脂肪组织工程室内YAP/TAZ参与脂肪再生负性调控的机制研究

Adipose tissue regeneration could be induced by adipose tissue engineering. However, the engineered adipose tissue is facing questions about the uncontrolled growth and tumorigenesis. Our previous study found that cells did not proliferate and differentiate without restriction during the process of adipose tissue regeneration, which is significantly related to the stiffness. YAP/TAZ shuttle between the cytoplasm and nucleus, and can sense and transduce mechanical cues. We hypothesized that the force in adipose tissue engineering chamber cell increases ECM exudation resulting in increase of adipose tissue stiffness, which promote the proliferation of ASCs. The good adipogenic microenvironment induces ASCs adipogenic differentiation, which decreases the ECM and tissue stiffness resulting in arrest of cell growth. The negative regulation of fat regeneration regulated by ECM stiffness is through the rearrangement of ASCs cytoskeleton, which leads to YAP/TAZ inactivation or degradation, and then fat regeneration arrests. The study demonstrated that the "ECM stiffness - cytoskeleton -YAP/TAZ- cell behaviors" chain of evidence in vitro through softwell culture system, and adipose tissue regeneration is inhibited by knockout of Yap gene in the EPAT model, which provides theoretical evidence for negative regulation of adipose tissue regeneration.

脂肪组织工程室技术可诱导脂肪组织的再生，但其一直面临对其增长的不可控性及脂肪瘤变性的质疑。本课题组的前期研究发现在脂肪组织再生过程中，细胞不会无限制增殖，且细胞的增殖分化与细胞接触的刚度呈显著相关。YAP/TAZ穿梭于细胞质和细胞核，能感受和传递刚度变化信号。我们猜测组织工程小室内应力改变使脂肪组织细胞外基质渗出增多，从而导致脂肪组织刚度增加促进ASCs增殖，脂肪瓣中良好的成脂微环境诱发增殖的ASCs成脂分化，细胞外基质减少、组织刚度降低，细胞生长停止。这种细胞外基质刚度降低对脂肪再生的负性调控，是通过ASCs细胞骨架的重排，进而导致YAP/TAZ失活或降解，脂肪再生停止。本研究通过采用softwell培养系统在体外证实“ECM刚度-细胞骨架-YAP/TAZ-细胞学行为”证据链条，并通过EPAT模型将Yap基因敲除后脂肪组织再生是否受到抑制，为脂肪再生过程中的负性调控提供理论证据。

课题组致力于探索脂肪组织原位再生的组织工程诱导，并探讨了力学诱导脂肪再生的作用机制，为实现缺损部位的软组织再生诱导提出了临床解决路径。为此，我们围绕力学刺激这一始动因素展开探索，聚焦细胞质骨架对力学信号的传导作用，结果证实：脂肪瓣在应力作用下将发生应变，应变通过激活“胞外基质（Extracellular matrix，ECM）-整合素β1 （Integrin β1）-细胞质骨架（F-actin）”轴促进脂肪来源间充质干细胞（adipose-derived stem cells, ASCs）增殖。应变激活“ECM-Integrin β1-F-actin-Lamin A/C”力学信号传导通路，下调Lamin A/C的表达并暴露与之结合的DNA motif片段，继而活化增殖相关转录因子并启动基因转录，从而促进ASCs增殖。本研究采用ChIP-seq、生物信息学分析、Protein/DNA芯片等筛选和验证Lamin A/C所结合的motif片段及所对应的增殖TF，并构建Lamin A/C过表达动物模型，验证Lamin A/C在应变调控脂肪组织再生中的作用及机制，为大体积工程化脂肪组织的临床应用提供理论依据。