甲状腺术后继发性低血钙导致甲状旁腺的“二次损伤”研究

Hypoparathyroidism is the most common complications of thyroid operation due to parathyroid injury, characterized by hypocalcemia, and has serious impact on patients’ postoperative recovery and the quality of life. It is of great importance to protect and promote the recovery of the injured parathyroid glands, so we established an novel model of parathyroid injury in rabbit, and found that cell apoptosis is the dominating pathological change and p38α was over-expressed. Both cell apoptosis and p38α can partly be inhibited by calcium supplement. Our previous findings indicate that calcium and p38α pathway play an important role in the process of parathyroid injury. In this project, we establish an in vitro model of parathyroid cell injury induced by hypoxia with low calcium in culture system. Calcium and specific inhibitor of p38MAPK are used as experimental intervention. The relationship between calcium and PTH secretion under condition of parathyroid injury will be studied in vivo and in vitro. p38α signal pathway, including death receptor pathway and mitochondrial pathway will be investigated. Western blotting, qPCR, flow cytometry and confocal microscope are used. Our study will help to understand the Calcium - PTH secretion regulation relationship under condition of injury, to validate the secondary injury hypothesis. This study will reveal the mechanism of parathyroid cell apoptosis, providing experimental theory for calcium supplement to protect and promote the recovery of parathyroid function following thyroid surgery, and inhibition of the p38α pathway can attenuate parathyroid cell apoptosis, providing new clues for further study.

甲状旁腺损伤导致的甲状旁腺功能低下是甲状腺手术最常见的并发症，表现为低钙血症，严重影响患者的术后康复和生活质量。如何保护和促进受损甲状旁腺功能恢复具有重要意义，于是我们建立了兔甲状腺术后甲状旁腺损伤动物模型，发现细胞凋亡是甲状旁腺损伤的主要病理改变，并有p38α蛋白的高表达，补钙治疗可以减轻细胞凋亡并抑制p38α的表达。本课题将在前期研究基础上，同时建立低氧低钙培养诱导甲状旁腺细胞损伤的细胞模型，利用钙和p38MAPK抑制剂进行实验干预，在体内外甲状旁腺损伤模型中，研究甲状旁腺损伤时的细胞PTH分泌功能，验证并阐述甲状旁腺损伤时继发性低血钙导致甲状旁腺的“二次损伤”假设，从基因、蛋白及细胞水平，系统检测死亡受体及线粒体凋亡信号通路，研究补钙治疗促进甲状旁腺功能恢复的分子机制，为甲状腺术后补钙治疗保护甲状旁腺提供实验理论依据，也为进一步研究p38α信号通路阻断保护甲状旁腺提供新的线索。

甲状旁腺损伤导致的甲状旁腺功能减退（甲旁减）是甲状腺手术最常见的并发症，表现为低钙血症，严重影响患者的术后康复和生活质量。如何保护和促进受损的甲状旁腺功能恢复具有重要意义。我们对体内外甲状旁腺损伤模型研究发现，细胞凋亡是甲状旁腺损伤的主要病理改变。本实验建立了新西兰大白兔甲状旁腺体内“缺血低钙损伤”模型、体外 “低氧低钙损伤”模型模型，利用钙和p38MAPK抑制剂进行实验干预，研究甲状旁腺损伤时的细胞PTH分泌功能，验证甲状旁腺损伤时继发性低血钙导致甲状旁腺的“二次损伤”假设，从基因、蛋白及细胞水平进行细胞凋亡信号通路检测，发现甲状旁腺损伤时补钙治疗通过 p38 MAPK / p53 途径抑制甲状旁腺细胞凋亡，并通过抑制p38 MAPK的表达抑制MMP降低，对线粒体起保护作用。为甲状腺术后补钙治疗保护甲状旁腺提供实验理论依据，也为进一步研究p38α信号通路阻断保护甲状旁腺提供新的线索。同时，本项目对甲状旁腺损伤时CaSR活化介导甲状旁腺细胞凋亡的机制进行了研究，发现甲状旁腺损伤时CaSR表达增加，在体内外甲状旁腺损伤模型中从正反两方面利用CaSR激动剂和抑制剂对CaSR活性进行干预，发现甲状腺旁腺损伤时CaSR活化导致细胞凋亡增加，而CaSR活性抑制时细胞凋亡减少。体外研究发现，甲状旁腺损伤时CaSR表上调，导致细胞凋亡增加，细胞周期被抑制，且细胞内游离Ca2+的水平增加，线粒体膜电位下降，线粒体ROS产生增多，并且上调线粒体凋亡途相关凋亡蛋白Cytc C、 C-Casepase-3和C-Casepase-9的表达。表明甲状旁腺损伤时CaSR活化并通过线粒体途经介导了甲状旁腺细胞的凋亡。为补钙治疗抑制CaSR活化减轻甲状旁腺损伤时低钙血症导致甲状旁腺的“二次损伤”提供了重要的实验理论依据。