起的牙槽骨吸收会导致牙齿脱落，给患者的生活造成诸多不便。研究表明乳牙牙髓干细胞（SHED）可以作为骨组织再生的种子细胞。然而干细胞移植修复骨组织的机制仍存在争议，移植的干细胞是通过直接的细胞分化还是通过旁分泌的方式促进了骨愈合。为了探讨SHED移植到体内的命运归转，本研究利用超顺磁性氧化铁颗粒Molday ION Rhodamine-BTM (MIRB)标记干细胞，通过磁共振成像（MRI）跟踪SHED的存活和迁移，探讨SHED在牙周骨组织修复中的机理。HE和免疫组化分析表明，标记和未标记MIRB的SHED都能促进牙周骨缺损的再生。免疫荧光显示人源细胞标记抗体hNUC和MIRB有共定位，这一结果表明移植的SHED可以在体内存活。部分MIRB阳性细胞可表达OCN、DMP1，但这些细胞主要分布在骨小梁间隙，不是真正的有生物学功能的成骨细胞及骨细胞，表明SHED通过直接分化的方式促进骨愈合的可能性较小。而Elisa检测显示SHED能持续分泌促进骨再生的蛋白因子IGF-1、OCN、ALP、IL-4、VEGF和bFGF，且免疫荧光显示在移植SHED周围聚集了大量的宿主源Runx2及ColⅡ阳性细胞，二者都是启动前期骨愈合的关键因素。这些结果表明移植的SHED很可能是通过旁分泌的方式调动宿主细胞促进了骨愈合。.本研究共发表期刊论文3篇，其中中文核心1篇，SCI收录2篇。发表会议论文1篇。项目主持人张男作为参与人获得山东省科技进步三等奖1项（第5位）。另有1篇SCI论文正在修稿中。