Sortilin在糖尿病血管钙化演进中的作用机制

Mechanism of Sortilin in the progression of diabetic vascular calcification

目的探究分拣蛋白（Sortilin）在糖尿病血管钙化中的作用机制。方法选取2015年6月至2017年6月在江苏大学附属医院骨科因糖尿病足截肢治疗的患者30例,根据钙定量检测胫前动脉钙化情况并分为对照组（钙定量〈2μmol/mg,n=15）和钙化组（钙定量≥2μmol/mg,n=15）。Western blot测定两组Sortilin水平,并对患者血清学及临床指标进行相关分析。基于人主动脉平滑肌细胞株（HA-VSMC）构建糖尿病血管钙化体外模型,利用茜素红染色检测钙化程度,Western blot检测该模型中Sortilin表达水平。而后用Sortilin及Sortilin中和性抗体处理该钙化模型,用钙盐染色和钙结节长径测定的方法显示Sortilin对于钙化模型的影响。结果钙化组胫前动脉钙化是对照组的4.9倍,同时Sortilin表达量也增加3.92倍。用含有羧甲基赖氨酸（CML）和氧化型低密度脂蛋白（oxLDL）的钙化培养液处理HA-VSMC后,钙化明显增加。在Sortilin重组蛋白刺激下,HA-VSMC钙化程度进一步加重,钙结节长径明显增加,同时组织非特异性碱性磷酸酶（TNAP）表达水平也显著提高。结论 Sortilin促进糖尿病血管钙化,并使其向大钙化发展。

Aim To investigate the mechanism of Sortilin in diabetic vascular calcification. Methods Thirty patients with diabetic foot amputation were enrolled from June 2015 to June 2017. The calcification of anterior tibial artery was measured by calcium quantification and divided into control group（ calcium concentration2 μmol/mg,n = 15）and calcification group（ calcium volume≥2 μmol/mg,n = 15）. Western blot was used to determine the expression of Sortilin in the two groups. And the serological and clinical indexes were also analyzed. The model of diabetic vascular calcification was established based on human aortic smooth muscle cell line（ HA-VSMC）. The degree of calcification was detected by alizarin red staining. The expression level of Sortilin in the model was detected by Western blot. The calcification model was then treated with Sortilin and Sortilin antibody,and the effect of Sortilin on the calcification model was shown by von Kossa staining,calcium quantification and calcium nodule diameter measurement. Results Calcification of the anterior tibial artery in the calcification group was 4.9 times that of the control group,and the expression level of Sortilin increased by 3.92 times. After treatment with calcified culture medium containing Nε-carboxymethyl-lysine（ CML）and oxidized low density lipoprotein（ ox-LDL）,HA-VSMC calcification increased significantly. The calcification of HAVSMC was obviously increased under the stimulation of Sortilin recombinant protein,and the diameter of calcium nodules was also significantly increased. Meanwhile,tissue non-specific alkaline phosphatase（ TNAP） expression levels were also significantly increased. Conclusion Sortilin promotes the formation of diabetic vascular calcification and develops it into macrocalcification.