HBV感染人胎盘滋养层细胞机制的初步探讨

目的 采用透射电镜观察HBV体外感染人胎盘滋养层细胞的超微结构变化.方法 HBV体外感染人胎盘滋养层细胞.ELISA检测培养上清中HBsAg,PCR检测细胞培养上清和滋养层细胞中的HBV DNA.HBV荧光定量PCR检测细胞培养上清中HBV DNA量(拷贝/ml).透射电镜观察滋养层细胞的超微结构.结果 感染组滋养层细胞培养上清中HBsAg在PBS清洗后12 h时A值为0.942,96 h时上升为1.264.PCR检测感染组细胞培养上清和感染组细胞HBV DNA均为阳性.PBS彻底清洗后0、12、36、60、84 h感染组细胞培养上清的HBV DNA分别为:＜103,3×104,6×105,5×105,3×105拷贝/ml.透射电镜观察到感染组滋养层细胞膜附近存在包涵素(Clathrin)形式的内吞小体形成,并且发现内吞小体内存在病毒颗粒样结构.在感染组细胞粗面内质网内发现HBsAg特异性的纤维丝状结构.结论 HBV可能经包涵素依赖的细胞内吞形式进入滋养层细胞,进而实现感染细胞或通过胞释作用将病毒排至细胞的对侧而实现穿越滋养层细胞屏障.     

HBV体外感染卵巢颗粒细胞

目的建立HBV体外感染颗粒细胞模型,研究HBV在颗粒细胞中的复制情况,为深入研究HBV经卵细胞母婴垂直传播提供研究平台。方法原代颗粒细胞体外培养后用HBV阳性血清感染。收集培养上清,在不同时点检测HBsAg、HBeAg定量,实时定量PCR检测HBVDNA。免疫组化检测培养细胞中的HBsAg和HBcAg。巢式PCR检测细胞中的HBVDNA及HBV-mRNA。原位杂交检测细胞内的HBVDNA。结果成功建立了HBV体外感染颗粒细胞模型,在培养上清中可以持续96h检测到HBsAg和HBV DNA,在细胞内检测到HBsAg和HBcAg的阳性信号,PCR扩增显示细胞内有HBVD-NA及HBV-mRNA的存在,原位杂交证实细胞内HBVDNA阳性。结论 HBV能够在体外感染颗粒细胞,并在其内复制,该结果为深入研究HBV经卵细胞传播机制提供了很好的研究平台。     

鸟苷结合蛋白1对柯萨奇病毒B3及乙型肝炎病毒体外介导的不同作用

目的构建人鸟苷结合蛋白1（hGBP-1）真核表达质粒，观察hGBP-1体外对柯萨奇病毒B3（CVB3）和乙型肝炎病毒（HBV）的抑制作用。方法长链RT-PCR扩增全长hGBP-1编码区基因，克隆到pCR2．1TA克隆载体，再亚克隆到pcDNA3．1（-）真核表达载体。体外转染HepG2细胞和Hela细胞，Western blot检测hGBP-1的表达。然后分别观察转染细胞中hGBP-1对HBV体外复制子pHBV1．3和CVB3的抑制作用。ELISA检测共转染HepG2细胞培养L清HBsAg、HBeAg水平；Southern blot检测细胞HBVDNA复制中间体。TCID50试验检测Hela细胞培养物中CVB3感染量。结果成功构建hGBP-1真核表达质粒，能在HepG2细胞和HeLa细胞进行高效表达。该质粒与pHBV1．3共转染HepG2细胞，不能抑制HBV复制，HBsAg、HBeAg及HBV DNA复制中间体水平与对照相比都无明显变化。转染质粒在HeLa细胞上对CVB3复制有明显的抑制作用，CVB3感染量显著降低，尤其在低剂量病毒攻击时能完全抑制CVB3复制。结论hGBP-1可能在IFN介导的抗CVB3中起重要作用，但不能抑制HBV的复制。     

基于PCR的乙型肝炎病毒体外中和试验

目的　建立一种新的基于PCR的乙型肝炎病毒(HBV)体外中和试验,将其用于HBV中和抗体的检测,为新型HBV疫苗的评估提供一种体外模型。方法　待检免疫血清与已知HBV作用后接种HepG2 细胞,吸附、洗涤、培养后提取细胞核酸,PCR检测HBVDNA以判断中和试验结果。结果　免疫血清与10倍最小PCR感染剂量的HBV作用后接种细胞,培养2 4h后检测表明,其中和滴度高,结果稳定。市售疫苗免疫小鼠血清、多数抗HBsELISA阳性人血清和2份含HBVS区的新型候选疫苗的免疫血清中和试验阳性,而正常小鼠血清、戊型肝炎病毒重组蛋白免疫血清和抗 HBs阴性人血清中和试验阴性。结论　基于PCR的HBV体外中和试验简单、快速、经济,具有良好的特异性和敏感性,可以用于新型HBV疫苗免疫效果的评估。     

