从IDO介导的免疫编辑途径研究理冲汤抑制子宫肌瘤免疫逃逸的作用及分子机制

Uterine myoma is the most common tumor which affects women's physical and mental health.The patients often have a pathological basis of “vitality-deficiency and blood stasis”. In clinical practice, Chinese medicine practitioners often carry out treatment from the perspective of strengthening the body resistance and removing blood stasis. Our previous studies found that Lichong decoction can inhibit angiogenesis of uterine myoma, and it has an anti-uterine myoma effect. We hypothesize that this effect is associated with the regulation of muscular tumor microenvironment of immunoediting and inhibition of immunoescape. In this study,we established an animal model of xenograft uterine myoma in order to clarify the molecular mechanism of Lichong decoction. The effect of Lichong decoction on IDO activity,Trp metabolism,T-cell function and related cytokines was observed around the related pathways of tumor immunoescape by using MSD hypersensitivity multiple-factor electrochemical luminescence, flow cytometry, real-time fluorescence quantitative PCR, immunoblotting test, immunofluorescence, HPLC and other technologies. At the same time, by the method of the vitro culture of uterine myoma cells, we will test the cytokine antibody microarray intervened by Lichong decoction and utilize the RNA interference, gene overexpression and ChIRP and other techniques to further explain the molecular mechanism of the Lichong decoction inhibiting uterine myoma immune escape through IDO-mediated immunoediting pathway, providing theoretical and experimental basis for clinical treatment of uterine myoma.

子宫肌瘤是影响女性身心健康的最常见肿瘤，患者多有“正虚血瘀”的病理基础。临床上中医学常从扶正祛瘀立法进行治疗。我们前期研究发现，扶正祛瘀中药—理冲汤可抑制子宫肌瘤的血管生成，有较好的抗子宫肌瘤作用，推测与其调控肌瘤微环境免疫编辑、抑制免疫逃逸有关。为明确其分子作用机制，本研究项目拟通过建立异种移植子宫肌瘤动物模型，采用MSD 超敏多因子电化学发光法及流式细胞术，结合实时荧光定量PCR、免疫印迹、免疫荧光、HPLC等技术，围绕肿瘤免疫逃逸途径，观察理冲汤对IDO活性、Trp代谢、T细胞功能及相关细胞因子的影响。此外，采用体外培养子宫肌瘤细胞的方法，结合细胞因子抗体芯片检测，以及RNA干扰、基因过表达和ChIRP实验等技术，进一步阐明理冲汤通过调控IDO介导的免疫编辑途径抑制子宫肌瘤免疫逃逸的分子机制，为临床治疗子宫肌瘤提供理论和实验依据。

子宫肌瘤是影响女性身心健康的最常见肿瘤，患者多有“正虚血瘀”的病理基础。本研究项目通过建立异种移植子宫肌瘤动物模型，采用MSD 超敏多因子电化学发光法及流式细胞术，结合实时荧光定量PCR、免疫印迹等技术，围绕肿瘤免疫逃逸途径，观察理冲汤对IDO活性、T细胞功能及相关细胞因子的影响。采用体外培养子宫肌瘤细胞的方法，进一步验证理冲汤抑制子宫肌瘤免疫逃逸的作用机制。通过CCK-8、流式细胞术、TUNEL等技术检测理冲汤对体外培养人子宫肌瘤细胞增殖和凋亡的影响。通过实时荧光定量PCR和Western blot检测免疫逃逸关键基因的表达情况。采用RNA干扰技术进行IDO等基因的体外功能验证。证明理冲汤通过影响IDO表达，进而阻断免疫逃逸而发挥作用。我们的体内实验研究显示，理冲汤可以改善子宫平滑肌细胞增生肥大及排列紊乱等现象，能够增加凋亡因子Caspase-3的表达、抑制增殖相关因子Ki67的表达。理冲汤能够促进抗免疫逃逸相关因子IL-2、IFN-γ、IL-12p70和CD28、CD80、CD86的表达，降低促免疫逃逸相关因子IDO、Foxp3以及IL-4、IL-5、IL-6、IL-1表达，可以抑制Trp沿Kyn途径分解。我们的体外实验结果表明，理冲汤可以有效抑制子宫肌瘤细胞增殖、促进肌瘤细胞凋亡，实时荧光定量PCR和Western blotting实验表明理冲汤在mRNA和蛋白水平显著降低了IDO、AKT1、PI3KCA、FOXO1的表达水平，增加了抑癌基因PTEN的表达水平。.由此我们得出结论，理冲汤具有抗子宫肌瘤的作用，其机制可能涉及：理冲汤通过调控PI3K/AKT通路进而抑制IDO诱导的子宫肌瘤免疫逃逸过程，并认为在此过程中，IDO基因起关键作用。理冲汤抑制IDO的表达，使Trp沿Kyn途径分解减少，从而限制了子宫肌瘤细胞的增殖与分化，另一方面理冲汤促进效应T细胞的表达，抑制调节性T细胞的表达，恢复免疫系统的正常功能。我们的研究进一步阐明了理冲汤通过调控IDO介导的免疫编辑途径抑制子宫肌瘤免疫逃逸的分子机制，为临床治疗子宫肌瘤提供理论和实验依据。