Objective: To explore the effect and mechanism of activation of peripheral blood mononuclear cell (PBMC) Toll-like receptor (TLR3) signaling pathway in recombinant HBsAg (rHBsAg) immune response. Methods: White blood cells were collected from peripheral blood of 13 healthy donors in the preparation of blood products. PBMC was isolated and treated with Poly I:C (Poly I:C group) and PBS (control group) respectively. 48 h later, some cells were collected and the expressions of TLR3 signaling pathway proteins were detected by flow cytometry. After activating (Poly I:C group)/inactivating (control group) TLR3 signaling pathway, rHBsAg was given to both groups for 72 h, and the proportions of DC, T, B cells and their subsets in PBMC were detected by flow cytometry. Paired t-test, paired samples wilcoxon signed-rank test and canonical correlation analyses were used for statistical analysis. Results: The percentage of TLR3 protein-positive cells (19.21%) and protein expression (8 983.95), NF-κB protein expression (26 193.13), the percentage of pNF-κB protein-positive cells (13.73%) and its proportion in NF-κB (16.03%), and the percentage of pIRF3 protein-positive cells (12.64%) and its proportion in IRF3 (21.80%) in Poly I:C group were higher than those in control group (11.54%, 8 086.00, 22 340.66, 8.72%, 9.71%, 9.57%, 19.12%) (P<0.05), and the percentage of TRIF protein-positive cells (89.75%) and protein expression (304 219.54) were higher in Poly I:C group than in the control group (89.64%, 288 149.72) (P>0.05). After PBMC stimulation by rHBsAg, the proportions of mDC (2.90%), pDC (1.80%), B cell (5.31%) and plasma cell (67.71%) in Poly I:C group were significantly higher than those in the control group (1.83%, 0.81%, 4.23%, 58.82%) (P<0.05). Results of canonical correlation analysis showed that the expression of TLR3 protein was positively correlated with the proportions of plasma cells, the expression of pIRF3 protein was positively correlated with the proportions of plasma cells and mDC, and the percentage of pNF-κB protein-positive cells and the percentage of pIRF3 protein-positive cells were positively correlated with the proportion of CD4+T cells. Conclusions: Poly I:C can activate TLR3/TRIF/NF-κB and TLR3/TRIF/IRF3 signaling pathway, promote the function of downstream signaling molecules, and then promote the maturation of DC, induce the immune responses of CD4+T cell, and promote the maturation and activation of B cells and the immune response of rHBsAg.
目的： 探讨外周血单个核细胞（PBMC）Toll样受体3（TLR3）信号通路的活化在重组HBsAg（rHBsAg）免疫应答中的作用及机制。 方法： 收集13名健康献血者外周血制备血液制品时滤除的白细胞，分离培养PBMC后分别给予TLR3激动剂聚肌苷酸-聚胞苷酸（Poly I：C组）及PBS（对照组）处理，48 h后收集部分细胞，采用流式细胞术检测TLR3信号通路蛋白水平；在活化（Poly I：C组）/未活化（对照组）TLR3信号通路后，采用rHBsAg处理两组PBMC 72 h，采用流式细胞术检测PBMC中树突状细胞（DC）、T、B淋巴细胞及其亚群比例。采用配对t检验、配对资料的符号秩和检验和典型相关分析进行统计学分析。 结果： Poly I：C组PBMC TLR3信号通路中TLR3蛋白阳性细胞百分比（19.21%）、TLR3蛋白表达量（8 983.95）、NF-κB蛋白的表达量（26 193.13）、磷酸化NF-κB（pNF-κB）蛋白阳性细胞百分比（13.73%）及其占NF-κB的比例（16.03%）、磷酸化IRF3（pIRF3）蛋白阳性细胞百分比（12.64%）及其占IRF3的比例（21.80%）均明显高于对照组（分别为11.54%、8 086.00、22 340.66、8.72%、9.71%、9.57%、19.12%）（P<0.05），TRIF蛋白阳性细胞百分比（89.75%）和蛋白表达量（304 219.54）均高于对照组（89.64%、288 149.72）（P>0.05）；经rHBsAg处理后，Poly I：C组髓样DC（mDC）（2.90%）、浆细胞样DC（1.80%）、B细胞（5.31%）比例及浆细胞占B细胞比例（67.71%）均明显高于对照组（1.83%、0.81%、4.23%、58.82%）（P<0.05）；TLR3信号通路相关蛋白阳性细胞百分比和蛋白表达量与免疫细胞之间均存在典型相关，TLR3蛋白表达量与浆细胞比例、pIRF3蛋白表达量与浆细胞和mDC比例、pNF-κB和pIRF3蛋白阳性细胞百分比与CD4+T细胞的比例均正相关。 结论： Poly I：C可以活化PBMC TLR3/TRIF/NF-κB和TLR3/TRIF/IRF3信号通路，促进下游信号分子发挥功能，进而促进DC的成熟，诱导CD4+T细胞免疫反应，促进B细胞的成熟分化，促进rHBsAg的免疫应答。.