白花香莲解毒颗粒调控Treg细胞Furin/TGFβ1正反馈环路促进慢乙肝HBeAg血清学转换的机制研究

HBeAg Conversion (HC) is critical events to determine prognosis and course of Chronic Hepatitis B(CHB). At Presnt, it is the commanding point of the Anti-HBV field in the international most frontier direction and technological competition. Furin/TGFβ1 Positive Feedback Loop of Treg cell is the Core Mechanism in the process of chronic HBV, which can format and maintain immune tolerance by HBeAg driving. And it is the key target to improve HBeAg Conversion rates. Previous study showed that Treg cell was the independent factor to have an effect on HBeAg Conversion rates of CHB patients. The Baihua Xianglian Detoxification Particle can significantly improve HC rates through break immune tolerance induced by Treg cells . However, the molecular mechanism is not yet clear. Based on this hypothesis, The Baihua Xianglian Detoxification Particle promotes HC rates by regulating the Furin/TGFβ1 Positive Feedback Loop of Treg cell. We will build a non-contact 3d cell co-culture system, and using slow virus plasmid interference technique and so on. We will develop deeply research from cell transmembrane dialogue, signal loop intervention, upstream transcriptional regulation and other steps. The purpose of this study has two purposes, one is to clarify the precise mechanism of Furin/TGFβ1 Positive Feedback Loop in inducing peripheral immune tolerance formation and maintenance after the Treg cells stimulated by HBeAg. The other one is to know the targets that the Baihua Xianglian Detoxification Particle with detoxification and deficiency promotes HC rates.

HBeAg血清学转换（HC）是决定慢性乙型肝炎（CHB）预后及疗程的关键性事件，是当前抗HBV领域国际最前沿方向及技术争夺的制高点。Treg细胞中Furin/TGFβ1正反馈环路是一个在HBV慢性化过程中由HBeAg驱动的免疫耐受形成与维持的核心机制，是实现提高HC率的关键靶点。前期研究表明，Treg细胞是影响CHB患者HC率的独立因素，白花香莲解毒颗粒能突破Treg细胞诱导的免疫耐受，显著提高HC率，其机制尚未明确。据此提出“白花香莲解毒颗粒通过调控Treg细胞中Furin/TGFβ1正反馈环路促进HC”的假说，将构建非接触式三维细胞共培养系统，利用慢病毒质粒干扰等技术，从细胞跨膜对话、信号环路干预、上游转录子调控等步骤开展深入研究，旨在明确Furin/TGFβ1正反馈环路在HBeAg刺激Treg细胞后诱导外周免疫耐受形成与维持的精确机制及白花香莲解毒颗粒“解毒补虚”促进HC的作用靶点。

围绕“白花香莲解毒颗粒通过调控Treg细胞中Furin/TGFβ1正反馈环路促进HBeAg血清学转换”的假说，课题组开展了以下三个步骤研究：（1）分析HBeAg对Treg细胞内Furin/TGFβ1正反馈环路活化的影响；（2）分析白花香莲解毒颗粒干预Treg细胞中Furin/TGFβ1正反馈环路，对Treg免疫耐受功能及HBeAg表达的影响；（3）分析白花香莲解毒颗粒对Treg细胞Furin上游转录机制的影响。结果：（1）在Treg细胞与HBV1.3P-HepG2稳转细胞的非接触式三维细胞共培养（TDCC）体系，干扰Furin表达可下调Treg细胞上清中TGFβ1含量；干扰TGFβ1表达可下调Treg细胞内Furin蛋白和mRNA表达。（2）在TDCC体系中，白花香莲解毒颗粒能通过抑制Treg中Furin/TGFβ1正反馈环路的信号扩增效应，促进效应性T细胞（Teff）增殖及提高IL-2和IFN-γ表达；恢复细胞毒性T细胞（CTL）对HBV的特异性杀伤活性；下调Treg功能蛋白FOXP3表达等功效，突破由HBeAg驱动的Treg免疫耐受状态，实现抑制HBVDNA复制，提高HBeAg、HBsAg血清学转换率的疗效作用。（3）预测出Furin启动子的潜在转录因子，采用QPCR对筛选出的18个转录因子进行检测与验证，结果NR3C1、ESR1、TP53表达上调，GTF2I、ETS1、TCF4、FOXA1、FOXP3、pax5、TFIID、gata1、CEBPA、XBP1表达下调，IRF2、RXRA、AR、TFAP2A、ELK1表达变化不大；选择表达下调明显的GTF2I转录因子构建过表达病毒，侵染Treg细胞，结果GTF2I过表达可使Furin表达上调。结论：白花香莲解毒颗粒可通过下调Treg细胞内Furin上游GTF2I等转录因子的表达，减少Furin表达量，抑制Furin/TGFβ1正反馈环路的信号扩增效应，继而通过促进Teff/CTL增殖、恢复特异性杀伤活性，下调Treg关键标志物FOXP3表达等多途径，突破由HBeAg驱动Treg细胞所诱导的免疫耐受状态，最终实现抑制HBVDNA复制，促进HBeAg，甚至HBsAg血清学转换的疗效作用。