基于容积敏感型氯通道研究糖尿病难愈创面表皮干细胞迁移的影响机制

The migration of epidermal stem cells (ESC) to participate in the epithelialization is the foundation of wound healing. The chronic diabetic wound non-healing is related to ESC's function impairment closely. The chloride channel regulates cell's volume changing and maintenance, and plays an important role in cell movement as well. In preliminary study of this project, we found that the migration abilities of Diabetes' ESC diminished, and epidermal cells cultured in high glucose medium showed decreased chloride channels' expression and activity, which prompted that the dysfunction of chloride channels that regulate cell's migration may be the important pathological basis of chronic diabetic non-healing wound formation. This project is based on the biological characteristics of the volume-sensitive chloride channels, using a major structural protein of the channel CLC-3 and regulating protein CLC-2 as target genes, combined with chloride channel blockers and activator, to study the effects of dysfunction of Diabetes' ESC volume-sensitive chloride channels on cell migration and wound healing, as well as the causes in the pathogenesis of chronic wounds. It can clarify that the enhancement of chloride channels' activity promotes the chronic wound healing, and to investigate the mechanisms of chloride channel dysfunction formation in diabetic state and the signal pathway of regulating ESC migration. It will reveal the principle of ion channels regulating the migration of stem cells to participate wound healing process. As an aim we can explore the new therapy of chronic non-healing wounds and strategies to promote tissue repair and regeneration.

表皮干细胞(ESC)向创面迁移参与上皮化进程是创面愈合的基础，糖尿病慢性创面不愈与ESC功能受损密切相关；细胞容积的改变和维持受氯离子通道的调节，并在细胞的迁移运动中起着重要的作用。本项目前期研究发现糖尿病ESC迁移能力减弱，高糖培养的表皮细胞氯通道蛋白的表达与活性下降，提示调节细胞迁移运动的氯通道异常可能是糖尿病慢性创面形成的重要病理基础。本项目基于容积敏感型氯通道的生物学特性，以该通道主要的结构蛋白CLC-3和调节蛋白CLC-2为目标基因，结合氯通道阻断和激活药物，研究糖尿病ESC容积敏感性氯通道的功能改变对细胞迁移和创面愈合的影响以及在慢性创面发病中的成因，明确提高氯通道活性对慢性创面愈合的促进作用，探讨其在糖尿病状态下功能异常的形成机制及对ESC迁移调控的信号作用途径，揭示离子通道调节干细胞迁移参与创面愈合的机理，以此为目标探索治疗慢性难愈性创面、促进组织修复和再生新的策略。

本课题基于容积敏感型氯通道对细胞迁移运动的调控作用，在糖尿病大鼠皮肤创面细胞和动物模型基础上，研究该通道最主要的结构蛋白CLC-3和主要的调节蛋白CLC-2对创缘表皮干细胞迁移的影响以及对糖尿病创面愈合的促进作用；结合应用特异性信号分子阻断剂，检测氯通道改变情况下体外细胞和在体组织内相关信号的表达变化，探讨糖尿病状态造成氯通道功能异常的形成机理，并揭示氯通道调控表皮干细胞迁移的具体分子机制。研究结果发现，糖尿病大鼠表皮干细胞 CLC-2通道的表达下降，同时细胞的容积敏感型氯电流及迁移能力受到抑制，三者之间可能存在相关性；慢病毒介导的 CLC-2 m RNA 对应的 shRNA 可以有效抑制 CLC-2 通道的表达，成功模拟了糖尿病状态下氯通道蛋白表达下降、容积敏感型氯电流及细胞体外迁移能力受抑制的情况，说明 ESCs 的氯通道活性与细胞容积敏感性氯电流及迁移能力有关；糖尿病状态通过抑制 PI3K 的磷酸化而抑制 CLC-2的表达，是ESC迁移能力减弱、创面愈合延迟的可能机制。氯通道CLC-3在ESC细胞上表达，抑制或激活CLC-3功能可改变细胞的迁移活性，从而影响其参与创面愈合和组织再生的作用，进而说明慢性创面难愈的机制可能与CLC-3氯通道活性下降有关。预期的研究结果可丰富趋化因子作用干细胞及离子通道调控干细胞迁移参与创面愈合的研究内容，为治疗慢性难愈创面提供新的策略和理论依据。