新型CpGODN对乙型肝炎病毒复制的抑制作用

目的:探讨我室自行设计的CpG ODN(CpG BW001)在体外对乙型肝炎病毒(HBV)复制的抑制作用.方法:CpG BW001刺激人外周血单个核细胞(PBMC)48小时,用VSV病毒保护实验及ELISA法检测培养上清的病毒保护能力及其中的IFN-α含量.将上述CpG BW001刺激人PBMC的上清与HepG2.2.15细胞共孵育12天后收集培养上清,用放射免疫法检测该培养上清液中HBsAg及HBeAg的含量,用点杂交法检测HBV DNA的含量.结果:CpG BW001刺激人PBMC产生以IFN-α为主的抗病毒物质;CpG BW001刺激人PBMC的培养上清作用于HepG2.2.15细胞后,HepG2.2.15细胞培养上清中HBsAg和HBeAg的含量明显减少,且细胞内HBV DNA的含量也显著降低.结论:CpG BW001能通过刺激细胞产生抗病毒物质如IFN-α等,从而在体外抑制HBsAg和HBeAg的表达及HBV的复制.     

雌三醇对乙型肝炎病毒转录和复制水平的影响

目的:研究雌三醇对乙型肝炎病毒(HBV)转录和复制水平的影响。方法:采用不同浓度(从640μmol/L到5μmol/L倍比稀释)的雌三醇作用于体外培养的HepG2. 2. 15和HepG2-NTCP细胞,MTS法检测雌三醇的细胞毒性。用10、20和40μmol/L雌三醇处理HepG2. 2. 15和HepG2-NTCP细胞,以恩替卡韦(25 nmol/L)作为阳性对照,6 d后采用RT-qPCR法检测细胞中HBV DNA和RNA水平,ELISA法检测上清中乙型肝炎表面抗原(HBsAg)和乙型肝炎e抗原(HBeAg)水平。对雄性HBV转基因小鼠腹腔注射雌三醇(5 mg/kg),以恩替卡韦(0. 02 mg/kg)作为阳性对照,28 d后取小鼠血清和肝脏,采用ELISA法检测血清中丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、HBsAg和HBeAg水平,RT-qPCR法检测血清和肝脏中HBV DNA水平及肝脏中HBV RNA水平。结果:与对照组相比,10、20和40μmol/L雌三醇可以显著降低HepG2. 2. 15和HepG2-NTCP细胞分泌的HBsAg和HBeAg水平及细胞中HBV DNA和RNA水平(P0. 05)。此外,雌三醇也可以显著降低HBV转基因小鼠血清中HBsAg、HBeAg和HBV DNA水平,以及肝组织中HBV DNA和RNA水平(P0. 05)。结论:在HepG2. 2. 15细胞、HepG2-NTCP细胞和HBV转基因小鼠模型中雌三醇均具有较强的抑制HBV转录和复制水平的作用。     

HBV感染HepG2细胞早期过程研究

目的 探讨HBV吸附穿入HepG2细胞的方式，明确HBV难以自然感染细胞系的受限因素，为建立HBV自然感染细胞模型奠定基础。方法 在4℃和37℃条件下，HBV吸附HepG2细胞，用PCR方法检测胰酶消化液和细胞内的病毒DNA。另一部分HepG2细胞经过低温同步化处理，接种HBV后，于不同时相分别收集细胞和培养上清液，其中部分细胞进行核浆分离，通过问接免疫荧光、ELISA和PCR方法分别检测其中的病毒抗原和DNA，并且通过选择性PCR检测HepG2细胞HBV cccDNA。结果 在4℃条件下，HBV只是吸附于HepG2细胞膜表面。37℃条件下，部分吸附于HepG2细胞表面的病毒颗粒发生穿人，进入细胞浆，HepG2细胞和胰酶溶液中均可检出HBV DNA。接种病毒后4h、8h、24h HepG2细胞HBV DNA均呈阳性，至48h转为弱阳性，而96h为阴性。各时相上清液HBV DNA和病毒抗原均为阴性。HBV穿入HepG2细胞后，首先主要分布于胞浆中，随后出现胞核聚集性，胞浆中的病毒逐渐减少直至消失(感染后24h)，而胞核中的病毒数量早期逐渐增加，从48h开始呈下降趋势，至96h消失。病毒接种后8h核膜与核浆中均可检出病毒DNA。各时相cccDNA检测始终阴性。结论 HBV可能以“共受体”模式感染靶细胞。病毒脱衣壳过程受阻可能是HepG2细胞不支持HBV复制型感染的主要受限因素。     

