中医“气滞血瘀”理论实证肿瘤干细胞起源的研究

The existence of cancer stem cell (CSC) is a major cause of tumor incidence and metastasis, but how it is originated remains unclear. Based on the traditional Chinese medicinal theory of "Qi stagnation and blood stasis", we propose here a hypothesis of "inflammation-triggered nitric oxide (NO) inducing metabolic hypoxia and tumorigenic mutation". To elucidate the molecular mechanism underlying that NO-induced hypoxia (Qi stagnation)leads to immature angiogenesis (blood stasis) and even tumorigenesis, a mouse model with tumor-like neoplasia will be established by injection of lipopolysaccharide (LPS), complete Freund's adjuvant (CFA) or the NO generator sodium nitroprusside (SNP), and verified inversely in iNOS gene knockout mice. We intend to reveal the relationship between CSC origin and Qi stagnation and blood stasis from aspects of tumor morphology, histopathology, activation of pro-inflammatory cytokines, regulation of angiogenesis, expression of mutagenesis signaling, and mutagenesis of tumorigenic genes. To prohibit CSC origin, anti-inflammation (rapamycin or benzoic acid), inhibition of NO production (artemisinin), suppression of TNFαfunction (anti-TNFαantibody), attenuation of HIF-1αactivation (betulinic acid), and activation of blood circulation to dissipate stasis (Chinese medicinal components) will be conducted, thereby establishing a new therapeutic approach that combines the traditional "pre-disease interventions" with the CSC-targeted anti-tumor strategy.

肿瘤干细胞(CSC)是肿瘤发生和转移的根源，但它究竟如何起源却不清楚。基于中医"气滞血瘀"理论，并受滑膜炎机理研究的启发，我们首次提出"炎症激发一氧化氮(NO)诱导代谢性缺氧及癌变"的CSC起源假说。为了阐明NO诱导缺氧(气滞)导致非健全血管增生(血瘀)乃至转化成瘤的分子机理，本研究拟通过注射脂多糖(LPS)、完全弗氏佐剂(CFA)、NO释放剂硝普钠(SNP)建立小鼠肿瘤样增生及浸润模型，并用iNOS基因敲除小鼠进行反向验证，从肿瘤形态学、组织病理学、促炎细胞因子激活、血管形成基因调控、癌变信号基因表达、肿瘤相关基因突变揭示CSC起源与气滞血瘀的关系。在造模过程中，经消炎(雷帕霉素、苯甲酸)、抑制NO合成(青蒿素)、阻断TNFα功能(抗TNFα抗体)、减弱HIF-1α活化(桦木酸)、活血化瘀(中药复方)等干预CSC的起源，由此建立融中医"治未病"与CSC靶向抗肿瘤的新疗法。

肿瘤病因不明且其诱发机制尚无定论。本项目围绕垂体腺瘤和乳腺癌开展了肿瘤逆转及模拟起源研究，从分子水平上初步阐明了肿瘤的成因及其演进。（1）体外逆转垂体腺瘤研究：胰岛素可通过降低细胞内葡萄糖浓度模拟热量限制效应，既抑制肿瘤细胞增殖，也下调肿瘤标志物（Cyclin D1与AFP）表达，并伴有ROS水平及细胞凋亡率降低，而高浓度葡萄糖在无胰岛素培养基中同样表现热量限制诱导的抗肿瘤效应。（2）乳腺癌逆转体外研究：以干扰RNA敲弱iNOS基因的乳腺癌细胞增殖明显降低，Cyclin D表达下调，而肿瘤抑制蛋白p21表达上调，且ROS水平及乳酸含量降低，ATP含量升高。（3）乳腺癌模拟起源研究：ERα基因敲弱的正常乳腺细胞中ATR基因和BRCA1基因表达上调，表明DNA损伤修复系统受到广泛激活，且ROS水平及乳酸含量升高，ATP含量降低，表现肿瘤样特征，但细胞增殖却意外降低，表明无氧代谢能效低下导致细胞生长速率减缓，暗示肿瘤可能依赖肿瘤微环境中的正常细胞提供能量。（4）肉食成分诱导肠道机会性感染致瘤研究：硫酸软骨素促进硫酸酯酶分泌细菌及硫酸盐还原细菌过度生长，破坏肠道完整性，造成LPS泄漏，诱发慢性低度炎症，并长出瘤状物，其Cyclin D1表达上调，p21表达下调。（5）非细胞毒性抗肿瘤研究：在利用内外源NO建立肿瘤样增生小鼠模型基础上，初步明确了缺氧导致组织增殖的因果关系，并验证了青蒿素、雷帕霉素、桦木酸和血府逐瘀片的抗缺氧抑制增殖的效果及其机理。