基于丝裂原活化蛋白激酶信号通路探讨从痰论治乳头状甲状腺癌的作用和机制

Combining Chinese medical theory and disease mechanism study would be helpful to get a new finding of medical science. The phlegm syndrome is a classic theory of traditional Chinese medical science, especially for thyroid tumor. To inhibit mutation of BARF gene and phosphorylation of MEK1/2 in MAKP has become the target for clinical research and treatment of papillary thyroid carcinoma(PTC). Based on previous studies we found: the prescription of expelling phlegm could improve clinical symptom and pathologic situations for PTC patients; the phosphorylation of MEK1/2 and expression of BRAF V600E reduced after intragastric administration of prescription of expelling phlegm for PTC mouse, which had the same meaning with clinical studies. According to these results, we expected whether there were some relations between "phlegm-expelling" in Chinese medical theory and "dephosphorylating of MEK1/2" in MAKP，which could probe the mechanism of treatment from Expelling Phlegm for PTC. Based on the previous clinical and experimental studies, we attempt to combine traditional Chinese and western medical sciences to carry out further researches: ① examining phosphorylated MEK1/2 and BRAF V600E expression of PTC mouse which received intragastric administration of prescription of expelling phlegm, to find whether there are relations between "phlegm-expelling" and "dephosphorylating"; ② comparing MEK1/2 and BRAF V600E expressions of PTC mouse，between the ones received intragastric administration of prescription and the ones received BARF, MEK1/2 antagonists abdominal injection, to confirm whether prescription and antagonists have a same effect to the pathway; ③ testing MEK1/2 and BRAF V600E expressions of PTC mouse that received both prescription and BARF, MEK1/2 agonists，to figure out whether prescription has depression effect to the over-activation of MAKP. All these may help to find the new targets for clinical treatment combined with both operation and prescription for PTC patients.

中医理论与西医发病机制研究的结合有助于获得新的医学认识。痰证是中医治疗甲状腺肿瘤的经典理论。MAKP通路BARF基因突变和MEK1/2磷酸化是乳头状甲状腺癌（PTC）发病机制研究热点。前期发现：消痰散结方治疗PTC能抑制患者肿瘤病变；PTC小鼠方剂灌胃后肿瘤结构改善，MEK1/2磷酸化和BARF V600E表达均下降。故假设中医之“痰”与MAKP通路“磷酸化产物”相映射，痰证之“祛除痰浊”与MAKP通路“去磷酸化”异曲同工。课题拟通过：①检测方剂灌胃后PTC小鼠BARF V600E和磷酸化MEK1/2表达水平，验证祛痰与去磷酸化的相关效应；②对比方剂灌胃和予BARF、MEK1/2抑制剂干预的PTC小鼠通路的变化，验证祛痰与去磷酸化的等效作用；③进一步检测方剂灌胃+BARF、MEK1/2激动剂干预的PTC小鼠通路变化，反向验证方剂对通路磷酸化激活的抑制效应，为中西医结合治疗PTC引进新思路。

消痰散结方是一种由十一味中草药制成的传统中药汤剂。之前我们有报道过其预防胃癌进展的体内和体外相关研究，其中一味半夏有明确的抑制肿瘤细胞生长增殖之效。本研究旨在研究消痰散结方以及半夏对于乳头状甲状腺癌的抗肿瘤作用机制。我们将研究主要分为乳头状甲状腺癌细胞系（TPC-1/BCPAP）干预培养的体外研究，和转染肿瘤细胞移植的基因敲除小鼠干预治疗的体内研究。通过研究发现：体外研究方面：消痰散结方剂和单一味半夏相对于去半夏方剂能更明显的抑制肿瘤细胞增殖，促进细胞凋亡，引起细胞自噬效应更显著。针对单一味半夏进行的研究发现，半夏具有剂量依赖性的抑制Nrf2基因表达的作用，同时，其还能抑制乳头状甲状腺癌细胞的克隆增殖；Nrf2基因表达的敲减和干扰转染能增强半夏对肿瘤细胞的自噬，增殖，凋亡，克隆和集落形成等方面的作用。针对MAPK信号通路下游因子的干预研究发现，结合自噬抑制剂3-MA的干预，半夏的干预和Nrf2基因的转染能显著的促进肿瘤细胞凋亡，抑制肿瘤细胞的增殖以及MAPK通路的激活。这些均说明消痰散结方以及单一味半夏对Nrf2表达和肿瘤细胞自噬作用的保护机制。体内研究方面：相对于对照组，半夏干预组以及Nrf2干扰基因转染细胞移植组均能明显的增加野生型小鼠的生存率。此外，半夏干预还能减小肿瘤体积，导致肿瘤细胞凋亡，能抑制MAPK通路的激活并引起肿瘤细胞的自噬，且半夏干预和转染细胞移植干预有叠加效应。此外，Nrf2干扰基因转染细胞的干预对照各项实验均进一步证明了半夏对乳头状甲状腺癌的抗肿瘤放大效应。而对于Nrf2基因敲除小鼠，半夏干预基本无效。综合所有实验结果分析和统计学处理，证明：消痰散结方/半夏在乳头状甲状腺癌的治疗中发挥抗肿瘤效应，其中，下调Nrf2基因表达能显著介导放大方剂作用，尤其在抑制MAPK通路激活的抗肿瘤作用机制方面效果明确。针对消痰散结方/半夏在乳头状甲状腺癌抗肿瘤机制的研究为临床治疗提供了理论支持，对临床治疗方案的制定具有指导性作用。