甲状旁腺嗜酸细胞参与尿毒症患者维生素D治疗抵抗的机制研究

Active vitamin D (1,25(OH)2D3) is the main treatment of secondary hyperparathyroidism (SHPT) in patients with renal disease, but the resistance to 1,25(OH)2D3 treatment is usually occurred in patients with end stage renal disease and ultimately lead to the failure of medical treatment. The underlying mechanisms have not been elucidated. Clinical studies showed that the percentage of oxyphil cells in parathyroid glands was significantly increased in SHPT patients compared to normal people. The relationship between raising proportion of oxyphil cells and resistance to vitamin D treatment was also established in uremic patients. In our previous study, vitamin D binding protein (DBP) , which is an important protein responsible for the transport of vitamin D in the circulation, was evidenced to be intracellular synthesized and may be associated with the transfer of vitamin D receptors (VDR) in the parathyroid cells. Moreover, the expression of DBP was significantly reduced in oxyphil cells compared to chief cells in the parathyroid glands from uremic patients. Therefore, we hypothesized that the intracellular synthesis of DBP in parathyroid cells might mediate nuclear transferring of 1,25(OH)2D3-VDR complex and participate in the binding of VDR complex to the VDRE in the promoter of target genes, thereby affecting the genomic regulation effects of vitamin D3. In current study, we plan to further investigate the association between parathyroid oxyphil cells proliferation and vitamin D3 treatment failure in uremic SHPT patients through a cohort study, and to verify whether the lack of DBP in the oxyphil cells is the decisive mechanism leading to the vitamin D3 resistance in both in vitro study of parathyroid oxyphil cells and in vivo study of animal model with SHPT.

活性维生素D（1,25(OH)2D3）是治疗继发性甲状旁腺功能亢进（SHPT）的主要方法，然而在终末期肾病患者中常出现维生素D治疗抵抗，导致内科治疗失败，其发生机制尚未阐明。临床研究发现SHPT患者甲状旁腺结节中嗜酸细胞比例明显升高，且与活性维生素D应用显著相关。本课题组前期工作证实甲状旁腺细胞可以合成维生素D结合蛋白（DBP），并可能影响维生素D受体（VDR）向核内转运，但嗜酸细胞中DBP的表达较主细胞显著减少，故我们推测：甲状旁腺局部合成的DBP有可能影响了1,25(OH)2D3-VDR复合物向核内的移位，参与VDR复合物与靶基因VDRE的结合，进而影响活性维生素D的基因调控功能。本研究拟通过SHPT患者队列研究明确尿毒症患者甲状旁腺嗜酸细胞异常增生是否导致维生素D治疗失败，通过甲状旁腺体外细胞研究和动物体内干预研究验证嗜酸细胞内DBP缺乏是否是导致活性维生素D作用抵抗的重要机制。

为了阐明甲状旁腺嗜酸细胞在尿毒症患者活性维生素D治疗抵抗中的作用，本课题组开展了如下工作：（1）通过SHPT患者队列研究明确尿毒症患者甲状旁腺嗜酸细胞增生与活性维生素D治疗抵抗相关；（2）通过蛋白组学质谱分析明确主细胞和嗜酸细胞结节蛋白表达差异，锁定DBP为目标蛋白；（3）通过免疫检测进一步证实主细胞和嗜酸细胞中维生素D作用通路的表达差异，嗜酸细胞DBP表达下调，且DBP与VDR存在蛋白质相互绑定作用；（4）通过给予SHPT患者甲状旁腺原代细胞活性维生素D或DBP干扰质粒体外干预，明确甲状旁腺细胞DBP缺乏可以抑制VDR向细胞核内的移位，使活性维生素D对VDR的激动作用减弱，降低VDR对PTH合成和细胞增生的抑制作用；（5）成功建立患者甲状旁腺组织移植裸鼠方法，计划一年内完成体内干预性研究。本研究从临床实践出发，以活性维生素D治疗抵抗的尿毒症患者甲状旁腺嗜酸细胞增生为切入点，揭示了嗜酸细胞DBP缺乏导致VDR向细胞核移位异常是引起活性维生素D治疗抵抗的重要机制。该理论不仅为临床防治尿毒症甲旁亢提供了新靶点，也为完善维生素D作用的分子机制提供新思路。研究成果已在国内核心期刊发表论文3篇，国际杂志2篇（SCI收录）。在项目实施过程中主要培养3名科研型博士生和2名科研型硕士生，另有2名科研型博士在读。该项目基本完成计划书中的考核目标。