CRISPR-Cas9系统介导的脂肪干细胞Sema3a表观遗传修饰用于促进2型糖尿病种植体骨结合的研究

Adipose-derived stem cells(ASCs) are the seed cells to better the osseointegration of the implant. However, the limited osteogenic capacity limits its efficacy and it is fundamentally important to improve the osteogenic potential of adipose-derived stem cells (ASCs) for the purpose to enhance the osseointegration of the implant in patients of type 2 diabetes mellitus(T2DM). We and other groups have proved that the low expression of Semaphorin3a (Sema3a) accounted for the poor osteogenic effect in terms of the attempt to accelerate the bone healing and to improve the osseointegration of T2DM patients with ASCs. Currently, there were few practical and safe approaches to increase Sema3a expression. With the advent of CRISPR/Cas9 in gene editing, we aim to remodel the epigenetic modification, especially the H3K27 methylation, on the promoter of Sema3a towards an active state via a modified CRISPR/Cas9 system. The epigenetic remodeling efficiency, effects, off-target effects, and underlying mechanism would also be analyzed with molecular biological and cytological approaches. Finally, the effects of the modified ASCs sheets established with Vitamin C on the osseointegration would be tested in T2DM rats. The study proposed here would be highly possible to build up a novel strategy to compensate the deficiency of Sema3a in the T2DM–derived ASCs, and thus provides a safe and effective method to improve the efficacy of ASCs for its application in oral implantation in the context of T2DM.

提高脂肪干细胞（ASCs）的成骨分化能力是应用脂肪干细胞解决2型糖尿病（T2DM）等种植体骨结合差的关键。课题组及国际同行研究发现Semaphorin3a (Sema3a)低表达是应用T2DM 患者ASCs促进骨缺损愈合、改善其种植体骨结合效果不理想的重要机制。然而，目前依然缺乏有效纠正Sema3a表达的安全高效的策略。为此，我们拟利用基因编辑技术CRRSPR/Cas9系统构建靶向Sema3a的表观遗传学修饰模型，通过分子生物学和细胞学手段明确T2DM来源的ASCs Sema3a异常表达的表观遗传学机制；探索Sema3a 基因H3K27去甲基化对ASCs形态、功能及多向分化潜能的影响；并通过Vit C 诱导法构建经表观遗传修饰的ASCs膜片，观察其对T2DM大鼠种植体骨结合的作用。本项目有望建立纠正ASCs中Sema3a表达的理想策略，为促进ASCs在口腔种植领域的应用提供新思路。

提高2型糖尿病大鼠来源脂肪干细胞（ADSCs）成骨分化能力是将其应用于骨组织工程最为关键的一步。课题组前期研究发现并验证了Sema3a能够促进2型糖尿病大鼠ADSCs成骨分化，ADSCs中过表达Sema3a搭载PLGA支架材料则能有效促进大鼠极限骨缺损的愈合。然而上述方式未从根本上解决其成骨分化能力弱，也未找到其成骨能力差的机制。综上我们设计如下实验：提取2型糖尿病和正常大鼠来源的ADSCs并进行启动子区和全基因甲基化检测，结果发现两组细胞在Sema3a启动子区和全基因段均未发现有甲基化差异。随后我们利用q-PCR检测待测基因在两组细胞中的差异表达，并结合DNA甲基化水平筛选两者成负相关的基因，并利用IGV软件进行基因差异显著性确定。最终确定待测基因：Calca（降钙素基因相关肽），检测了不同浓度Calca能够促进ADSCs成骨分化，增殖和大鼠极限骨缺损；随后提取了ADSCs和骨髓间充质干细胞并进行鉴定，分别观察其增殖、成骨分化能力上的差异，并分析了其在全基因组甲基化水平的差异，确定了Wnt10b基因，构建了针对CRISPR/Cas9表观遗传修饰系统并发现其体内外提高ADSCs成骨分化。