金复康干预肺癌细胞外泌体介导的外周细胞免疫逃逸分子机制研究

Lung cancer is the leading cause of death among all malignancy diseases. Recent study revealed that tumor cell could manipulate the peripheral cellular immunity through the releasing of exosomes(tumor derived exosomes, TEXs) which carrying the genomic information of its parental cells. The TEXs could cause the apoptosis and/or dysfunction of immunologic effector cells (including NK and CD8+T cells), through Fas/fasl or TGFβ/Smad pathway respectively. The event led to the occurrence of peripheral immune tolerance, which resulted in the escape of circulating tumor cells(CTCs) from the host immune system. The counts of CTCs in the sera is closely related to the remote metastasis. The previous clinical and experimental studies have confirmed repeatedly that Jinfukang could hinder the distant metastasis and prolong the survival of patients with lung cancer, by improving the patients's peripheral cellular immunity. Jinfukang is composed of many Chinese medical herbs under the guidance of the principles of nourishing the body resistance Based on these results, we hypothesized that Jinfukang may interfere with the pathological process of peripheral tolerance induced by tumor-derived exosomes. Under the guidance of the wholism concept of traditional chinese medicine, we will develop the in vivo and vitro models involving CTCs, TEXs and immunologic effector cells. Many cutting-edge technologies including orthotropic lung cancer model, Living imaging technology, flow cytometry as well as signal transduction pathways chip will be applied simultaneously. The role of "the deficiency of essential qi" in the etiology of lung cancer will be studied at the same time,from a point of peripheral cellular tolerance induced by tumor-derived exosomes.

肿瘤细胞可主动分泌携带其自身遗传信息的外泌体（Tumor derived exosomes,Texs），后者通过Fas/Fasl和TGFβ/Smad通路分别介导免疫效应细胞的凋亡和失活，最终导致外周细胞免疫耐受的发生，弱化宿主对循环肿瘤细胞的清除而导致转移的发生。基于前期研究证实以"扶正"中药为主制成的金复康有明确地抗肺癌转移、提高细胞免疫功能和延长患者生存期的作用，提出"干预肺癌外泌体介导的外周免疫耐受，促进循环肿瘤细胞清除是金复康抗转移和延长生存的生物学基础"的假说。课题在"整体观"指导下，创建肺癌外泌体+循环肿瘤细胞+淋巴细胞体内外研究平台，综合运用肺癌原位移植瘤模型、活体成像、流式细胞术、免疫荧光、生物芯片等技术，从整体与细胞、分子等水平系统探讨金复康干预肺癌外泌体介导外周细胞免疫耐受的分子机制和信号转导通路，阐释肺癌发病"正气亏虚"的科学本质，丰富"扶正治癌"理论的科学内涵。

转移是导致肺癌患者死亡的最主要原因，而目前化疗、分子靶向和免疫治疗均缺乏精准干预而疗效不佳。循环肿瘤细胞是转移发生的种子，其命运受到所处外周环境的影响，决定转移的发生与否。申请人以肿瘤细胞与外周免疫细胞的细胞间通讯干预为切入点，提出“干预肺癌外泌体介导的外周免疫耐受，促进循环肿瘤细胞（Circulating tumor cells，CTCs）清除是金复康抗转移和延长患者生存期的生物学基础”的假说。临床研究发现早期肺癌患者即高表达CTCs，提示早期干预的重要性，并将CTCs归入“伏毒”以丰富中医病因内涵。同时发现髓源抑制细胞和调节性T细胞的表达与临床分期成正相关，将其归入中医“正虚”的范畴。为进一步探索CTCs与外周免疫抑制之间的关系，肺癌患者外周血中的外泌体检测发现其粒径大，浓度低，与健康者相比存在显著差异。从肺癌患者外周血中提取并成功建立人肺癌循环肿瘤细胞系，体外研究发现该细胞具备干细胞表型、耐药性强、低表达淋巴细胞招募相关因子CX3CL1等特征，体内研究发现该细胞具有体内潜伏及形成肺转移能力，从而揭示其生物学特性与原发灶肿瘤细胞的区别。扶正药物金复康可显著抑制CTCs增殖，诱导CTCs衰老及失巢凋亡，抑制CTCs转移，其机制可能为调控p16-RB和MEK/ERK信号通路；金复康干预CTCs后提取外泌体并进行蛋白质组学分析，发现差异蛋白主要参与调控糖酵解途径和ECM受体相互作用等。并进一步构建了免疫衰老小鼠肺癌移植瘤模型，发现金复康对免疫衰老小鼠肺癌移植瘤具有预防及抑制肿瘤增殖的作用，其可能的机制为干预氧化损伤介导的免疫衰老。本研究初步揭示肿瘤细胞分泌囊泡诱导外周免疫耐受可能是外周免疫抑制发生的关键环节，而干预外周免疫抑制、延缓免疫衰老可能是金复康防治肺癌的重要机制。本课题揭示了中医药防治肺癌的关键机制，阐释肺癌发病“正气亏虚”的科学本质，在“扶正治癌”理论指导下提出“正虚伏毒”致癌的学术观点。