继发性甲状旁腺功能亢进患者血清对内皮细胞的影响及Klotho蛋白的保护作用

Effect of serum from secondary hyperparathyroidism patients on the endothelial cells,and the intervention of Klotho

目的：观察继发性甲状旁腺功能亢进（SHPT）患者血清对人脐静脉内皮细胞（HUVEC）增生、凋亡、一氧化氮（NO）合成的影响,并探讨Klotho蛋白的保护作用及机制. 方法：分别收集15例健康人、10例不伴SHPT的CKD 5期患者、15例SHPT拟手术治疗的患者混合血清,体外培养HUVEC.以健康人血清为正常对照,观察SHPT患者血清、不伴SHPT的CKD5期患者血清处理细胞24h后对细胞增生的影响（CCK-8）,观察不同浓度、不同时间SHPT血清对HUVEC增生的影响.在10％ SHPT血清环境下,以不同浓度Klotho蛋白干预24h,检测HUVEC增生、凋亡（流式细胞术）、NO合成（硝酸盐还原法）.加/不加PD98059[细胞外调节蛋白激酶（ERK） 1/2抑制剂],检测总ERK（t-ERK）、磷酸化ERK（p-ERK）（Western印迹法检测）. 结果：不伴SHPT的CKD 5期患者血清和SHPT患者血清均可抑制细胞增生,且SHPT血清对于细胞增生的抑制作用较不伴SHPT的CKD5期患者血清显著（P＜0.05）.在一定浓度范围内（5％～20％）,随着SHPT血清浓度增加,细胞增生受抑制（P＜0.05）,呈现浓度依赖性.10％ SHPT血清处理细胞6h、12h、24h,随着处理时间延长,细胞增生受抑制（P＜0.05）,呈现时间依赖性.50～ 100 ng/ml Klotho蛋白干预可部分恢复10％ SHPT血清处理后HUVEC的增生活力（P＜0.05）、抑制其凋亡,并上调p-ERK表达,且可被PD98059阻断.SHPT血清作用下,HUVEC中NO合成减少（P＜0.05）,Klotho蛋白干预可促进NO合成（P＜0.05）. 结论：SHPT血清抑制HUVEC增生和NO合成.Klotho蛋白可部分拮抗SHPT血清对HUVEC增生的抑制作用,并促进NO合成,其促进HUVEC增生的机制可能与其抗凋亡作用及上调p-ERK有关.

Objective：To investigate the effect of secondary hyperparathyroidism （SHPT） patients＇ serum on the endothelial cells and to explore the protection of Klotho and its possible mechanisms in this process.Methodology：Three types of mixed serum from fifteen patients with SHPT scheduled for parathyroidectomy,10 CKD patients at stage 5 without SHPT and 15 healthy volunteers were collected.Human umbilical vein endothelial cells （HUVECs） were incubated in vitro with SHPT serum or serum of stage 5 CKD patients without SHPT or healthy serum as control.First,the effects of the above three types serum on the proliferation of HUVEC after 24h treatment （CCK-8） were compared.Second,HUVECs were divided into 3 groups：control group （10％ healthy serum medium）,SHPT group and Klotho treatment group （SHPT serum and Klotho）.The proliferation and apoptosis of endothelial cells were evaluated respectively by CCK-8 and Flow Cytometry.The synthesis of NO was measured by nitrate reduction method.The levels of extracellular signal-regulated kinase （ERK1/ 2） and phosphorylated forms of ERK1/2 （p-ERK1/2） were detected by Western blotting （with or without ERK inhibitor PD98059）.Results：The proliferation of HUVEC were both inhibited by the serum of SHPT patients and CKD-5 patients without SHPT,and the inhibition of SHPT serum was greater than that of the other （P ＜ 0.05）.With the increase of concentration in a certain range （5％ ～20％）,the proliferation of HUVEC was inhibited by SHPT serum in a concentrationdependent manner （P ＜ 0.05）.Compared with HUVEC incubated with SHPT serum for 6h,12h,the proliferation of HUVEC was significantly decreased when incubated for 24h （P ＜ 0.05）.The proliferation was partly restored and the apoptosis was inhibited when added 50 ng/ml ～ 100 ng/ml of Klotho into 10％ SHPT serum （P ＜ 0.05）.In the meantime,p-ERK was also upregulated and could be inhibited by PD98059.The synthesis of NO was decreased in SHPT group （P ＜0.05） and increased in the treatment of Klotho （P ＜ 0.05）.Conclusion：The proliferation of HUVEC was inhibited by the serum of SHPT patients,which could be partly antagonized by Klotho.The NO synthesis of the endothelial cells in the SHPT serum was also increased by klotho.The promotion of HUVEC proliferation by Klotho might be due to the inhibition of HUVEC apoptosis and upregulation of p-ERK.