CAJ | 학술논문

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.
목적 관찰계발성갑상방선공능항진증(SHPT)환자혈청대인제정맥내피세포(HUVEC)조망솔、반광안산천동안산단백매3(Caspase-3)활성적영향,탐토klotho단백적보호작용급궤제.방법 분별수집15례건강인、10례불반SHPT적CKD5기환자、15례엄중SHPT의수술치료환자적혼합혈청.체외배양HUVEC,분별이건강인혈청(혈청H)위정상대조,관찰SHPT환자혈청(혈청S)、불반SHPT적CKD5기환자혈청(혈청C)부육HUVEC 24h후,이류식세포술검측기대세포조망솔적영향.이불동농도klotho단백간예24 h,검측HUVEC조망솔,가혹불가LY294002(PI3K/AKT특이성억제제),검측총AKT(t-AKT)、린산화AKT(p-AKT)적표체(Western인적법)급Caspase-3활성(분광광도법).결과 혈청C、혈청S균유도세포조망,차혈청S유도세포조망작용교혈청C현저(P＜0.05).50～ 100 μg/L klotho단백가부분억제10％ SHPT혈청작용하HUVEC적조망(P＜0.05),병상조p-AKT표체、억제Caspase-3활성,차가피LY294002조단.결론 여건강인혈청상비,SHPT혈청、불반SHPT적CKD5기환자혈청균가유도내피세포조망,차SHPT혈청유도세포조망적작용경위현저.klotho단백가부분길항SHPT혈청소치HUVEC조망적작용.기항조망적궤제가능여상조p-AKT、억제Caspase-3활성유관.
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.