基于TGF-β1-Smad2信号途径研究补肾方抑制耐受性自然杀伤细胞分化治疗慢性乙型肝炎的作用机制

The persistent infection of hepatitis B virus activates the TGF-β1-Smad2 signaling pathway and induces differentiation of natural killer cells (NK) into tolerant NK, which is one of the reasons for the low HBeAg seroconversion rate in patients with chronic hepatitis B (CHB).Bushen Recipe is an effective prescription for the treatment of CHB, which can increase the number of NK cells in CHB patients, promote the secretion of IFN-γ, and improve the serological response of HBeAg.Therefore,we infer that Bushen Recipe can down-regulate the TGF-β1-Smad2 signaling pathway, inhibit tolerant NK differentiation, restore NK cell function, and improve host immune response to HBV.This study is intended to: 1. Analyze the effect of Bushen Recipe on TGF-β1 content in serum, tolerance NK ratio, NK cell Smad2 phosphorylation, activated receptor expression (NKG2D, 2B4) and functional changes (IFN-γ secretion, cytotoxic) ; 2. Isolation of NK cells from healthy human peripheral blood, pre-incubation with patient’s serum, healthy human’s serum and TGF-β1, respectively, to observe the target of Bushen Recipe to inhibit tolerance NK differentiation and improve NK cell function, and clarify the mechanism of Bushen Recipe in improving the serological response of HBeAg in CHB patients and revealing the advantages of Traditional Chinese Medicine in regulating CHB immunity.

乙肝病毒持续感染可活化TGF-β1-Smad2信号途径，诱导自然杀伤细胞（NK）分化为耐受性NK，是慢性乙型肝炎（CHB）患者HBeAg血清转换率低的原因之一。补肾方是治疗CHB的有效验方，可增加CHB患者NK细胞数量，促进IFN-γ分泌，提高HBeAg血清学应答。据此提出假说：补肾方可下调TGF-β1-Smad2信号途径，抑制耐受性NK分化，恢复NK细胞功能，改善宿主对HBV的免疫应答。本研究拟：1.分析补肾方对CHB患者外周血TGF-β1水平、耐受性NK比例、NK细胞Smad2磷酸化、活化受体表达（NKG2D、2B4）及功能变化（IFN-γ分泌、细胞毒性）的影响；2.分离健康人外周血NK细胞，分别与患者血清、健康人血清及TGF-β1预孵育，明确补肾方抑制耐受性NK分化、改善NK细胞功能的作用靶点，阐明补肾方提高CHB患者HBeAg血清学应答的分子机制，揭示中医药调控CHB免疫的优势环节

乙肝病毒持续感染可活化TGF-β1-Smad2信号途径，诱导自然杀伤细胞（NK）分化为耐受性NK，是慢性乙型肝炎（CHB）患者HBeAg血清转换率低的原因之一。补肾方是治疗CHB的有效验方，可增加CHB患者NK细胞数量，促进IFN-γ分泌，提高HBeAg血清学应答。本研究观察了：1.补肾方治疗慢性乙型肝炎的临床疗效；2.补肾方对CHB患者耐受性NK分化的影响；3.补肾方对人NK细胞增殖率、细胞毒性、细胞表面活化受体表达的影响；4.基于蛋白质组学技术探讨补肾方有效调控HBV相关免疫应答的机制。结果证实补肾方可抑制NKtolerant分化，一定程度上促进NKcytotoxic/总NK的比值升高；证实补肾方药物血清可促进NK细胞增殖，提高细胞毒功能，提高细胞表面活化受体NKG2D的表达；推测补肾方可通过上调NK细胞Integrin（αVβ3）表达，激活PI3K/Akt信号通路，活化免疫细胞功能从而改善了宿主清除HBV的能力。