基于核苷酸切除修复通路参麦方对奥沙利铂治疗结直肠癌的增敏机理

Abstract: Oxaliplatin is a first-line chemotherapy for colorectal cancer, but only 60% of patients are sensitive to oxaliplatin. “Supporting the Healthy Energy” is one of the important TCM therapy of cancers, famous Shenmai formula has been confirmed to have a synergistic function for adjuvant chemotherapy by regulating immune function and improving tumor microenvironment. We previously found that the effect of oxaliplatin in the treatment of colorectal cancer was associated with the nucleotide excision repair (NER) signal pathway. Accordingly, we propose a hypothesis that Shenmai formula can contribute to the sensitivity of tumor cells to oxaliplatin by regulating the NER pathway key genes . Therefore, this project will establish the colon subcutaneous fluorescent orthotopic transplantation model, and clarify Shenmai formula in the treatment of colorectal cancer effect of oxaliplatin. We will investigate the Shenmai formula adjuvant chemotherapy effect on NER pathway key genes by using RT-PCR and Western-blot. We will aslo test the influence of Shenmai formula on the molecular regulation of NER sensitive miRNA target gene activity mechanism by using luciferase reporter gene experiment , immunoprecipitation and immunoblotting methods simultaneous purification of main material Shenmai, explore the active components of NER pathway regulated and detailed mechanism. This study will illustrate the molecular mechanism of Shenmai formula treatment in colorectal cancer, and regulation of NER pathway genes. The results will provide theoretical basis for the clinical application of the combination of Shenmai formula and Oxaliplatin for colorectal cancer treatment.

奥沙利铂是结直肠癌靶向治疗的一线药物，但只有60%左右病人对其敏感。扶正培本法是中医治疗肿瘤的重要治法之一，经典名方参麦方已被证实对辅助化疗具有一定的增效功能，主要是通过调节机体免疫和改善肿瘤微环境发挥作用。课题组前期发现奥沙利铂治疗结直肠癌的疗效与核苷酸切除修复（NER）信号通路密切相关。我们提出科学假说：参麦方可通过调节肿瘤细胞NER通路关键基因而影响肿瘤细胞对奥沙利铂的敏感性。因此，本项目拟通过建立结肠癌皮下荧光原位移植模型，观察参麦方对奥沙利铂治疗结直肠癌疗效的影响；采用双荧光素酶报告基因实验、RT-PCR、免疫共沉淀以及免疫印迹等方法研究参麦方影响miRNA调节NER通路靶基因活性的增敏分子机理。本研究将阐明参麦方调节NER通路基因影响奥沙利铂增效的分子机理，为参麦方和奥沙利铂联用抗结直肠肿瘤的临床应用提供理论依据。

参麦方具有临床化疗增效作用。NER通路是清除大量铂类药物，导致DNA螺旋结构扭曲的主要机制，也是影响奥沙利铂疗效的重要因素。本研究科学假说为：参麦方可以改善肿瘤微环境增强机体抗癌能力，并影响NER 通路，进一步改变机体内DNA 损伤修复的能力，最终增加奥沙利铂治疗结直肠肿瘤的疗效。我们构建了NER通路关键基因RPA1敲降及过表达的结直肠癌细胞株，发现RPA1具有影响细胞增殖及单克隆的能力，且干扰RPA1能增加奥沙利铂对细胞增殖及单克隆的抑制程度。进行参麦注射液联合奥沙利铂对结肠癌LoVo细胞裸鼠移植瘤的实验，发现参麦注射液能增强奥沙利铂抗结肠恶性肿瘤的作用，同时能减轻奥沙利铂对小鼠免疫器官与肝功能的影响，其机制可能与抑制b-FGF、HIF-1α介导的血管生成信号通路相关；另外，进行参麦方活性成分人参皂苷Rb1及人参皂苷Rg1对炎症相关结直肠癌小鼠作用及机制的研究，发现人参皂苷Rb1及人参皂苷Rg1均能抑制肿瘤生长，其机制可能与减少促炎因子、增加抑炎因子、增强免疫以及调节肠道微环境有关；此外，进行人参皂苷Rg1对炎症肠道类器官的研究，发现人参皂苷Rg1对TNF-α诱导的肠道炎症有一定抑制作用，同时，对正常组织无明显损伤；最后，进行人参皂苷Rg1联合奥沙利铂对炎症相关结直肠癌小鼠作用及机制研究，发现Rg1能够缓解肿瘤及化疗药物带来的体重下降，抑制肿瘤生长，机制可能与调节炎症因子、增强免疫及调节肠道菌群有关，进一步针对肠道菌群功能分析发现人参皂苷Rg1对结直肠癌小鼠的作用与NER通路高度相关。本研究结果提示，参麦方及其活性成分能够改善肿瘤微环境增强机体抗癌能力，对奥沙利铂治疗结直肠癌具有增效减毒作用，可联合奥沙利铂运用于临床，治疗结直肠癌。