RUNX2靶向E-cadherin对ADSCs表皮分化及烧伤创面修复影响及其机制

Plenty of sources of epidermis are critical to promote burn wound healing. ADSCs can differentiate into epidermis, but the efficiency of epidermal differentiation is pretty low. Our previous data indicated that over-expression of RUNX2 in ADSCs can promote epidermal differentiation of ADSCs and enhance the expression of E-cadherin. RUNX2 Knockout by CRISPR-Cas9 system can significantly inhibit epidermal differentiation of ADSCs and E-cadherin expression. By means of software analysis, we found that the promotor of E-cadherin may have potential binding sites for RUNX2. Based on the above data, we made the assumption that RUNX2 improves epidermal differentiation of ADSCs through the enhanced expression of E-Cadherin. So we chose to focus on this pathway for our investigatin, and we intend to clarify the possible mechanisms by which RUNX2 enhances the epidermal differentiation of ADSCs. This study may offer a novel target for effective enhancement in ADSCs differentiation into epidermal cells and improvement of burn wound healing.

寻找充足的表皮来源是促进烧伤创面愈合关键。ADSCs可向表皮分化，但分化效率偏低。申请者前期发现。ADSCs过表达RUNX2，细胞表皮分化能力及E-Cadherin水平显著提高，CRISPR-Cas9基因敲除RUNX2， ADSCs表皮分化能力和E-Cadherin表达下降；ADSCs过表达E-Cadherin，细胞表皮分化能力显著提高。同时，软件分析发现，RUNX2可结合E-cadherin核心启动子。因而提出：RUNX2通过E-Cadherin调控ADSCs表皮分化。 .因此，我们拟应用过表达、基因敲除、点突变和CRISPR-Cas9系统等技术，从细胞、分子和整体水平阐明RUNX2在ADSCs表皮分化中的作用及可能机制，并探讨CRISPR-Cas9系统靶向激活E-Cadherin启动子的可行性及其可能的治疗作用，为提高ADSCs表皮分化效率、促进烧伤创面表皮修复奠定基础。

完整的表皮重建是烧伤创面修复的首要前提，众多研究发现ADSCs细胞具有向表皮细胞分化的潜能，但是，ADSCs表皮分化效率偏低。我们在前期工作基础上，应用过表达、基因敲除、基因突变和CRISPR-Cas9系统等分子生物学技术，以ADSCs向表皮细胞方向分化效率为切入点，分别从整体、细胞、分子和基因水平阐明RUNX2在ADSCs表皮分化及烧伤创面修复中的作用及其机制，并探讨靶向激活E-Cadherin启动子促进ADSCs表皮分化的可行性及潜在治疗作用。结果发现1.人脂肪组织可成功获得脂肪干细胞株 ADSCs；2. ADSCs 向表皮诱导后，RUNX2、E-cadherin、CK18、 ZO-1的mRNA 及蛋白的表达量显著提高；3.过表达RUNX2慢病毒转染后的ADSCs以上指标均显著高于单纯诱导组和阴性对照组；干扰RUNX2慢病毒转染后的ADSCs以上指标均低于单纯诱导组和阴性对照组；过表达RUNX2及干扰E-cadherin共转染的ADSCs CK18和ZO-1表达水平均低于单纯诱导组；干扰RUNX2及过表达E-cadherin共转染的ADSCs CK18和ZO-1表达水平均高于单纯诱导组；4.CHIP及双荧光素酶报告基因证明RUN2和E-cadherin处于同一条基因链的上下游；5注射过表达RUNX2修饰过后的ADSCs的裸鼠烧伤创面愈合水平明显优于普通ADSCs组和空载体病毒转染的ADSCs组，干扰RUNX2修饰的ADSCs组的创面愈合水平明显劣于普通ADSCs组和空载体ADSCs组。通过研课题我们证实1. 成人脂肪可成功提取人 ADSCs，并可在体外向表皮细胞诱导分化；2.RUNX2与E-cadherin协同促进ADSCs表皮分化；3.RUNX2与E-cadherin位于同一条基因链的上下游，RUNX2靶向正向调控E-cadherin促进ADSCs表皮分化；4.RUNX2和E-cadherin在ADSCs的表达水平与烧伤创面的愈合速度成正比。本课题证实了RUNX2可以直接与E-cadherin启动子结合，提高 E-cadherin转录活性，同时证实RUNX2通过提高ADSCs中E-cadherin表达，促进ADSCs表皮分化及烧伤创面愈合；结果为提高ADSCs表皮分化的效率提供干预靶点，并为探寻有效的的烧伤创面治疗方法提供新的理论依据。