基于肿瘤微环境和基因多态性探究益气活血解毒方调控Tregs治疗晚期卵巢癌的个体化免疫机制

Ovarian cancer has the highest mortality rate of the women's cancers, The mainstay of treatment is surgery followed by platinum-based adjuvant chemotherapy. Despite much progress has made in cheomtherapy ,the cure rate has not improved in decades Immunotherapy is widely viewed as one of the most promising approaches, Unfortunately,a natural immune response to cancer is not typical, because of various molecular mechanisms by which tumors either hide from or suppress any effective immune response. With understanding the pivotal role of T regulatory cells(Tregs) in tumor immune escape and angiogenesis in tumor microenvoriment and inherited variants in Tregs genes are related to outcome of ovarian cancer. the time of tailored immunotherapy is coming. Our research showed that Yiqihuoxuejiedu Formula improved time-to-relapse among advanced ovarian cancer patients with the sydrome of qi deficiency and blood stasis, it could modulate Tregs in advanced ovarian cancer and Treg-related immunosuppression in mircroenviroment. So deep research of Chinese herbs on immunosuppressin pathway in microenviroment and Treg-related genes inherited single nucleotide polymorphisms might offer immunological evidence of tailored-treatment to ovarian cancer. we will start our research from Tregs and take advantages of SNPs and mRNA chips to investigate the impact of Yiqihuoxuejiedu Formula on Tregs-VEGF pathway of immune net in tumor microenviroment, and the correlation of Treg-related genes SNPs and survival as well as syndrome, and how the formula will influence the mRNA expressions of specific genes, then we will understand the mechanisms of Yiqihuoxuejiedu Formula and the inherited characteristics of syndromes to some extent.The results of this work will provide scientific evidence for improving efficacy of individulized therapy of traditional chinese medicine characterized by syndrome differentiation and treatment.

卵巢癌是女性肿瘤中致死率最高的肿瘤，手术+化疗是标准治疗，尽管化疗药有了长足进展,但生存率近三十年未见提高。免疫治疗被认为是突破现有治疗的最有希望疗法，但肿瘤微环境中复杂的免疫抑制分子机制及缺乏对卵巢癌个体免疫差异的认识，使得其在临床未取得明显进展。最新研究发现：调节T细胞（Tregs）是卵巢癌肿瘤微环境复杂免疫网络中调控免疫逃逸和血管生成的关键免疫抑制因子，其相关基因的多态性(SNPs)与卵巢癌预后密切相关。而我们前期工作显示"益气活血解毒方延长晚期气虚血瘀型卵巢癌生存,可能与调控Tregs有关"，故拟以Tregs为切入点，采用SNPs及mRNA芯片，研究该方对肿瘤微环境免疫网络中Tregs-VEGF通路的影响，并探究与辨证论治疗效有关的Tregs相关基因的多态（SNPs）特点及该方对对应标志基因表达的影响，方证结合疗效，探索中药干预机制及证型的基因多态性特点，为中药个体化治疗提供依据。

卵巢癌是女性肿瘤中致死率最高的肿瘤，益气活血解毒方可延长晚期气虚血瘀型卵巢癌生存,前期工作显示可能与调控Tregs有关。本课题以Tregs为切入点，通过体外基础实验（人脐静脉内皮细胞血管生成试验、CCK8），证实益气活血解毒方体外对小鼠卵巢上皮癌 ID-8 细胞株的增殖过程有显著的抑制作用，同时能够干预血管的形成，这也许与本方影响 Tregs 促进肿瘤新生血管的作用相关；通过体内基础实验（C57BL/6小鼠ID8卵巢癌抑瘤实验），证实了益气活血解毒方能抑制C57BL/6小鼠ID8卵巢癌，抑瘤率32%，与对照组相比，下调CD4+CD25+Tregs水平（P＜0.05），iTRAQ蛋白组得到与疗效、Tregs、VEGF表达有关的差异蛋白，western blot验证得到Gimap4、Orm1的差异表达具有统计学意义（P＜0.05）。临床试验，服药半年后患者血样制备SNPs及mRNA芯片，得到与益气活血解毒方疗效相关的SNPs位点有1773个，这些位点可能是对中药敏感的SNPs位点，得到益气活血解毒方疗效的差异标志基因可能为： CD40、AFF3、EIF2AK3、WNK1、FYB、FBXL13、PLXNC1、BCL11A、IFI16、GNG7。. 本课题从基础和临床两个层面，证明了益气活血解毒方对免疫抑制分子Tregs的调控，基础方面证明了益气活血解毒方从Tregs角度调控卵巢癌肿瘤微环境，并得到与Tregs-VEGF通路相关的潜在核心基因，为研究本方的有效机制提供了新的思路。从临床层面探究与辨证论治疗效有关的Tregs相关基因的多态（SNPs）特点，且本方对对应标志基因表达的亦有影响，方证结合疗效，探索中药干预机制及证型的基因多态性特点，为中药个体化治疗提供依据。