激素性股骨头坏死中医证型的蛋白质组学研究

Proteomics Research of Chinese Medical Syndromes of Steroid-induced Osteonecrosis of the Femoral Head

【目的】应用蛋白质组学技术,筛选激素性股骨头坏死(SONFH)筋脉瘀滞证、肝肾亏虚证患者差异表达蛋白质,为SONFH中医证候分型寻找物质基础。【方法】分别收集SONFH筋脉瘀滞证患者、肝肾亏虚证患者及健康人各10例,应用双向电泳结合质谱检测技术,寻找并鉴定差异表达的蛋白质。对筛选获得的候选蛋白应用Western blot法验证。【结果】共筛选出血红蛋白、肌动蛋白、补体C4、抗凝血酶Ⅲ、载脂蛋白A-Ⅳ、富亮氨酸α-2-糖蛋白、血清淀粉样蛋白A-2共7种差异蛋白质。其中,抗凝血酶Ⅲ、血清淀粉样蛋白A-2在筋脉瘀滞组特异性表达,补体C4、富亮氨酸α-2-糖蛋白在肝肾亏虚组特异性表达。Western blot验证结果显示:与正常组比较,筋脉瘀滞组与肝肾亏虚组中补体C4、抗凝血酶Ⅲ蛋白表达下调(P<0.05);与筋脉瘀滞组比较,肝肾亏虚组中补体C4蛋白表达下调(P<0.05);与肝肾亏虚组比较,筋脉瘀滞组抗凝血酶Ⅲ蛋白的表达下调(P<0.05),与蛋白质组学研究结果一致。【结论】抗凝血酶Ⅲ、血清淀粉样蛋白A-2可能是SONFH筋脉瘀滞证潜在的血清特异性蛋白标志物;补体C4、富亮氨酸α-2-糖蛋白可能是SONFH肝肾亏虚证潜在的血清特异性蛋白标志物。

Objective To screen the differentially expressed proteins of blood stasis blocking tendon and vessel syndrome and liver-kidney deficiency syndrome of the steroid-induced osteonecrosis of the femoral head （ SONFH） by proteomic technology, so as to supply evidence for Chinese medical syndrome classification. Methods The serum was taken separately from 10 patients with blood stasis blocking tendon and vessel syndrome, 10 patients with liver-kidney deficiency syndrome and 10 healthy volunteers. The two dimensional electrophoresis combined with mass spectrometric method was applied to screen and identify the differentially expressed proteins, and then the obtained protein candidates were verified by Western blotting method. Results Seven proteins were identified from differentially expressed protein spots, including hemoglobin subunit delta, actin, complement C4, antithrombin-Ⅲ, apolipoprotein A-IV, leucine-rich alpha-2 glycoprotein and serum amyloid A-2 protein. We found that antithrombin-Ⅲ and serum amyloid A-2 protein had specific expression in the SONFH patients with blood stasis blocking tendon and vessel, and complement C4 and leucine-rich alpha-2-glycoprotein had specific expression in the SONFH patients with liver-kidney deficiency syndrome. The results of Western blot method showed that the expression levels of complement C4 and antithrombin-Ⅲ were down-regulated in SONFH patients （ P〈0.05 compared with the healthy volunteers） , the down-regulation of complement C4 was more obvious in SONFH patients with liver-kidney deficiency syndrome （P〈0.05） and the down-regulation of antithrombin-Ⅲ was more obvious in the patients with blood stasis blocking tendon and vessel （P〈0.05）, the results being accorded with those of proteomic detection. Conclusion Antithrombin-Ⅲand serum amyloid A-2 protein may be the specific serum protein markers of SONFH patients with blood stasis blocking tendon and vessel syndrome, and complement C4 and leucine-rich alpha-2-glycoprotein may be the specific serum protein markers of SONFH patients with liver-kidney deficiency syndrome.