Kir2.1通道在剪切应力促EPCs向内皮细胞分化中的门控效应研究

Vascular endothelial cell injury has been implicated in the onset of atherosclerosis. A number of previous studies have demonstrated that endothelial progenitor cells (EPCs) play important roles in maintaining of vascular integrity. The evidence has revealed shear stress is a key regulator for EPC differentiation, which contribute to vascular repair. However, the detailed events about the regulation of the shear stress-induced EPC differentiation, in particular the mechanisms of mechanotransduction, remain to be identified. Because of their special localization, ion channels are therefore ideally situated to convert mechanical forces into electrical or intracellular biochemical signals. In particular, Kir2.1 channel has been explored in terms of their ability to function as endothelial mechanotransducer. Moreover, recent study has been shown that EPCs functionally express Kir2.1 channel which may modulate the differentiation of EPCs. Therefore, it would be interesting to establish the role of Kir2.1 channel in shear stress-induced EPC differentiation. Herein, using a variety of experimental tools, such as the patch-clamp techniques, RNA interference and EMSA, we try to elucidate: ① whether Kir2.1 channel is activated by shear stress in EPCs; ② if so, how shear stress regulates EPC differentiation via Kir2.1-associated signal pathways; ③ how Kir2.1 channel, which is activated by shear stress, contribute to the nuclear events that lead to EPC differentiation toward endothelial cells. These studies would have a high probability of revealing the mechanism of vascular endothelial repair and providing new directions that lead to the development of clinical strategies toward the prevention and/or treatment of atherosclerosis.

内皮祖细胞（EPCs）分化为成熟内皮细胞是血管损伤后再内皮化的有效途径。国外资料和我们前期研究均发现：剪切应力可促进EPCs向内皮细胞分化。然而其核心问题-EPCs如何感知胞外力学信号并将其转化为胞内生物化学信号所知甚少。最新研究表明，EPCs细胞膜上分布有Kir2.1通道，且其表达量与EPCs分化程度相关。鉴于Kir2.1所处位置的特殊性，本课题推测"Kir2.1可能是剪切应力促EPCs分化的'力感受装置'"。据此，我们将利用膜片钳、RNA干扰等技术探讨：①EPCs上Kir2.1的力学敏感性；②Kir2.1及激活后引发的胞内信号对剪切应力促EPCs分化的影响；③Kir2.1介导的剪切应力促EPCs分化的核内调控机制。从而揭示剪切应力促EPCs向内皮细胞分化的离子通道跨膜信号转导网络，完善受损血管内皮修复的力学、生物学机制，为AS等心血管疾病的发病机理及剪切应力介导的细胞疗法提供理论依据。

内皮损伤是动脉粥样硬化（AS）的始动因素， 内皮祖细胞（EPCs）分化为成熟内皮细胞是血管损伤后再内皮化的有效途径。前期研究发现：剪切应力可促进EPCs向内皮细胞分化。然而其核心问题——EPCs如何感知胞外力学信号并将其转化为胞内生物化学信号所知甚少。有文献指出，EPCs细胞膜上分布有Kir2.1通道，且其表达量与EPCs分化相关。据此，我们以EPCs的分化为突破点，运用膜片钳、siRNA及细胞移植等方法和技术，阐明剪切应力对Kir2.1通道的影响及该通道在剪切应力促EPCs分化中的作用。结果显示：(1) EPCs上记录到的内向电流具有整流特性，可被100 μmol/L Ba2+阻断，也可被10 μmol/L ML133(特异性Kir2.1 离子通道阻断剂)阻断；(2) ML133 可促进EPCs的分化，增强EPCs粘附、迁移及成血管能力，加速EPCs对受损血管内皮的修复，减缓新生内膜的形成，其机制可能与ML133促进EPCs 的自噬有关；(3) 随着EPCs的分化，Kir2.1基因及蛋白表达逐步降低；(4) 剪切应力明显增大EPCs及表达有Kir2.1的HEK293细胞的内向整流K+电流；(5)干预Kir2.1的活性或表达可调控剪切应力促EPCs向内皮细胞分化的效应；(6) 剪切应力下调Kir2.1表达，细胞骨架、P38、ERK1/2等均不同程度地影响了剪切应力调节的Kir2.1表达；(7) 剪切应力升高EPCs内Ca2+浓度，Bacl2明显抑制该效应；(8) ML133、Kir2.1 siRNA均不同程度抑制了剪切应力导致的Akt、p53及p21的活化；(9) Akt抑制剂(tricribine)、p53抑制剂(PFT-α)及p21 siRNA均不同程度抑制了剪切应力导致的EPCs内皮分化；(10) 剪切应力促进核转录因子KLF2表达，增加KLF2与DNA的结合；(11) 过表达Kir2.1通道可抑制剪切应力促EPCs中KLF2基因及蛋白的表达，而Kir2.1 siRNA则促进剪切应力促EPCs中KLF2基因及蛋白的表达。以上结果，进一步揭示剪切应力促EPCs向内皮细胞分化的离子通道跨膜信号转导网络，完善了受损血管内皮修复的力学、生物学机制，为AS等心血管疾的发病机理及剪切介导的细胞疗法提供理论依据。