肝脏内源性microRNA调控乙型肝炎病毒基因的表达与复制

目的探讨肝脏内源性microRNA对乙型肝炎病毒(HBV)复制与基因表达的影响。方法通过miRNA靶点分析软件寻找与HBV序列之间相关联的肝脏内源性microRNA,体外化学合成相应的microRNA分子,将合成寡核苷酸及对照与1.3倍HBV全基因组真核表达质粒pUC18-HBV1.3采用Lipofectamine2000共转染HepG2细胞,转染48h后收集细胞培养上清;通过ELISA检测HBsAg、HBeAg的表达水平;Western印迹检测HBcAg的表达水平;Trizol抽提转染细胞RNA,逆转录后用荧光定量PCR检测HBVmRNA的水平;提取细胞基因组DNA,Southern印迹检测HBV的复制中间体。经以上检测从HBV蛋白表达、转录和复制水平评价相应的microRNA作用效应。结果生物信息学方法提示miR-16和miR-122存在与HBV基因组作用的可能结合位点。经试验初步证实miR-16可下调HBV蛋白的表达及HBVDNA水平;miR-122可下调HBsAg、HBeAg的表达,上调HBVmRNA的水平。结论肝脏内源性microRNA可以调节HBV的复制与基因表达。     

乙型肝炎病毒表面抗原突变体稳定表达细胞系的建立和抗原性鉴定

目的 探讨乙型肝炎(乙肝)病毒(HBV)表面抗原(HBsAg)阴性HBV感染的分子机制。方法 在对46例HBsAg阴性但血清HBVDNA阳性感染者的S基因序列进行分析的基础上，构建了6株新的HBsAg变异株(4株联合点突变株，2株插入变异株)的EBO-plpp真核表达载体，并转染了COS7细胞，建立了这6株HBsAg变异株的稳定表达细胞系。分别用酶联免疫法(ELISA)和放射免疫法(RIA)对细胞表达的HBsAg抗原性进行检测。结果 除1例插入变异株可检测到较弱的阳性外，其余5株均检测不到HBsAg的存在。结论 新发现的HBsAg联合点突变和氨基酸插入变异，均对HBsAg的抗原性有负面的影响，是造成HBsAg阴性HBV感染的直接原因。     

CpG-ODN体外抑制乙型肝炎病毒复制的研究

目的:探讨CpG-ODN体外对HBV复制和HBV抗原表达的抑制作用。方法:CpG-ODN体外刺激人外周血单个核细胞(PBMC),用ELISA检测培养液中IFN-α及IFN-γ的水平。将不同比例的PBMC培养上清与HepG2. 2. 15细胞共孵育 2、4、6、8d后,用ELISA和荧光定量PCR法,分别检测培养上清液中HB sAg、HBeAg和HBVDNA的水平。结果:CpG-ODN可诱导PB MC分泌IFN-α和IFN-γ。CpG-ODN本身虽不能直接抑制HBV的复制,但经刺激活的化的PBMC的培养上清,却能显著抑制 2. 2. 15细胞产生HBsAg、HBeAg和HBVDNA,在培养的第 8天,抑制率最高分别达 90. 8%、31. 3%和 32. 2%。结论:CpG-ODN作为一种新型的免疫调节剂,可诱发机体的免疫效应,抑制HBV复制和HBV抗原的表达。     

Hepatitis B virus infection and replication in primary cultured human granulosa cells

To investigate the infection and replication of hepatitis B virus (HBV) in primary cultured human granulosa cells. Human granulosa cells were cultured with HBV-positive serum. Media were collected and assayed for HBsAg and HBeAg by ELISA, and HBV DNA by quantitative PCR. HBsAg and HBcAg were detected by immunocytochemistry in cultured cells. HBV DNA and RNA were extracted and amplified by nested PCR. Intracellular HBV DNA was localized by in situ hybridization. By co-cultivation of human GCs with HBV-positive serum, a system was established to study HBV infection and replication in GCs. HBsAg in medium could be detected from 4 to 96 h, and HBV DNA could be detected from 12 to 96 h after exposure. HBsAg and HBcAg showed positive signals by immunocytochemistry. A 206-bp fragment was amplified by nested PCR to detect HBV DNA and RNA in granulosa cells. HBV DNA was detected in GC nuclei by in situ hybridization. HBV can infect and replicate in human primary granulosa cells. This culture system could enable us to study infection of ova by HBV.     

