扶正解毒法对Ang-Tie2介导的TEMs在肿瘤免疫抑制微环境下血管重塑的调控研究

Angiogenesis in the tumor microenvironment is the important influencing factor of tumor metastasis and relapse to treatment failure. The Tie2 expressing monocyte/macrophage(TEMs) are the important subset of the tumor- associated macrophage(TAM), and relate to the tumor immune escape and micro- angiogenesis. The Ang-Tie2 mediated Rheb/mTOR/p70S6K1 signal pathway is vital for the carcinogenesis functions of TEMs. Principle of Strengthening the body resistance and disintoxication plays an important role in cancer prevention and treatment of metastasis and relapse, previous study shows Fuzheng Jiedu Formula (FZJD) remodels TAM's phenotypes and reverses tumor immunomicroenvironment suppression and hypoxia station via mTOR pathway. Acting on TEMs and inhibiting their tumor angiogenesis function could be a neo-antitumor mechanism.We design this study by using laser Doppler perfusion imaging(LDPI), hypoxyprobe, flow cytometry, high content screening(HCS) analysis, immunohistochemistry assay, co-culture technique and hypoxia workstation to observe the intervention of FZJD on micro-angiogenesis around the tumor immune suppressive microenvironment, from the multi-aspect of vivo and vitro,tumor bearing and recurrence models, TEMs and tumor cells. On the new sight of regulation on immune suppressive cells and tumor cell unfold protein reaction(UPR)，this study will provide scientific evidence for the application of FZJD formula in cancer prevention and treatment guided by the strengthening the body resistance and disintoxication theory of TCM, and explore its molecular mechanisms in preventing and treating tumor metastasis and recurrence.

肿瘤微环境下血管生成是肿瘤治疗失败和转移复发的重要影响因素。Tie2表达的单核巨噬细胞（TEMs）是肿瘤相关巨噬细胞（TAM）的重要亚群，与肿瘤微环境下血管生成密切相关。Ang-Tie2介导的Rheb/mTOR/p70S6K1信号通路是TEMs促进肿瘤微血管生成的主要途径。扶正解毒法是临床防治肿瘤转移复发的重要法则，前期研究显示：扶正解毒方是通过mTOR介导的信号通路重塑TAM表型，逆转肿瘤微环境的免疫抑制和乏氧状态，干预TEMs功能及抑制血管生成可能是其新的作用机制，本课题拟通过LDPI、Hypoxyprobe、FCM、高内涵分析等技术及低氧工作站、共培养等手段，从体内外，带瘤和术后复发转移模型观察扶正解毒法对Ang-Tie2介导的TEMs在肿瘤免疫抑制微环境下血管重塑的影响，从调控免疫抑制细胞和肿瘤细胞非折叠蛋白反应抑制血管生成的新视角，进一步揭示扶正解毒法防治肿瘤复发转移的分子机制。

肿瘤微环境下血管生成是肿瘤治疗失败和转移复发的重要影响因素。TEMs细胞是肿瘤相关巨噬细胞（TAM）的重要亚群，与肿瘤微环境下血管生成密切相关。扶正解毒法是临床防治肿瘤转移复发的重要法则。.本课题通过建立移植性前胃癌小鼠荷瘤模型、术后复发转移小鼠模型及M-CSF刺激下移植性前胃癌荷瘤小鼠模型，系统观察了扶正解毒中药组、化疗组（5-FU）及中西医结合组(扶正解毒中药+5-FU)对肿瘤抑制率、复发转移率；通过LDPI、Hypoxyprobe等观察了扶正解毒方对肿瘤组织微血管及肿瘤组织乏氧状态的影响；从小鼠CD4+/CD8+T细胞比值、Treg细胞、TEMs细胞含量、免疫抑制因子IL-6、IL-10、CCL-17、Arg-1等表达水平观察了扶正解毒方对肿瘤免疫微环境的影响；通过检测体、内外VEGFa、MMP-9、COX-2基因转录水平及Ang-1、Ang-2蛋白水平观察了扶正解毒方调控肿瘤微血管的作用；通过检测UPR中心调控因子GRP-78蛋白及ATF-6、IRE1α、PERK及PI3K、Rheb、p70S6K1蛋白、AKT、TSC1、mTOR、Tie-2等蛋白含量观察扶正解毒方调控免疫抑制微环境及血管重塑的分子机制。.实验的结果显示：扶正解毒方可抑制肿瘤的生长、复发及转移；改善肿瘤组织灌注、乏氧状况及微血管生成，下调TEMs细胞表达，降低促血管生成因子VEGFa、MMP-9、COX-2、血管生成素Ang1、Ang2及免疫抑制因子L-6、IL-10、CCL-17、Arg-1、UPR中心调控因子GRP-78蛋白的表达，抑制PERK和IRE1蛋白磷酸化，下调PI3K、Rheb、mTOR、p70S6K1、AKT、Tie-2蛋白含量，从而表明扶正解毒方可通过调节Ang-Tie2介导的Rheb/mTOR/p70S6K1信号通路及抑制UPR反应来发挥改善免疫抑制微环境及重塑肿瘤微血管作用，从而阐释了以扶正解毒法为主的中药复方防治肿瘤转移新的科学内涵，为进一步应用中医药防治肿瘤转移提供了新的思路和科学理论依据。