摘要
目的:以诱导多能干细胞(iPSC)和倒置胶体晶体聚乙二醇支架(ICC)为基础构建乙型肝炎病毒(HBV)感染的肝脏类器官系统,并验证核苷类药物的抗病毒作用。方法:将iPSC分化诱导成肝细胞样细胞(HLC),并接种至ICC中建立肝脏类器官系统。采用实时荧光定量聚合酶链反应(RT-qPCR)检测Nanog同源框(NANOG)、性别决定区Y框(SOX)2、SOX17、叉头框蛋白A2(FOXA2)、甲胎蛋白、白蛋白的mRNA相对表达水平;应用激光共聚焦显微镜对类器官三维结构进行摄片;采用蛋白质印迹法和免疫荧光分析HLC中钠离子-牛磺胆酸共转运蛋白(NTCP)表达水平。HepG2.2.15细胞提取HBV病毒颗粒感染肝脏类器官,RT-qPCR法检测细胞内HBV前基因组RNA(pgRNA)相对表达量;激光共聚焦显微镜下观察细胞质中乙型肝炎核心抗原(HBcAg)和乙型肝炎表面抗原(HBsAg)表达。分别以0.5μmol/L恩替卡韦、0.5μmol/L拉米夫定干预HBV感染细胞,采用RT-qPCR法检测感染与未感染细胞内HBV pgRNA相对表达量。统计学分析采用独立样本t检验和单因素方差分析。结果:iPSC分化21 d内,干细胞标志物NANOG、SOX2 mRNA表达水平降低(F=158.90、8.31,P<0.001,P=0.002),内胚层SOX17、FOXA2 mRNA表达水平先升高后降低(F=37.23、82.57,均P<0.001);分化后期,肝细胞中甲胎蛋白、白蛋白mRNA表达水平升高(F=4.65、34.64,P=0.012,P<0.001),差异均有统计学意义。蛋白质印迹法和荧光显微镜下显示分化后细胞中NTCP高表达,其蛋白质相对表达水平为0.803±0.099,激光共聚焦显微镜显示分化后肝细胞表达白蛋白,在ICC中呈现三维球体结构。HBV pgRNA表达和HBsAg、HBcAg的免疫染色证实HBV成功感染肝脏类器官系统。核苷类药物作用类器官系统3 d后,恩替卡韦组(0.665±0.220)和拉米夫定组(0.503±0.117)的HBV pgRNA水平较未感染细胞(3.347±0.454)明显下降,差异均有统计学意义(t=10.53、12.72,均P<0.001)。结论:iPSC分化后表现出肝脏特异性基因白蛋白和NTCP,以iPSC和ICC构建的肝脏类器官系统具有人体肝脏功能,HBV能感染该系统,恩替卡韦、拉米夫定能有效抑制肝细胞中HBV复制。
Objective To establish the hepatic organoid of hepatitis B virus(HBV)infection on the basis of induced pluripotent stem cells(iPSC)and an inverted colloidal crystal polyethylene glycol scaffold(ICC),and to evaluate the antiviral effect of nucleoside drugs.Methods iPSC was differentiated into hepatocyte-like cells(HLC),and inoculated into ICC to construct a hepatic organoid.The relative mRNA expressions of Nanog homeobox(NANOG),sex determining region Y-box(SOX)2,SOX17,forkhead box protein A2(FOXA2),alpha fetoprotein(AFP)and albumin(ALB)were detected by real time quantitative polymerase chain reaction(RT-qPCR).Confocal laser microscopy was used to photograph the three-dimension(3D)structure of organs.The expression of sodium taurocholate cotransporting polypeptide(NTCP)in HLC was analyzed by Western blot and immunofluorescence.HepG2.2.15 cells were used to extract HBV virus particles to infect hepatic organoid.The relative expression of HBV pregenome RNA(pgRNA)in cells was detected by RT-qPCR.The expressions of hepatitis B core antigen(HBcAg)and hepatitis B surface antigen(HBsAg)in cytoplasm were observed under confocal laser microscopy.A total of 0.5μmol/L entecavir and 0.5μmol/L lamivudine were used to treat the infected cells respectively.The relative expression of HBV pgRNA in infected and uninfected cells was detected by RT-qPCR.Independent sample t test and one-way analysis of variance were used for statistical analysis.Results Within 21 days of iPSC differentiation,the mRNA expressions of NANOG and SOX2 in stem cells markers decreased(F=158.90 and 8.31,respectivley;P<0.001 and P=0.002,respectively),while the mRNA expressions of SOX17 and FOXA2 in the endoderm increased first and then decreased(F=37.23 and 82.57,respectively,both P<0.001).In the later stage of differentiation,the mRNA expressions of AFP and ALB in liver cells increased(F=4.65 and 34.64,respectively,P=0.012 and P<0.001,respectively),and all differences were statistically significant.NTCP was highly expressed in differentiated cells detected by Western blot and fluorescence microscopy,the protein expression level was 0.803±0.099.Confocal laser microscopy confirmed that the differentiated cells expressed ALB and presented spherical structure in ICC.The expression of HBV pgRNA and the immunostaining of HBsAg and HBcAg confirmed that HBV successfully infected hepatic organoid.Three days after the application of entecavir and lamivudine,the HBV pgRNA level decreased significantly both in entecavir group(0.665±0.220)and lamivudine group(0.503±0.117)compared to the uninfected cells(3.347±0.454),and the differences were both statistically significant(t=10.53 and 12.72,respectively,both P<0.001).Conclusions HLC display hepatic specific genes ALB and NTCP.Hepatic organoids constructed with iPSC and ICC have human liver function and can be infected by HBV.Entecavir and lamivudine could effectively inhibit the replication of HBV in hepatic organoids.
作者
陈金梅
沈乐而
陈洁
马思远
臧国庆
陈小华
Chen Jinmei;Shen Leer;Chen Jie;Ma Siyuan;Zang Guoqing;Chen Xiaohua(Department of Infectious Diseases,Shanghai Sixth People′s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine,Shanghai 200233,China)
出处
《中华传染病杂志》
CAS
CSCD
2023年第4期263-268,共6页
Chinese Journal of Infectious Diseases
基金
国家自然科学基金(82070615)
上海市科技计划项目(21140901100)。
关键词
诱导多能干细胞
肝脏类器官
HBV感染
核苷类药物
Induced pluripotent stem cells
Hepatic organoid
Hepatitis B virus infection
Nucleoside analogues
