ECM膜片介导Ln332-Integrin-FAK信号轴在种植体牙龈生物学封闭形成中的作用及机制

Peri-implantitis is one of the major reasons for implant failure. Studies have shown that the biological sealing of implants is weaker than that of natural teeth, mainly due to that HDs mainly form in the apical portion of the implant-gingival interface. Our achieved work has proved that the overexpression of LAMA3 can accelerate the secretion of Ln332 , which is conducive to the formation of HDs and improves biological sealing . Therefore, we propose a hypothesis that the combination of Ln332 and the extracellular domain of Integrin induces the formation of HDs and activates the intracellular domain of Integrin to phosphorylate FAK (Ln332-Integrin-FAK), promoting the formation of biological sealing in implants. On the basis of this concept, an emerging tissue engineering material, ECM sheet, was chosen as carrier. We will introduce LAMA3 gene into three kinds of stem cells by adenovirus, and prepare Ln332-rich ECM sheet on the surface of implants. Then evaluations of in vitro and in vivo experiments will be taken to explore the effect of Ln332-Integrin-FAK signal axis on the biological sealing of ECM sheet modified implant-gingival interface and underlying regulatory mechanisms. This study can provide a new experimental basis for the formation of implant-gingival biological sealing and a new therapeutic method for the promotion of gingival cuff formation in clinical practice.

研究表明种植体牙龈生物学封闭较天然牙薄弱，主要原因是附着结构半桥粒(HDs)在种植体牙龈界面的上2/3较缺乏。我们最新研究发现LAMA3过表达可促进细胞分泌Ln332及HDs在种植体牙龈界面形成，改善种植体牙龈生物学封闭。由此本项目提出假设:Ln332与Integrin的胞外段结合诱导HDs形成并激活Integrin胞内段使FAK磷酸化(Ln332-Integrin-FAK)，促进种植体牙龈生物学封闭形成。为验证该设想，我们选用新兴组织工程学材料ECM膜片为载体，通过腺病毒将LAMA3基因导入3种干细胞中，在种植体表面制备富含Ln332的ECM膜片，通过体内外实验探讨不同来源的富含Ln332的ECM膜片介导Ln332-Integrin-FAK信号轴在种植体牙龈生物学封闭形成中的作用及调控机制，为更深入地认识种植体牙龈生物学封闭形成机理提供实验基础，也为临床上促进种植体袖口形成提供新思路。

研究表明种植体周牙龈生物学封闭较天然牙周薄弱，主要原因是附着结构半桥粒在种植体牙龈界面的上2/3较缺乏。本项目通过构建钛片表面牙龈间充质干细胞膜片，与牙龈上皮细胞共培养并通过早期粘附、免疫荧光、RT-qPCR、western blot、透射电镜等实验方法检测半桥粒相关基因、蛋白的变化；通过高通量测序技术对共培养后的差异基因进行富集分析。结果发现牙龈间充质干细胞膜片可以显著提高牙龈上皮细胞的粘附数量和形态的铺展；免疫荧光、RT-qPCR、western blot结果显示牙龈上皮细胞半桥粒相关基因、蛋白的表达增强，差异具有统计学意义；透射电镜结果表明膜片组可以观察到成熟的半桥粒结构。转录组测序结果富集分析发现PI3K-AKT信号通路具有统计学差异，加入通路抑制剂后发现半桥粒相关蛋白显著下调。本项目为种植体周牙龈生物学封闭的形成提供了新的治疗思路和靶点。