Expression of core gene cDNA of Chinese hepatitis C virus in HepG2 cell line

AlthoughthegenomeofseveralstrainsofhepatitisCvirus(HCV)havebeenclonedandscquenced,theHCVparticleshavenotbeenobservedsofar.Currently,becauseofverylowInfectionefficiency,oneofthemajorimpedimentstothestructuralanalysisofHCVgenomeandgeneticanalysisofviralreplicationisthelackofareliablecellculturesystempermissiveforH(IVreplicanon.Inthisstudy,usingrecomblnantDNAtechnlque,weconstructedarecombinantplasmidbysubcloningcoregenecDNAofChineseH(7VisolateIntoaeukaryoticexpressionvectorPTM3andexpres…     

Therapeutic effectiveness of interferon alpha on hepatitis B virus DNA in peripheral blood mononuclear cells

ＴｈｅｒａｐｅｕｔｉｃｅｆｆｅｃｔｉｖｅｎｅｓｓｏｆｉｎｔｅｒｆｅｒｏｎａｌｐｈａｏｎｈｅｐａｔｉｔｉｓＢｖｉｒｕｓＤＮＡｉｎｐｅｒｉｐｈｅｒａｌｂｌｏｏｄｍｏｎｏｎｕｃｌｅａｒｃｅｌｌｓＧｕｏＹａｂｉｎｇ（郭亚兵）；ＺｈａｎｇＪｉｓｕｏ（张继锁）；...     

Single-cell analysis of IL-4 and IFN-γ production by CD4+ T cells in peripheral blood from chronic hepatitis B virus-infected individuals

Objective To investigate the composition of Th[0]/Th(1)/Th(2) in chronic Hepatitis B Virus (HBV)-infected individuals by determining the expression of IL-4/IFN-γ in single CD4(+) T cell from peripheral blood, and to analyze the role of Th(1)/Th(2)cytokines in chronic HBV-infection. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from 3 ml fresh heparinized blood by Ficoll density-gradient centrifugation.The interphase PBMCs were suspended in RPMI 1640 culture medium supplemented with 10% heat-inactivated fetal calf serum (FCS), glutamine and antibiotics.Furthermore, in order to isolate CD4(+) cell rich population, anti-CD8 monoclone antibody (mAb) was added to the cell suspension at a final dilution of 1/120.After 60 minutes incubation at 37 fresh rabbit sera as a source of complement were added.Freshly isolated CD8(+) free PBMCs were enriched for lymphocytes by removal of adherent cells after 1.5 hours adherence to plastic culture flasks.Cells were stimulated with 1 ng/ml PMA, 1 μmol/L Ionomycin and 1 μmol/L monensin for 4 hours at 37℃.Cells were washed with PBS containing 0.5% (w/v) BSA.Then washing with PBS, cells were fixed with PBS containing 4% (v/v) paraformaldehyde for 10 minutes on ice.Cells were washed twice with PBS and permeabilized with PBS containing 0.1% (w/v) saponin, 0.5% BSA and 10% pooled human serum for 10 minutes on ice.Cells were then incubated with PE-labled antibodies against IFN-γ and FITC-labled antibodies against IL-4 for 45 minutes at 4℃.Before, between and after incubations with mAb, cells were washed with PBS containing 0.5% BSA and 0.1% saponin.Cell samples were analyzed on a fluorescence activated cell sorter (FACS).The percentage of IL-4/IFN-γ cytokines-producing cells was expressed.In each experiment, cryopreserved PBMC from the same healthy donor were analyzed in parallel to serve as an internal control.Results We developed an assay for intracellular staining of cytokines.Cells were able to produce IFN-γ (but not IL-4) which were defined as Th(1); cells were able to produce IL-4 (but not IFN-γ) which were defined as Th(2); Cells were able to produce IFN-γplus IL-4 which were defined as Th[0].Cells cultured in medium alone do not give a signal with any of the Abs used; whereas upon stimulation with PMA and ionomycin , part of the cells were positive for IL-4 and IFN-γ, but not the control Ab.Monensin was added to the cultures, which caused intracellular accumulation of newly synthesized proteins by arresting their transport principally in the Golgi complex.These results in a higher intracellular concentration for both cytokines, while in non-stimulated cells the cytokines remained undetectable in the presence of monensin.The percentage of IFN-γ-producing T cells and IL-4-producing T cells ranged from 2.3% to 18.6% and from 1.1% to 8.7% respectively in CD4(+) cells in non-infected individuals.The majority of CD4(+) T cells in peripheral blood from chronic HBV-infected individuals were Th[0] cells.The percentage of Th(1) cells increased significantly with the hepatic inflammation activity.The percentage of Th(1) cells in advanced period of chronic hepatitis B was higher than in stable period of chronic hepatitis B.The percentage of Th(2) cells in CD4(+) T cells from HBV-infected individuals did not differ significantly, but was higher than controls.Conclusions Th(1) cells are associated with hepatic inflammation activity of chronic hepatitis B, and Th(2) cells may be associated with the chronicity of HBV infection.     

