BMSCs通过调节巨噬细胞极化促进自体脂肪移植成活的机制研究

The clinical application of autologous fat grafting is greatly restricted by the low survival rate of fat grafts. Recent studies demonstrated that M1 macrophages could cause detriments to the grafted fat tissue, and M2 macrophages could improve autologous fat graft survival by stimulating angiogenesis. In addition, increasing evidences showed that BMSCs could elicit the polarization of macrophages toward an anti-inflammatory M2 phenotype. Our previous study found that BMSCs could increase the vascular densities and enhance the survival of grafted fat. However, the underlying mechanism is not well elucidated. Hereby we hypothesize that BMSCs’ beneficial effect possibly plays via promoting the transformation of M1 type macrophages to M2 type ones, thus reducing detrimental effects of M1 and inducing neovascularization effects of M2. In the present study, we will take the advantages of transgenic mice, which allows for conditional ablation of macrophage, to test the hypothesis that infiltration of pro-inflammatory M1 macrophages contribute to fat detriment and M2 macrophages infiltration may rescue the destruction effects of M1 and stimulate angiogenesis. By using this mouse model in vivo and cell co-culture experiments in vitro, we will also study extensively that polarization of M1 macrophages to M2 may participate in the BMSCs promotion effects of neovascularization and the survival of grafted fat. We will further clarify the molecular mechanisms of BMSCs’ regulation on macrophage polarization, and the molecular effector and target that influence fat graft survival and vascularization. This study is expected to clarify the mechanism of BMSCs’ promotion on fat graft survival as well as the role of macrophage activation state, providing new theory and novel therapeutic target for the clinical application of fat grafting.

自体脂肪移植临床应用最大瓶颈问题是移植后成活率低。研究表明，巨噬细胞在脂肪移植后微环境中起重要作用，M1型巨噬细胞对移植脂肪有破坏作用，M2型可促进移植脂肪血管化和成活，而BMSCs具有促进M1型巨噬细胞向M2型极化的作用，最近课题组发现BMSCs可以提高移植成活率，但具体机制不清。据此提出假设：BMSCs可通过促进M1型巨噬细胞向M2型转化，减少M1对移植脂肪的破坏，促进M2的血管化作用，从而提高移植脂肪成活率。本项目拟利用条件诱导的巨噬细胞剔除小鼠，验证巨噬细胞向M2型极化有利于移植脂肪的成活；利用该小鼠模型和体外共培养实验，深入研究BMSCs参与巨噬细胞向M2极化后分泌促血管新生的效应因子；通过信号通路进一步明确BMSCs调控巨噬细胞向M2极化的分子机制。本研究有望阐明BMSCs促进移植脂肪成活的机制以及巨噬细胞活化状态对脂肪成活的影响，为脂肪移植在临床的广泛应用提供新理论和新靶点。

自体脂肪移植因其优势明显，近年来成为整形外科最热门的研究领域之一，但成活率低，成活质量差，效果难以预测，这些仍是限制其在临床广泛应用的瓶颈因素。研究表明，巨噬细胞在脂肪移植后微环境中起着重要作用，M1型巨噬细胞对移植脂肪有破坏作用，M2型可促进移植脂肪血管化和成活率，BMSCs可以促进M1向M2极化，但其机制不清。为了探讨机制，首先我们建立裸鼠脂肪移植模型，分别将BMSCs和PBS与脂肪颗粒进行混合移植，并于3个月后观察到BMSCs组脂肪保留率明显高于PBS对照组。通过免疫荧光和qPCR分析，我们发现移植早期招募更多的巨噬细胞，且M2抗炎巨噬细胞占主导，接着我们通过体外巨噬细胞和骨髓间充质干细胞共培养体系研究发现，骨髓间充质干细胞促使巨噬细胞M0向M2极化，提高抗炎以及血管相关生长因子IL-10，VEGF等，并降低TNF-α，IL-12等促炎因子，通过转录组我们比较BMSCs+脂肪颗粒和PBS+脂肪颗粒的基因差异表达，所得的结果进行GO分析、KEGG通路分析，我们发现BMSCs相较于PBS组的脂肪组织在AMPK、分泌因子受体配体结合、脂肪因子分泌、以及氧化磷酸化等通路内功能靶分子上有上调趋势，这提醒BMSCs可激活移植脂肪早期的炎症相关的通路。在此基础上，我们通过收集缺氧和常氧来源的BMSCs作用在裸鼠脂肪移植模型上，从缺氧诱导外泌体角度找到提高移植脂肪保留率的新方法，此外，我们在临床上进行了骨髓干细胞辅助自体脂肪移植治疗颜面部凹陷畸形后移植脂肪体积保留率的差异，通过三维数字化技术评估移植脂肪保留率的差异，以客观、科学的数据为临床提供佐证。