SHPT患者血清对内皮细胞凋亡的影响及klotho蛋白的保护作用  

Effects of hyperparathyroidism patients＇ serum and klotho protein on the apoptosis of human umbilical vein endothelial cells

作　　者：陈铖[1] 毛慧娟[1] 孙彬[1] 俞香宝[1] 曾鸣[1] 王宁宁[1] 张波[1] 刘佳[1] 赵秀芬[1] 钱军[1] 邢昌赢[1] 

机构地区：[1]南京医科大学第一附属医院肾内科,210029

出　　处：《中华肾脏病杂志》2014年第11期856-862,共7页Chinese Journal of Nephrology

基　　金：“十二五”国家科技支撑计划项目（2011BAI10B08）;江苏省卫生厅医学科研基金项目（Z201002）

摘　　要：目的 观察继发性甲状旁腺功能亢进症（SHPT）患者血清对人脐静脉内皮细胞（HUVEC）凋亡率、半胱氨酸天冬氨酸蛋白酶3（Caspase-3）活性的影响,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CKD5期患者、15例严重SHPT拟手术治疗患者的混合血清.体外培养HUVEC,分别以健康人血清（血清H）为正常对照,观察SHPT患者血清（血清S）、不伴SHPT的CKD5期患者血清（血清C）孵育HUVEC 24h后,以流式细胞术检测其对细胞凋亡率的影响.以不同浓度klotho蛋白干预24 h,检测HUVEC凋亡率,加或不加LY294002（PI3K/AKT特异性抑制剂）,检测总AKT（t-AKT）、磷酸化AKT（p-AKT）的表达（Western印迹法）及Caspase-3活性（分光光度法）.结果 血清C、血清S均诱导细胞凋亡,且血清S诱导细胞凋亡作用较血清C显著（P＜0.05）.50～ 100 μg/L klotho蛋白可部分抑制10％ SHPT血清作用下HUVEC的凋亡（P＜0.05）,并上调p-AKT表达、抑制Caspase-3活性,且可被LY294002阻断.结论 与健康人血清相比,SHPT血清、不伴SHPT的CKD5期患者血清均可诱导内皮细胞凋亡,且SHPT血清诱导细胞凋亡的作用更为显著.klotho蛋白可部分拮抗SHPT血清所致HUVEC凋亡的作用.其抗凋亡的机制可能与上调p-AKT、抑制Caspase-3活性有关.Objective To investigate the effects and underlying mechanism of secondary hyperparathyroidism （SHPT） patients＇ serum and klotho protein on the apoptosis of human umbilical vein endothelial cells （HUVECs）.Methods Three types of mixed serum from 15 patients with SHPT （serum S）,10 CKD stage 5 patients without SHPT （serum C） and 15 healthy volunteers （serum H） were collected.HUVECs were incubated with 10％ serum H,10％ serum C,10％ serum S and 10％ serum S plus klotho respectively.The apoptosis rate of endothelial cells was evaluated by flow cytometry.The activity of Caspase-3 was measured by spectrophotometry.The levels of AKT and phosphorylated forms of AKT （p-AKT） were detected by Western blotting （with or without PI3K/AKT inhibitor LY294002）.Results The apoptosis of HUVECs was both induced by the serum S and serum C.The apoptosis rate was greater in serum S group than that in serum C group （P ＜ 0.05）.The apoptosis was partly inhibited when klotho protein （50-100 μg/L） was added （P ＜ 0.05）,accompanying the up-regulation of p-AKT.The above effects could be blocked by LY294002.The activity of Caspase-3 was up-regulated in SHPT group compared to healthy control group （P ＜ 0.05） and the up-regulation could also be inhibited by klotho protein （P＜ 0.05）.Conclusions The apoptosis of HUVECs is induced by the serum from CKD stage 5 patients without SHPT and SHPT patients.Klotho protein can protect the HUVECs from apoptosis by up-regulating p-AKT and inhibiting Caspase-3.

关 键 词：甲状旁腺功能亢进症 继发性 细胞凋亡 KLOTHO蛋白 

分 类 号：R582.1[医药卫生—内分泌] R692[医药卫生—内科学]