Development of a rapid, single-step procedure using protein G affinity chromatography to deplete fetal calf serum of its IgG and to isolate murine IgG1 monoclonal antibodies from supernatants of hybridoma cells

Fetal calf serum (FCS) was depleted of its immunoglobulin G (IgG) in a rapid procedure using protein G affinity chromatography. 20 ml of FCS was depleted of its IgG in less than 80 min by applying 5 ml of FCS to a 1 ml HiTrap protein G Sepharose column followed by appropriate elution. Various concentrations of IgG-depleted FCS (G-FCS) were used in RPMI-1640 medium to grow the mouse hybridoma cell lines CAy-G (anti-HBs IgG1 mAb producing hybridoma cell) and CAy-M (anti-HBs IgM mAb producing hybridoma cell), which secreted hepatitis B virus surface antigen (HBsAg)-reactive IgG1 and IgM monoclonal antibodies (mAbs), respectively. Antibody production and cell growth were used as indices to compare the efficacy of RPMI/G-FCS with that of RPMI/FCS and serum/protein-free Hybri Max (Sigma, MO, USA) hybridoma medium. MAb production and cell growth of CAy-G and CAy-M hybridoma cell lines in RPMI/G-FCS were similar to culture in RPMI/FCS and significantly better than culture in Hybri Max. We found that G-FCS was superior to whole FCS as a culture supplement for the purification of IgG1 mAbs. IgG1 mAbs were isolated in a single-step procedure using protein G affinity chromatography, from the supernatant of CAy-G hybridoma cells cultured in RPMI/10% G-FCS (RPMI-1640 medium supplemented with 10% G-FCS). SDS-PAGE analysis revealed that the purity of IgG isolated from the supernatant of CAy-G cells cultured in RPMI/10% G-FCS was more than 99%.     

细胞人基因组DNA对荧光实时定量PCR检测细胞培养液中HBV DNA的影响

 目的 研究细胞人基因组 DNA 对荧光实时定量 PCR（FQ-PCR）检测细胞培养上清液中 HBV DNA 含量的可能干扰影响，提高细胞培养上清液中 HBV DNA 定量检测的准确性。方法 取 HBV DNA 阳性血清，以 DMEM 培养基分别配制出 HBV DNA 拷贝数为 5 × 107/ml（高拷贝组）、5 × 105/ml（中拷贝组）、5 × 103/ml（低拷贝组）的不同样本组；各组内再分 5 个亚组，分别加入终浓度为 0（对照组）、12.5、25、50、100 μg/ml 的细胞人基因组 DNA（提取自人肝癌细胞系 HepG2）。以不含 HBV DNA 阳性血清的 DMEM培养基加入上述浓度细胞人基因组 DNA 为空白组。采用基于 TaqMan 技术的 FQ-PCR 检测各组样本的 HBV DNA 拷贝数并绘制 HBV DNA 定量曲线。每组均重复检测 10 次。根据 HBV DNA 定量检测结果确定受干扰样本和未受干扰样本，分别计算其定量曲线线性期斜率和平均扩增效率。 结果 高拷贝组中加入不同浓度细胞人基因组 DNA 的各个亚组与相应对照组比较，HBV DNA 拷贝数差异均无统计学意义。中拷贝组中加入细胞人基因组 DNA 浓度为 50 和 100 μg/ml 的 2 个亚组，其 HBV DNA 拷贝数结果明显高于相应对照组，增高可达 50 ~ 100 倍，且重复性差。低拷贝组中只有加入细胞人基因组 DNA 浓度为 12.5 μg/ml的亚组可通过提高基线值取得与相应对照组接近的定量检测结果，而其他亚组 HBV DNA 定量曲线指数扩增期斜率明显异常，无法确定其定量检测结果。空白组各亚组均未观察到明显的 HBV DNA 指数扩增期。受干扰样本线性期斜率（-1.01 ± 0.06）和平均扩增效率（90.0% ± 2.1%）与对照组样本（分别为 -1.52 ± 0.06，97.0% ± 0.4%）比较，差异均有统计学意义（P < 0.01）。 结论 细胞人基因组 DNA 对应用 FQ-PCR 技术进行的HBV DNA 定量检测可产生非特异性干扰，尤其在 HBV DNA 拷贝水平较低时。在细胞培养上清液作 HBV DNA 定量检测时应尽量去除细胞人基因组 DNA。     

