Hepatitis C virus infection of human hepatoma cell line 7721 in vitro

AIM: To establish a cell culture system with long-term replication of hepatitis C virus in vitro. METHODS: Human hepatoma cell line 7721 was tested for its susceptibility to HCV by incubating with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various phases during the culturing periods. The presence of HCV RNA, the expression of HCV antigens in cells and/or supernatant were examined by RTPCR, in situ hybridization and immunohistochemistry respectively. RESULTS: The intracellular HCV RNA was first detected on d2 after infection and then could be intermittently detected in both cells and supernatant over a period of at least three months. The expression of HCV NS3,CP10 antigens could be observed in cells. The fresh cells could be infected by supernatant from cultured infected cells and the transmission of viral genome from HCV-infected 7721 cells to PBMCs was also observed. CONCLUSION: The hepatoma line 7721 is not only susceptible to HCV but also supports its long-term replication in vitro.

Hepatitis C virus may infect extrahepatic tissues in patients with hepatitis C

AIM To explore the status of extrahepatichepatitis C virus(HCV)infection and replicationin hepatitis C patients,and its potentialimplication in HCV infection and pathogenicity.METHODS By reverse-transcriptase poly-merase chain reaction(RT-PCR),in situhybridization(ISH)and immunohistochemistry,HCV RNA,HCV replicative intermediate(minus-strand of HCV RNA),and HCV antigens weredetected in 38 autopsy extrahepatic tissuespecimens(including 9 kidneys,9 hearts,9pancreas,5 intestines,2 adrenal glands,2spleens,1 lymph node,and 1 gallbladder)from 9hepatitis C patients,respectively;and thestatus of HCV replication in extrahepatic tissueswas studied.RESULTS By RT-PCR,all 9 patients werepositive for HCV RNA in kidney,heart,pancreas,and intestine,but only 6(66.7%)patients were positive for HCV replicativeintermediate.HCV RNA and HCV antigens weredetected in kidney,heart,pancreas,intestine,adrenal gland,lymph node,and gallbladder in 5(55.6%)and 6(66.7%)patients by ISH andimmunohistochemistry,respectively.HCV RNA and HCV antigens were not detected in theseextrahepatic organs in 3(33.3%)patients,although their livers were positive for HCV.HCVreplicative intermediate detected by RT-PCR wasconsistent with HCV RNA and HCV antigensdetected by ISH and immunohistochemistry(Kappa=0.42-0.75).HCV RNA and HCVantigens were detected in myocardial cells,epithelial cells of intestinal gladular,interstitialcells of kidney,epithelial cells of tubules andglomerulus,pancreas acinar cells and epithelialcells of pancreatic duct,epithelial cells ofmucous membrane sinus of gallbladder,cortexand medulla cells in adrenal gland,andmononuclear cells in lymph node.HCV RNA wasalso detected in bile duct epithelial cells,sinusoidal cells,and mononuclear cells in livertissues by ISH.CONCLUSION HCV can infect extrahepatictissues,and many various tissue cells maysupport HCV replication;extrahepatic HCVinfection and replication may be of'concomitantstate'in most of patients with hepatitis C.Theinfected extrahepatic tissues might act as areservoir for HCV,and play a role in both HCVpersistence and reactivation of infection.HCVas an etiologic agent replicating and expressingviral proteins in extrahepatic tissues itselfcontributes to extrahepatic syndromeassociated.HCV infection in a few patients withchronic HCV infection.

丙型病毒性肝炎患者肝组织Fas抗原的表达与HAI积分关系的研究

目的：研究丙型病毒性肝炎患者肝组织Ｆａｓ抗原的表达状况与肝细胞病理损伤的关系，探讨细胞凋亡在丙型肝炎发病机制中的作用和意义。方法：６９例丙型肝炎患者，均为血清抗－ＨＣＶＩｇＧ阳性或ＨＣＶＲＮＡ（ＰＣＲ）阳性，肝组织均为快速肝穿，福尔马林固定，连续切片，常规ＨＥ染色，按Ｋｎｏｄｅｌ方法计算ＨＡＩ积分前三项，根据Ｋｉｙｏｓｈｉ方法进行Ｆａｓ抗原半定量分析。结果：Ｆａｓ抗原在丙肝的阳性率为７４％，其中ＣＡＨ（８４．５％）。急肝（８３．４％）明显高于ＣＰＨ（６４．９％，Ｐ＜０．０５）。ＨＡＩ积分与Ｆａｓ抗原半定量分析显示，肝组织病理炎症积分越高，Ｆａｓ抗原表达量越高（Ｐ＜０．０５）。结论：Ｆａｓ抗原在丙型病毒性肝炎患者肝组织的表达与肝细胞的病理损伤明显相关。

慢性丙型肝炎患者肝组织中HCV-RNA和HCV、NS_3抗原的研究

为了研究丙型肝炎的发病机理应用原位杂交和免疫级化方法检测了38例慢性肝炎患者肝内HCV－RNA和HCV、NS3。11例丙型肝炎患者肝细胞中8例HCV－RNA阳性，6例HCV、NS3阳性；17例乙、丙肝重叠感染者7例阳性；10例慢性乙肝患者中求发现阳性结果。肝内HCV－RNA和HCV、NS3阳性与否同肝组织学病理改变的严重性无关。结果表明HCV可能无直接致病作用。

Fas和FasL在丙型肝炎肝组织中的表达及意义

目的了解丙型肝炎患者肝细胞损伤与Fas抗原表达的关系。方法采用免疾组织化学方法显示Fas抗原与Fas配体（FasL）在丙型肝炎肝组织的分布，并同时运用原位杂交方法，检测肝组织中的丙型肝炎病毒（HCV）RNA。结果Fas抗原主要位于肝细胞胞浆。Fas抗原表达阳性细胞在肝小叶中多呈散在或灶状分布，汇管区周围和碎屑样坏死区内阳性表达较强。FasL大多位于肝内浸润的淋巴细胞，某些病例肝细胞胞浆也可呈弥漫强阳性。Fas抗原表达的阳性率与肝组织炎症程度相关。结论在丙型肝炎患者，Fas抗原表达水平与炎症活动度有关，未发现（HCV）RNA原位杂交阳性细胞与表达Fas抗原的肝细胞之间的对应关系。