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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CKD 5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CKD 5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 observe the effects of serum of secondary hyperparathyroidism (SHPT) on the proliferation, apoptosis and nitric oxide synthase (HUVEC) in human umbilical vein endothelial cells (HUVECs) and to explore the protective effect and mechanism of Klotho protein. Methods: Fifteen healthy people, 10 CKD stage 5 patients without SHPT, and 15 patients undergoing SHPT surgery were enrolled in this study. HUVECs were cultured in vitro. Normal human serum was used as the control group. The effect of serum-treated cells in CKD stage 5 without SHPT on the proliferation of cells was observed (CCK-8). The effects of SHPT serum at different concentrations and times on the proliferation of HUVEC were observed . Under the condition of 10% SHPT serum, the Klotho protein was treated with Klotho at different concentrations for 24 h. HUVEC proliferation, apoptosis (flow cytometry) and NO synthesis (nitrate reduction) were detected. Total ERK (t-ERK) and phosphorylated ERK (p-ERK) were detected by Western blotting with and without PD98059 [inhibitor of extracellular regulated protein kinase (ERK) 1/2]. Results: The serum of CKD stage 5 patients without SHPT and the serum of SHPT patients all inhibited the cell proliferation. The inhibitory effect of SHPT serum on cell proliferation was significantly higher than that of CKD stage 5 patients without SHPT (P <0.05). In a certain concentration range (5% ~ 20%), with the increase of SHPT serum concentration, cell proliferation was inhibited (P <0.05) in a concentration-dependent manner. Cells treated with 10% SHPT serum for 6h, 12h, 24h, with prolonged treatment, cell proliferation was inhibited (P <0.05), showing a time-dependent. 50-100 ng / ml Klotho could partially restore the proliferation of HUVECs treated with 10% SHPT (P <0.05), inhibit its apoptosis and up-regulate the expression of p-ERK, and could be blocked by PD98059. Under the action of SHPT serum, the NO synthesis in HUVEC decreased (P <0.05), Klotho protein intervention promoted NO synthesis (P <0.05). Conclusion: SHPT serum inhibits HUVEC proliferation and NO synthesis. Klotho protein can partially antagonize the inhibitory effect of SHPT serum on HUVEC proliferation and promote NO synthesis, and its mechanism of promoting HUVEC proliferation may be related to its anti-apoptotic effect and up-regulation of p-ERK.