活血化瘀法治疗腰椎间盘突出症血瘀证的血清蛋白质组学研究

Lumbar intervertebral disk protrusion (LIDP), which is well known as the most predominant disease in spinal disorders, and it is seriously harmful to the health of young adults. However, majority of LIDP patients are suitable for nonoperative treatment, so it is very important to apply Chinese Traditional Medicine (TCM) treatment and improve its efficacy. Based on numbers of previous clinical practices and research results of syndrome of blood stasis of LIDP at genetic and proteomes level, this project is undertaken using isobaric tags for relative and absolute quantitation（iTRAQ）,liquid chromatography matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer (LC-MALDI TOF/TOF?) and multiple reaction monitoring(MRM) new technologies to screen the value marker serum proteins with Blood Stasis Syndrome of LIDP patients whose treatment was Method of Huoxue Huayu. Our study is aimed to analyze the serum proteomes by method of Huoxue Huayu with Blood Stasis Syndrome of LIDP, and this research is intended to lay the foundations for differentiating syndromes via biomarkers and improve curative effect of herbal treatment by searching for their targets. It will be in favor of the health care of people who live in the Guangxi Zhuang region and promote the progress with family and social harmony when we perform the process of this research. It also provides a new idea for doing research about LIDP treatment with TCM.

腰椎间盘突出症(Lumbar Intervertebral Disk Protrusion,LIDP)是脊柱骨科最常见的疾病，并严重危害青壮年的健康，临床上绝大部分患者适合选择非手术治疗，故提高其中医治疗的疗效意义重大。本项目是在前期大量临床实践并从基因、蛋白质组水平探讨LIDP血瘀证的基础上，运用蛋白质同位素标记相对和绝对定量(iTRAQ)新技术、纳流LC-MALDI TOF/TOF?定量分析和鉴定蛋白质新技术，筛选和鉴定经活血化瘀法治疗LIDP 血瘀证患者血清中的差异表达蛋白质，再采用质谱多反应监测(MRM)新技术对筛选出的蛋白质进行验证及确认，以阐明活血化瘀法治疗LIDP的蛋白质组学基础，为建立LIDP分子水平的辨证论治、寻找靶标中草药治疗以提高其疗效打下良好基础。项目研究的开展将有利于推动广西民族地区人口健康事业的进步，增进家庭和社会和谐；同时也为中医治疗LIDP的研究提供新思路。

本项目运用蛋白质同位素标记相对和绝对定量(iTRAQ)、液相色谱与串联质谱(LC-MS/MS)及质谱多反应监测(MRM)等技术，筛选、鉴定及验证活血化瘀法治疗腰椎间盘突出症(Lumbar Intervertebral Disc Protrusion，LIDP)患者血清中的差异表达蛋白。项目纳入样本总量120例，即随机抽取临床确诊为LIDP并中医辨证为血瘀证的患者60例为观察组，并将其随机分为A1、A2两个亚组，每组30例；同期就诊并与观察组性别、年龄及临床症状等相匹配的非血瘀证LIDP患者60例为对照组（B组）。首先，项目组筛选观察组中A1亚组30例LIDP血瘀证患者经活血化瘀治疗的血清差异表达蛋白；随即开展了观察组中A2亚组30例LIDP血瘀证患者经西医非手术综合治疗前后的血清差异表达蛋白研究。然后，项目组按病例对照研究方法筛选60例LIDP非血瘀证患者（B组）经西医非手术综合治疗的血清差异表达蛋白，并开展了不同证型两组间共90例（A2组与B组）LIDP患者经西医非手术综合治疗的血清比较蛋白质组学研究。最后，在以上研究的基础上，项目组采用同法开展了60例血瘀证患者经活血化瘀法治疗（A1亚组）与西医非手术综合治疗（A2亚组）的血清比较蛋白质组学研究。在进行实验研究的同时，观测各组患者疗效等相关指标。研究结果显示LIDP血瘀证患者的血清中存在多种差异表达蛋白，并筛选出12个差异蛋白可区分LIDP血瘀证与非血瘀证，以及2个差异蛋白可成为活血化瘀法治疗LIDP的血清标志蛋白。项目研究过程无不良事件发生，各组患者疗效优良率均在90%以上。. 本研究首次应用iTRAQ结合LC-MS/MS及生物信息学技术筛选活血化瘀治疗LIDP血清标志蛋白，为中医辨证研究提供新模式，丰富了中医血瘀证的证候及治疗学内涵。项目研究总体进展顺利，基本完成了原定筛选出活血化瘀治疗LIDP的血清差异蛋白并鉴定及确认、以及阐述其机制和辨证规律的蛋白质组学水平研究、培养研究生3名等量化目标，已完成研究论文7篇，其中已发表及接收待发表中文核心期刊各1篇、研究生学位论文1篇、会议交流1篇、已投SCI收录期刊1篇、2篇待投稿。取得了预期研究成果。