大鼠大脑皮质星形胶质细胞的体外培养及其促神经突起生长作用研究

取生后１周以内ＳＤ大鼠大脑皮质，用胰蛋白酶消化结合机械吹打使细胞分散，制成较大量的初细胞悬液。将初细胞悬液先行粘附处理３０ｍｉｎ以排除其中的成纤维细胞，再制成较小量的次细胞悬液。将之接种到预先涂有鼠尾胶原的培养瓶中，令其贴壁３～５ｈ，细胞密度约１×１０５个／ｃｍ２。补加培养液，继续培养。培养液为含２０％小牛血清的ＤＭＥＭ／Ｆ１２混合培养液。俟细胞铺满瓶底后传代。届时细胞悬液亦行３０ｍｉｎ的粘附处理。以０．５×１０５个／ｃｍ２的细胞密度种植于培养瓶中。传代后先培养４８ｈ；换成无血清培养液再培养４８ｈ并收集条件培养液。对部分传代细胞进行胶质原纤维酸性蛋白免疫细胞化学杂色鉴定，证明传代后的细胞中星形胶质细胞比例达９４．７１％。取条件培养液及单纯ＤＭＥＭ／Ｆ１２培养液各１份，分别与２份有血清培养液混合，制成实验组与对照组两种培养液，以悬滴法培养鸡胚背根节。４８ｈ后，比较两组背根节神经突起的平均长度。经统计学分析揭示，实验组背根节神经突起平均长度显著大于对照组者，说明星形胶质细胞具有明显的促神经突起生长作用。     

Morphologic, immunohistochemical, and ultrastructural studies of the production of hepatitis B virus in vitro

Well differentiated human hepatoblastoma Hep G2 cells after transfection with cloned hepatitis B virus (HBV) genomes produce replicative HBV DNA intermediates, high levels of HBsAg, HBeAg and HBcAg as well as mature Dane particles. To analyze the replication cycle of HBV, we studied the expression of HBV antigens with monoclonal antibodies by immunomorphologic methods in the transfected cells at various time intervals after plating. HBcAg and HBeAg were detected in the cytoplasm and less frequently in the nuclei of transfected cells. The percentage of positive cells increased with time after plating and reached a plateau of about 50% positive cells at 10 days. HBsAg and the large and middle HBsAg polypeptides were observed in the cytoplasm of transfected cells and a maximum of 20 to 30% positive cells was reached during the 3rd week after plating. Examination of viable cells in suspension revealed HBcAg/HBeAg and HBsAg expression on the cell surface. Electron microscopy demonstrated characteristic core particles in the nuclei and cytoplasm and Dane particles in cytoplasmic vesicles and culture media of transfected cells. The HBV producing cells did not show any evidence of a cytopathic effect. These observations demonstrate significant similarities between the HBV DNA transfected cells and infected human hepatocytes which support active HBV replication in vivo. Taken together, the results suggest that the cultured cells may serve as a model to elucidate a number of unsolved problems of the molecular and cellular pathobiology of hepatitis B.     

着色性干皮病基因D和p53对HBV复制的影响

 目的:探讨人着色性干皮病基因D(XPD) 和p53对乙型肝炎病毒（HBV）复制的影响。方法:使用脂质体转染法把重组质粒pEGFP-N2/XPD和空载质粒pEGFP-N2转染进入人肝癌细胞株HepG2.2.15细胞,转染后的第2天用20 μmol/L pifithrin-α（p53抑制剂）孵育细胞24 h。实验共分为空白对照组、pEGFP-N2组、pEGFP-N2/XPD组、pEGFP-N2/XPD+pifithrin-α组和pifithrin-α组。使用RT-PCR法检测XPD、乙型肝炎表面抗原（HBsAg）、乙型肝炎e抗原（HBeAg）及乙型肝炎病毒X蛋白（HBx） mRNA表达的变化;使用ELISA法检测培养上清液中HBsAg和HBeAg含量的变化;用荧光定量PCR法检测培养上清液中HBV-DNA含量的变化;用bDNA法检测细胞内核心颗粒中HBV-DNA含量的变化。结果:RT-PCR结果显示,重组质粒pEGFP-N2/XPD的转染可使得XPD mRNA表达增高,XPD表达增高能使得HBsAg、HBeAg和HBx mRNA表达明显减少,而pifithrin-α能抑制XPD的这一作用（均P<0.01）。ELISA结果显示,XPD表达增高能使得培养上清液中HBsAg和HBeAg含量明显减少,而pifithrin-α能抑制XPD的这一作用（均P<0.01）。荧光定量PCR结果显示,XPD表达增高能使得培养上清液中HBV-DNA含量明显减少,而pifithrin-α能抑制XPD的这一作用（均P<0.01）。bDNA结果显示,XPD表达增高使得细胞内核心颗粒中HBV-DNA含量明显减少,而pifithrin-α能抑制XPD的这一作用（均P<0.01）。结论: XPD能通过p53通路抑制HBV复制,所以XPD和p53可能成为乙型肝炎抗病毒治疗的作用靶点。     

