腰椎间盘突出症活血化瘀治疗的目标蛋白筛选及鉴定

Screening and Identifying the Target Protein of Blood Activating and Stasis Removing for Lumbar Intervertebral Disk Protrusion

背景活血化瘀属于中医药特色疗法,也是临床上非手术治疗腰椎间盘突出症(LIDP)疗效优良的主要方法之一,既往有学者从病理、影像、免疫及基因等角度开展了大量研究,但有关活血化瘀治疗LIDP机制仍未明了。目的通过同位素标记相对和绝对定量技术(i TRAQ)结合液相色谱与串联质谱(LC-MS/MS)筛选和鉴定LIDP活血化瘀治疗前后差异表达蛋白,筛选LIDP活血化瘀治疗的目标蛋白。方法选取2013年7月—2014年12月于广西中医药大学第一附属医院治疗的血瘀证LIDP患者30例为研究对象,予活血化瘀治疗4周,观察患者临床疗效。分别于治疗前后提取患者外周血血清,通过去除高丰度蛋白、测定蛋白浓度、酶解蛋白、肽段i TRAQ标记、高效液相色谱分离、LC-MS/MS分析等程序和方法,筛选及鉴定治疗前后的差异表达蛋白。结合基因本体论注释及KEGG通路分析相关生物信息学,对差异蛋白的功能和代谢通路进行分析。结果治疗后临床控制19例,显效8例,有效2例,无效1例,总有效率为96.7%(29/30)。i TRAQ联合LC-MS/MS鉴定到300个蛋白质组,其中209个蛋白各通道标记标签均有定量信息,筛选出20个差异表达蛋白,其中呈上调表达及下调表达的差异蛋白各10个。本研究对20个差异蛋白的KEGG富集结果显示,9个差异蛋白富集到27条信号通路。补体和凝血级联反应富集到3个差异蛋白,PPAR信号通路、百日咳、美洲锥虫病、金黄色葡萄球菌感染及系统性红斑狼疮富集到2个差异蛋白。百日咳、美洲锥虫病、金黄色葡萄球菌感染及系统性红斑狼疮等11条信号通路富集到补体C3,PI3K-Akt信号通路、黏着斑、肌动蛋白细胞骨架调节等9条信号通路富集到纤维连接蛋白1。结论活血化瘀治疗血瘀证LIDP效果显著;载脂蛋白A1、载脂蛋白M、载脂蛋白C3、纤维连接蛋白1及补体C3很可能是血瘀证LIDP选用活血化瘀治疗的潜在血清靶蛋白。

Background Promoting blood circulation and removing blood stasis treatment is the characteric therapy used by traditional Chinese medicine,and it＇s also one of the main effective non-surgical therapy for lumbar intervertebral disk protrusion（ LIDP） in clinic. In the past, scholars had carried out a great deal of researches from the pathology, imaging,immunology and gene about the disease,but the mechanism of LIDP treated by blood circulation was still unknown. Objective To obtain the target protein by screening and identifying the differentially expressed proteins before and after promoting blood circulation and removing blood stasis treatment for LIDP patients,using i TRAQ isobaric tags coupled with LC-MS / MS. Methods30 LIDP patients diagnosed as blood stasis syndrome by Chinese differentiation clinically from July 2013 to December 2014 in the First Affiliated Hospital of Guangxi University of Chinese Medicine were selected as the experiment subjects. Promoting bloodcirculation and removing blood stasis treatment were given for 4 weeks and clinical efficacy of patients were observed. The serum of the peripheral blood was extracted before and after the treatment. To screen and identify the differentially expressed proteins before and after the treatment through removing the high abundance protein, measuring the protein concentration, enzymatic hydrolysis of proteins, peptide i TRAQ labeling, high performance liquid chromatography and LC-MS / MS analysis. The functional and metabolic pathways of the differentially expressed proteins were analyzed by the combination of gene ontology annotation and KEGG pathway-related bioinformatics. Results The results showed that 19 cases were clinical controlled,8cases were excellent,2 cases were effective,1 case was invalid,and the total effective rate of treatment was 96. 7%（ 29 /30）.In total,300 non-redundant proteins were identified by using the technique of i TRAQ and LC-MS / MS. There were quantitative information on each channel tag in 209 of these proteins. Twenty differentially expressed proteins were screened out,10 upregulated proteins and 10 downregulated proteins. In this study,the KEGG enrichment results of 20 differentially expressed proteins showed that 9 differential proteins were enriched to 27 related signal pathways. The complement and coagulation cascades were enriched to three differential proteins. PPAR signal pathways,pertussis,trypanosomiasis,staphylococcus aureus infection and systemic lupus erythematosus were enriched to two differentially expressed proteins. 11 signal pathway,such as pertussis,trypanosomiasis,staphylococcus aureus infection and systemic lupus erythematosus,et al. were enriched to Complement C3.Nine signal pathways,such as PI3K-Akt signal pathway,focal adhesion,actin cytoskeleton regulation,et al. were enriched to Fibronectin 1. Conclusion The therapy of promoting blood circulation and removing blood stasis is remarkable on treating LIDP patients with blood stasis syndrome. Apolipoprotein A1,Apolipoprotein M,Apolipoprotein C3,Fibronectin 1 and Complement C3 may be the potential target serum protein of therapy of promoting blood circulation and removing blood stasis for the LIDP patients with blood stasis syndrome.