尿毒症血清诱导的磷酸钙晶体对人主动脉平滑肌细胞的钙化作用

目的 探讨尿毒症患者血清诱导的磷酸钙晶体对人主动脉平滑肌细胞(HASMCs)钙化的影响.方法 尿毒症患者血清在37℃下孵育3d,超速离心法从尿毒症血清中分离磷酸钙晶体和无晶体血清,采用扫描电子显微镜和能量色散X射线光谱仪分析晶体的形态及化学特征.体外培养HASMCs,分为以下4组:对照组、尿毒症血清组、磷酸钙晶体组和无晶体血清组.茜素红染色及甲氧酚酞络合酮法检测HASMCs钙化结节的形成及细胞内钙含量.实时荧光定量PCR法检测骨形态发生蛋白2(BMP-2)、骨桥蛋白(OPN)、核结合因子α1亚基(Cbfα1)、碱性磷酸酶(ALP)、基质γ羧基谷氨酸蛋白(MGP)的mRNA表达.Cbfα1、OPN和BMP-2的蛋白表达用Westcrn印迹和ELISA法检测.结果 尿毒症血清可诱导磷酸钙晶体形成.与对照组相比,尿毒症血清明显促进HASMCs钙化结节形成,增加细胞内钙含量(P＜0.05),并上调细胞BMP-2、OPN、Cbfα1的mRNA和蛋白表达(均P＜ 0.01)；而磷酸钙晶体亦促进HASMCs钙化结节的形成,增加细胞内钙含量(P＜0.05),上调BMP-2、OPN、Cbfα1的mRNA和蛋白表达(均P＜0.01).无晶体血清组的HASMCs胞内钙含量和上述成骨蛋白的表达与对照组差异无统计学意义(均P＞ 0.05).结论 尿毒症血清可能通过诱导磷酸钙晶体的形成促进血管钙化的发生.

Effect of calcium phosphate crystals induced by uremic serum on calcification of human aortic smooth muscle cells

Objective To investigate the impact of calcium phosphate crystals induced by uremic serum on calcification of human aortic smooth muscle cells (HASMCs). Methods Uremic serum was incubated at 37℃ for 3 days. Calcium phosphate crystals and uremic supernatant were isolated from uremic serum by ultracentrifugation. Scanning electron microscope (SEM) and energy dispersive X - ray spectroscopy (EDX) were performed for analysis of morphological and chemical characteristics of the crystals. HASMCs were treated in vitro with control medium, uremic serum - medium, calcium phosphate crystals - medium and uremic supernatant - medium. Calcification was visualized by Alizarin red staining. Calcium loads in cells were quantified by o - cresolphthalein complexone method. Gene expression of bone morphogenetic protein-2 (BMP-2), osteopontin (OPN) and core-binding factor α1 (Cbfα1), alkaline phosphate (ALP) and matrix gamma carboxyglutamic acid protein (MGP) were quantified by real - time PCR. Cbfα1, OPN and BMP - 2 protein levels were determined by Western blotting or ELISA. Results Calcium phosphate crystals which induced by uremic serum displayed laminated shapes containing crystallized needle- like projections and ranged from 30 - 500 nm, with Ca/P ratios of 1.41±0.05. Compared with the cells in control group, uremic serum induced HASMCs calcification，increased calcium loads (P＜0.05), up-regulated BMP-2, OPN , Cbfα1 mRNA and protein expression (all P＜0.01). Similar to uremic serum, calcium phosphate crystals also induced HASMCs calcification, increased calcium loads (P＜0.05), and up-regulated BMP-2, OPN , Cbfα1 mRNA and protein expression (all P＜0.01). However, there was no significant difference between HASMCs growing in uremic supernatant and control medium in calcium loads or the expression levels of these osteogenic proteins (P＞0.05). Conclusions Calcium phosphate crystals induced by uremic serum promote HASMCs calcification, which might be one of the mechanisms of uremic vascular calcification.