Cellular chromosome DNA interferes with fluorescence quantitative real-time PCR detection of HBV DNA in culture medium

Fluorescence quantitative real-time PCR (FQ-PCR) is a recently developed technique increasingly used for clinical diagnosis by detection of hepatitis B virus (HBV) DNA in serum. FQ-PCR is also used in scientific research for detection of HBV DNA in cell culture. Understanding potential FQ-PCR interference factors can improve the accuracy of HBV DNA quantification in cell culture medium. HBV positive serum was diluted with culture medium to produce three test groups with HBV DNA levels of 5 x 10(7) copies/ml (high), 5 x 10(5) copies/ml (medium), and 5 x 10(3) copies/ml (low). Chromosome DNA was extracted from HepG2 cells and then added to high, medium, and low group samples at final concentrations of 0, 12.5, 25, 50, and 100 microg/ml. The samples were quantified by FQ-PCR and data were evaluated using statistical software. No marked changes were seen in the quantitative curves for high level HBV DNA samples when the samples were supplemented with 0-100 microg/ml of chromosome DNA. Interference was observed in medium level samples when 50 and 100 microg/ml of chromosome DNA was added. Interference was also observed in low level HBV DNA samples when the concentration of added chromosome DNA was greater than 25 microg/ml. The interference was eliminated when samples were digested by DNase I prior to PCR detection. In Conclusions, the presence of cellular chromosome DNA can interfere with the detection of HBV DNA by FQ-PCR. Removal of cellular chromosome DNA from culture media prior to FQ-PCR is necessary for reliable HBV DNA quantitative detection.     

Inhibition of human hepatocellular carcinoma and hepatoblastoma cell lines by deferoxamine.

Inhibition of human hepatocellular carcinoma (PLC/PRF/5 and Hep3B) or hepatoblastoma (Hep G2) cell lines by inclusion of deferoxamine mesylate (desferrioxamine) (DFX) in the culture medium was evaluated. When PLC/PRF/5 cells were maintained for 7 days in 30 or 60 microM DFX, the cell number was decreased by 30-60%, little or no alpha-fetoprotein (AFP) was produced, and supernatant endpoint dilution titers of hepatitis B surface antigen (HBsAg) were reduced 1-2 logs. PLC/PRF/5 cells maintained for 7 days without DFX (simultaneous controls) grew to confluence, produced AFP that reached 10-60 ng/ml in the supernate, and the HBsAg titer remained constant or increased 1 log. Similar effects were observed in Hep3B and Hep G2 cells maintained in DFX (except that Hep G2 cells do not produce HBsAg), compared to simultaneous control cells grown in the absence of DFX. The growth of a human embryonic lung fibroblast cell line (Wl 38) was not significantly inhibited by DFX, although it grew at a slower rate than simultaneous control cells grown without DFX. Subsequent growth in FeSO4 of PLC/PRF/5, Hep3B, and Hep G2 cells that previously had been maintained in DFX did not reverse the effects of DFX. PLC/PRF/5 cells were also inhibited when maintained in medium containing equimolar concentrations of DFX and FeCl3 and in medium containing equimolar concentrations of DFX and FeSO4. PLC/PRF/5 cells were not inhibited by maintenance in up to 60 microM of another chelating agent that has a similar affinity for iron, calcium disodium versenate (EDTA). These studies show that DFX inhibits the growth of human hepatocellular carcinoma and hepatoblastoma cell lines regardless of the presence (PLC/PRF/5, Hep3B) or absence (Hep G2) of integrated hepatitis B virus DNA. The findings also suggest that the inhibition may have been due to mechanisms other than iron chelation.