摘要目的:构建丁型肝炎病毒(HDV)感染的肝脏类器官系统，并探讨钠离子-牛磺胆酸共转运蛋白(NTCP)受体抑制剂布乐韦肽对HDV复制的抑制作用。方法:将由诱导多能干细胞(iPSC)分化的肝细胞样细胞(HLC)接种于倒置胶体晶体聚乙二醇支架(ICC)，构建肝脏类器官系统。质粒转染人肝癌细胞(HuH7)后，收获细胞上清中的HDV颗粒，同时提取HepG2.2.15细胞上清液中的乙型肝炎病毒(HBV)颗粒，将HBV和HDV颗粒共同感染肝脏类器官，构建HDV感染的肝脏类器官，同时以未感染HDV的肝脏类器官作为阴性对照组。采用免疫荧光法在激光共聚焦显微镜下观察肝脏类器官单元的结构及丁型肝炎抗原(HDAg)和乙型肝炎表面抗原(HBsAg)的表达。蛋白质印迹法检测肝脏类器官中NTCP和HDAg的蛋白质水平。布乐韦肽Pre组为感染HDV前在肝脏类器官中加入布乐韦肽进行预处理，布乐韦肽Post组为感染24 h后加入布乐韦肽，IFN-α组为感染24 h后加入α干扰素，并设未经药物处理的空白对照组，比较4组的HDV复制情况。采用实时荧光定量聚合酶链反应(RT-qPCR)检测iPSC分化过程中Nanog同源框(NANOG)、性别决定区Y框(SOX)2、SOX17、叉头框蛋白A2(FOXA2)、肝细胞核因子4α(HNF-4α)、白蛋白、甲胎蛋白、NTCP的mRNA相对表达量，以及药物干预后4组HDV mRNA表达量。统计学分析采用两独立样本 t检验。 结果:iPSC分化为HLC的21 d内，NANOG的mRNA表达量逐渐下降，SOX17、FOXA2的表达量先升后降，HNF-4α、白蛋白、甲胎蛋白、NTCP的表达量逐渐升高。iPSC中NTCP的蛋白质水平为0.118±0.003，低于HLC的1.315±0.073，差异有统计学意义( t＝11.92, P<0.001)。HDV感染后肝脏类器官中HDAg的蛋白质水平高于未感染HDV的阴性对照组(1.284±0.128比0.157±0.040)，差异有统计学意义( t＝23.27, P<0.001)。感染第14天激光共聚焦显微镜下观察到三维球体结构，HDAg与HBsAg高表达。用药干预后第3天，分别与空白对照组(1.000±0.077)比较，IFN-α组(0.453±0.028)和布乐韦肽Pre组(0.136±0.012)的HDV mRNA相对表达量均下降，差异均有统计学意义( t＝19.95、33.15，均 P<0.001)。而布乐韦肽Post组(0.968±0.069)与空白对照组的HDV mRNA相对表达量差异无统计学意义( t＝0.94， P>0.05)。 结论:iPSC衍生的HLC与ICC构建的肝脏类器官能模拟人体肝脏功能，并成功感染HDV颗粒。经布乐韦肽早期阻断能有效降低HDV感染肝脏类器官系统中病毒的复制水平。

abstractsObjective:To construct the liver organoid infected with hepatitis D virus (HDV), and to investigate the role of the sodium taurocholate cotransporting polypeptide (NTCP) receptor inhibitor bulevirtide in inhibiting viral replication.Methods:Hepatocyte-like cells (HLC) differentiated from induced pluripotent stem cells (iPSC) were seeded onto inverted colloidal crystal polyethylene glycol scaffolds (ICC) to construct liver organoids. After transfecting human hepatocelluar carcinoma cells (HuH7 cells) with plasmids, HDV particles were harvested from the supernatant, while HBV particles were extracted from the HepG2.2.15 cell supernatant. The liver organoids were infected with both HBV and HDV particles, and the negative control group without HDV infection was set up. The microstructure of the liver organoid units and the expression of hepatitis D antigen (HDAg) and hepatitis B surface antigen (HBsAg) were observed under laser scanning confocal microscope by immunofluorescence method. The protein levels of NTCP and HDAg in the liver organoids were detected by Western blotting. Bulevirtide was added before HDV infection (bulevirtide pre group) and 24 hours after infection (bulevirtide post group), and interferon-alpha (IFN-α) was also added after 24 hours infection (IFN-α group), and a control group without drug treatment was set up. HDV replication was compared among the four groups after drug intervention. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was used to measure the relative mRNA expression levels of Nanog homeobox (NANOG), sex determining region Y-box (SOX)2, SOX17, forkhead box protein A2 (FOXA2), hepatocyte nuclear factor 4 alpha (HNF-4α), albumin (ALB), alpha-fetoprotein (AFP), NTCP during the differentiation of iPSC, and the mRNA expression of HDV after the drug intervention of the four groups. Statistical analysis was performed using two independent sample t tests. Results:Within 21 days of the differentiation of iPSC into HLC, the mRNA expression level of NANOG gradually decreased, while the expression levels of SOX17, FOXA2 initially increased then decreased, and the expression levels of the HNF-4α, ALB, AFP and NTCP progressively increased. The protein level of NTCP in iPSC (0.118±0.003) was lower than that in HLC (1.315±0.073), and the difference was statistically significant ( t=11.92, P<0.001).The protein level of HDAg in the liver organoids after HDV infection was higher than that in the negative control group without HDV infection (1.284±0.128 vs 0.157±0.040), and the difference was statistically significant ( t=23.27, P<0.001).Laser scanning confocal microscopy showed three-dimensional spheroid structures and high expressions of HDAg and HBsAg at the 14th day of infection.Compared with the control group (1.000±0.077), the HDV mRNA expressions in both IFN-α group (0.453±0.028) and bulevirtide pre group (0.136±0.012) decreased after three days of drug intervention. The differences were statistically significant ( t=19.95 and 33.15, respectively, both P<0.001). However, there was no significant difference in HDV mRNA expressions between the bulevirtide post group (0.968±0.069) and the control group ( t=0.94, P>0.05). Conclusions:The liver organoids constructed from iPSC-derived HLC and ICC can simulate human liver functions and successfully be infected by HDV particles. Early blockade with bulevirtide can effectively reduce the level of viral replication in the HDV-infected liver organoids.