养阴扶正解毒方逆转T细胞功能耗竭治疗乙肝相关原发性肝癌的机制研究

T cell exhaustion is an important cause of tumor immune escape and is closely related to tumor progression. The T cell dysfunction is more prominent in patients with Hepatitis B virus related primary hepatocellular carcinoma (HBV-HCC), but the mechanism of T cell exhaustion is still unclear. Our clinical studies showed that Yangyinfuzhengjiedu Decoction can reduce the expression of Notch1 on T cells, reverse T cell exhaustion and improve survival rates in patients with HBV-HCC. Therefore, we hypothesize that Yangyinfuzhengjiedu Decoction can reinvigorate exhausted PD-1+ T cells and delay the progression of HBV-HCC by regulating Notch1-RBP-Jκ-PD-1 signal pathway. We intend to: 1) in individual level, evaluate the efficiency of the decoction and the major components through P21/HBx transgenic mice model; 2) in cytological level, sort CD8+T cells from tumor tissue of transgenic mice and peripheral blood of patients with HBV-HCC by flow cytometry, explore the regulation of drugs to the quantity and function of exhausted T cells; 3) in molecular level, investigate the expression of key molecules in Notch1-RBP-Jκ-PD-1 signal pathway by MHCC97H cell line and CD8+T cells in HBV-HCC patients. Clarify the key target will shed lights on new immunotherapeutic strategies for hepatocellular carcinoma by the traditional Chinese medicine.

T细胞功能耗竭是肿瘤免疫逃逸的重要原因，与肿瘤进展密切相关。乙肝相关原发性肝癌患者T细胞功能受损，但机制不明。我们初步研究发现经验方养阴扶正解毒方能够下调乙肝相关原发性肝癌患者T细胞Notch1表达，恢复PD-1+耗竭T细胞功能，进而提高生存率。据此提出：“养阴扶正解毒方通过调控Notch1-RBP-Jκ-PD-1信号通路逆转T细胞功能耗竭，延缓肝癌进展”。本课题拟：1）在整体水平采用P21乙型肝炎病毒转基因小鼠肝癌模型，验证全方及拆方的疗效，筛选出逆转T细胞耗竭治疗肝癌的关键药物；2）在细胞水平分选转基因小鼠肿瘤组织及肝癌患者外周血CD8+T细胞，明确药物对耗竭T细胞数量及功能的调节作用；3）在分子水平应用MHCC97H肝癌细胞系及肝癌患者CD8+T细胞，检测Notch1-RBP-Jκ-PD-1信号关键分子，探讨全方及关键药物的作用靶点，为临床中医药参与肝癌免疫治疗提供新策略。

T细胞功能耗竭是原发性肝癌发生免疫逃逸的重要原因，是肝癌免疫微环境的显著特征之一。前期研究发现经验方养阴扶正解毒方能够下调乙肝相关原发性肝癌患者T细胞Notch-1表达，恢复PD-1+耗竭T细胞功能，改善肝癌患者生存率。在此基础上，本团队通过构建H22肝癌荷瘤小鼠模型、T细胞与肿瘤细胞体外共培养模型，发现：1）养阴扶正解毒方可抑制肝癌进展，刺激宿主免疫应答，其发挥作用的关键拆方为扶正方；2）养阴扶正解毒方及关键拆方可改善CD8+T细胞耗竭表型，恢复其增殖、凋亡、杀伤、细胞因子产生等免疫功能，改善免疫抑制微环境，并通过肝癌临床样本加以验证；3）采用体外CD8+T细胞与肝癌细胞系共培养模型，确证本方是通过调节Notch-PD-1信号通路逆转CD8+T细胞的耗竭。综上所述，本课题证实了养阴扶正解毒方的抗肿瘤药效，并筛选出关键拆方为扶正方；阐述了全方和关键拆方发挥抗肿瘤作用的机制在于逆转CD8+T细胞耗竭状态，恢复其免疫功能，改善肝癌免疫抑制微环境；进一步明确了养阴扶正解毒方发挥其抗肿瘤、免疫调节作用的通路机制为Notch-PD-1信号通路，为临床中医药参与肝癌免疫治疗提供新策略。