补肾健脾方通过调节DC模式识别受体的表达诱导CD4+T细胞亚群分化平衡治疗慢乙肝的机制

The imbalance of CD4+T cell subsets is a major cause of chronicity of hepatitis B, which is associated with the abnormalities of DCs function. Large sample study showed that spleendeficiency and dampness heat and kidney deficiency is the basic pathogenesis of chronic hepatitis B, and the clinical RCT research confirm that Bushen Jianpi Recipe is an effective prescription for the treatment of chronic hepatitis B, its efficacy is associated with the improvement of the function of DC and the balance of CD4 + T cell subsets differentiation. Our previous studies showed that HBV regulated the expression of PRRs in DCs and inhibited the differentiation of CD4+T cells to Th1, and promoted the differentiation of of CD4+T cells to Th2/Treg. Based on the mechanism of the differentiation of CD4+T cell subsets induced by PRRs expressed on DC, the hypothesis was presented: "the recipe treat chronic hepatitis B by balancing immune cell network through the" PRRs on DCs - cytokine - key transcription factor during the differentiation of CD4+T cell "pathway. Sample library of clinical RCT study (serum and peripheral blood mononuclear cells) will be used in this study, PRRs on DCs，cytokines and key transcription factor during the differentiation of CD4+T cell were screened. The coculture system of DC and naive CD4+T cells will be used to build the immune model”HBV- PRRs on DCs - cytokine - key transcription factor during the differentiation of CD4+T cell”, which will help to clarify the target point of traditional Chinese medicine and to explain the immune mechanism of the treatment of chronic hepatitis B with the Bushen Jianpi Recipe

CD4+T细胞亚群失衡是乙肝慢性化的主要原因，与DC功能异常有关。大样本研究表明脾虚湿热兼肾虚是慢乙肝基本病机，临床RCT研究证实补肾健脾方是治疗慢乙肝的有效方剂，其疗效与改善DC功能、平衡CD4+T细胞亚群分化有关。前期证实，HBV调节DC模式识别受体（PRRs)表达，抑制CD4+T细胞向Th1分化，促进Th2/Treg分化。基于DC的PRRs诱导CD4+T细胞亚群分化的机制，提出假说:补肾健脾方通过“DC的PRRs-细胞因子-CD4+T细胞分化关键转录因子"途径，平衡免疫细胞网络治疗慢乙肝。应用RCT研究样本库（血清和外周血单个核细胞），筛选与补肾健脾方疗效有关的DC表达的PRRs、细胞因子及CD4+T细胞分化转录因子；应用DC /CD4+T细胞共培养体系建立“HBV→DC的PRRs→细胞因子→关键转录因子→细胞亚群”免疫模型，明确中药作用靶点，阐释补肾健脾方治疗慢乙肝的免疫机制。

CD4+T细胞亚群失衡是乙肝慢性化的主要原因，与DC功能异常有关。大样本研究表明脾虚湿热兼肾虚是慢乙肝基本病机，临床RCT研究证实补肾健脾方是治疗慢乙肝的有效方剂，其疗效与改善DC功能、平衡CD4+T细胞亚群分化有关。前期研究证实，HBV调节DC模式识别受体（PRRs)表达，抑制CD4+T细胞向Th1分化，促进Th2/Treg分化。基于前期结果，首先收集“十二五”RCT临床试验患者外周血以及PBMC样本。其次，通过流式细胞术分析治疗前后患者血清中13个TH细胞因子的变化情况，结果表明，与治疗前相比，治疗后患者血清IL-10水平明显降低，差异有统计学意义。结合临床疗效，以HBsAg治疗后较治疗前下降10%作为临床有效标准将样本分组，应用的信号通路磷酸化广谱筛选抗体芯片（PEX100），筛选了31条信号通路的584个磷酸化位点。找出组间差异磷酸化蛋白。在通过组间两两比较，得到与补肾健脾方疗效相关的磷酸化水平上升的位点8个。随后，ELISA的方法验证了对照组与实验组各40例患者PBMC的NFkB1的磷酸化水平。结果显示，与对照组相比，补肾健脾方组p-NFkB1的OD值显著升高。将THP -1诱导得到的DC细胞先予补肾健脾方血清及正常大鼠血清干预48小时后，加入大三阳慢乙肝患者血清刺激细胞8h，结果表明，慢乙肝患者的血清可明显上调TLR3、TLR7、TLR9、RIG-1和MDA-5的表达，补肾健脾方可明显降低TLR3和RIG-1的表达水平。明确此方对DC细胞先天免疫模式识别受体表达以及DC细胞对辅助性T细胞亚群分化的的调节作用。比较HBV转基因小鼠以及高压微静脉注射质粒C57小鼠的构建模型情况，确定适合的动物模型进行进一步机制研究。