SDF-1/CXCR4生物轴对钙敏感受体介导的大鼠游离皮瓣缺血/再灌注损伤通路的拮抗作用及机制的研究

Partial or total necrosis of free flaps after ischemia/reperfusion injury will severely affect the quality of life in patients. However, the exact mechanism of ischemia/reperfusion injury has long been unclear. Furthermore, the reciprocal effect of stromal-cell derived factor and calcium-sensing receptor in ischemia/reperfusion injury of free flaps belongs to the “New World” in research field. This project has successfully proved the vascular endothelial cells expressing calcium-sensing receptor, and a purified cell culture model of vascular endothelial cells. Then, a combination method of multi-levels and multi-means, for example, high performance liquid chromatography, laser scanning confocal microscopy, transmission electron microscopy, patch clamp, etc. was used to detect four important aspects in the function and mechanisms of calcium-sensing receptor in ischemia/reperfusion injury of free flaps. Firstly, this study will detect the physiological role of calcium-sensing receptor of vascular endothelial cells in rat free flaps model. Secondly, this project will observe the expression impact of ischemia/reperfusion on calcium-sensing receptor of free flaps. Thirdly, this experiment will reveal the irreversible damage mechanism caused by the calcium-sensing receptor activation to tissue cells of free flaps. Lastly, this work to explore the protecting mechanism of cells free to programmed cell death through stromal-cell derived factor to reverse the effect of calcium-sensing receptor pathway. And finally, this project hope to test and verify our hypothesis, “the protective effect caused by SDF-1/CXCR4 biological axis to reverse the effect of calcium-sensing receptor in ischemia/reperfusion injury pathway”, and to provide a new idea, new target and new technology platform to prevent ischemia/reperfusion injury of free flaps.

游离皮瓣缺血/再灌注损伤后发生部分或全部坏死将严重影响患者的生活质量。但是其缺血/再灌注损伤的确切机制并不明确。另外SDF-1与钙敏感受体在游离皮瓣缺血/再灌注损伤过程中的交互作用，属于科研领域的"荒芜地"。本课题在成功验证游离皮瓣血管内皮细胞表达钙敏感受体及纯化培养血管内皮细胞的基础上，采用多层次（器官、细胞、分子）和多手段（高效液相色谱仪、激光扫描共聚焦显微镜、透射电镜、膜片钳仪等)相结合的方法，检测大鼠游离皮瓣血管内皮细胞钙敏感受体的生理作用；观察缺血/再灌注对钙敏感受体表达的影响；揭示钙敏感受体激活引起组织细胞发生不可逆性损伤的机制；并探讨SDF-1拮抗钙敏感受体通路,保护细胞免于发生凋亡的机制。最终多角度验证我们提出的“SDF-1/CXCR4生物轴对钙敏感受体介导的游离皮瓣缺血/再灌注损伤通路的拮抗作用”观点，为游离皮瓣缺血/再灌注损伤的防治提供一个新思路、新靶点和新的技术平台。

游离皮瓣缺血再灌注损伤后发生部分或全部坏死将严重影响患者的生活质量。但是游离皮瓣缺血再灌注损伤的确切机制并不明确。另外基质细胞衍生因子（SDF）-1α与钙敏感受体（CaSR）在游离皮瓣缺血再灌注损伤过程中的交互作用亦亟待阐明。本课题组在成功验证游离皮瓣血管内皮细胞表达CaSR及纯化培养血管内皮细胞的基础上，成功地构建了以右腹壁下浅动静脉为血管蒂的改良型大鼠游离皮瓣缺血再灌注损伤模型；检测了缺血再灌注对CaSR及SDF-1α受体CXCR4表达的影响；并通过分子生物学方法检测了CaSR及SDF-1α/CXCR4生物轴对凋亡通路相关蛋白的调控机制。研究结果表明：CaSR在缺血再灌注游离皮瓣的血管内皮细胞表面广泛表达，其表达水平与游离皮瓣缺血再灌注损伤程度密切相关，即CaSR高表达水平表明游离皮瓣发生严重缺血再灌注损伤，而CaSR低表达水平表明游离皮瓣遭遇轻度缺血再灌注损伤。游离皮瓣重度损伤，与之相对应的是皮瓣坏死面积大；TUNEL染色检测的凋亡细胞多；有较少的微血管密度；免疫组化染色检测显示较多的促凋亡蛋白如Caspase-3表达，蛋白质印迹法检测显示促凋亡蛋白如Caspase-9等的高表达，以及qPCR检测显示CaSR mRNA高表达；而抑制凋亡蛋白如Bcl-2的低表达。缺血再灌注能够诱导CaSR激活，CaSR激活能呈现“瀑布效应”加重上述的游离皮瓣缺血再灌注损伤；SDF-1α/CXCR4生物轴能够通过拮抗CaSR激活减轻游离皮瓣缺血再灌注损伤，其机制可能与抑制凋亡的发生发展以及促进新生血管形成有关。结论：本课题组首次证实CaSR在游离皮瓣血管内皮细胞中广泛表达，其表达水平和游离皮瓣缺血再灌注损伤特别是早期缺血再灌注损伤程度密切相关；SDF-1α/CXCR4生物轴的激活可以通过增加血管新生和抑制细胞凋亡来减轻CaSR激活介导的游离皮瓣缺血再灌注损伤；因此CaSR可能成为防止游离皮瓣缺血再灌注损伤的治疗新靶点，并且不含Ca2+的灌注液将成为游离皮瓣缺血再灌注时的必要条件。