Prospective study of mono-strand DNA virus (TTV) transmission by blood transfusion

AlthoughsensitiveandspecificimmunoassayandmolecularbiologicaltechniquesoftheknownhepatitisA-Evirusareavailable,theetiologyofasubstantialfractionofpost-transfusionandcom-munity-acquiredhepatitiscaseshaveremainedun-defined[1'2],hepatitisGvirus(HGV)couldbetheagentsofpartnonA-Ehepatitis,butthepathogenicityofHGVisstillremainedtobeidenti-fied,suggestingtheexistenceofadditionalcausativeagentsL"'.'].Anewhumanhepatitisre-latedviruswasisolatedbyagroupofJapanesesci-entists[6.7]-Thenewvirusisprovisio…     

Clinical significance of peripheral blood CD4＋ natural killer T cells in chronic hepatitis B virus infection

CD4+Thlymphocytesplayanimportantroleintheimmuneresponseagainstintracellularviral,bacterial,andparasiticpathogens.Onthebasisoftheircytokineprofile,Thcellscanbecategorizedinto3functionallypolarizedpopulations.Th1cells,capableofproducingIFN-γ,IL-2andTNF-β,areinvolvedprincipallyincell-mediatedimmunityandareintheprotectionagainstintracellularpathogens,suchasavarietyofviruses.Th2cells,secretingIL-4,IL-5,IL-6andIL-10andmostlyfunctioningtoregulatehumoralimmuneresponse,canexertbeneficialeffec…     

血浆置换治疗肝功能衰竭临床研究

目的观察血浆置换治疗肝功能衰竭的疗效。方法 148例患者在内科综合治疗的基础上给予血浆置换治疗,观察临床疗效和并发症发生情况,监测治疗前后肝功能生化指标的变化情况。结果 148例患者经血浆置换治疗后临床症状有不同程度缓解,有效率为62.2%（92/148）;临床症状无好转48例;死亡8例。治疗后TBil、谷丙转氨酶、谷草转氨酶、PT均低于治疗前,白蛋白和PTA均高于治疗前,差异均有统计学意义（P〈0.05和P〈0.01）。早期患者临床好转率为85.7%均高于中期的59.5%和晚期的38.0%,差异有统计学意义（P〈0.05）。连续3次以上（包含3次）血浆置换的患者TBil下降幅度为65.93%高于连续3次以下的43.49%,差异有统计学意义（P〈0.01）。148例患者共进行血浆置换治疗436次,出现不良反应58次（13.30%）。结论血浆置换治疗肝功能衰竭疗效明显,连续多次治疗对降低TBil效果更好,治疗早、中期肝功能衰竭最为适宜。     

T helper cells in patients with chronic hepatitis B virus infection

Objective To investigate the compositions of Th1/Th2/Th3 cells in chronic hepatitis B viru s (HBV)- infected individuals by determining the expression of interleukin- 4 (I L- 4), inetrferon-γ (IFN-γ), and transform growth factor-β (TGF- β) in si ngle CD4(+)T cells isolated from peripheral blood mononuclear cells (PBMCs) and the role of polarized Th cell populations in chronic HBV- infection was discuss ed. Methods PBMCs from chronically infected HBV individuals were isolated, stimulated by PMA /Ionomycin/Monensin, and IL- 4, IFN-γ and TGF-β production by CD4(+)T cells was determined by using fluorescence activated cell sorter (FACS) analysis. Results The percentage of IFN- γ- producing T cells, IL- 4- producing T cells and TGF- β- producing T cells ranged from 2. 3%-18. 6%, 1. 1%-8. 7% and 0. 7%-7. 1% resp ectively in CD4(+) T cells from non- infected individuals. Most of CD4(+)T cel ls from PBMCs in chronically infected HBV individuals were Th0 cells. The propo rtion of Th1 cells increased significantly with hepatic inflammatory activity, and in the active period of chronic hepatitis B infection were higher than those in the non- active period (P<0. 05). Th2 cell percentage in CD4(+)T c ells from HBV- infected individuals did not differ significantly (P>0. 05), but were higher than that from controls (P<0. 05). Th3 cell percentage in CD4(+) T cells from asymptomatic carrier (AsC) group was higher than that in the chronic hepatitis B (CHB) and control groups (P<0. 05). Conclusions Th1 phenotype cytokines were positively correlated with hepatic inflammatory act ivity in chronic hepatitis B and Th2 cells may be associated with the persistenc e of HBV infection. Th3 cells cooperating with Th2 cells can negatively regulat e immune responses and may be associated with the immune tolerant state of chron ic HBV infection.     

长效与常规干扰素治疗慢性乙型肝炎引起骨髓抑制的特点及其对疗效的影响

目的分析聚乙二醇干扰素α(Peg-IFN-α)与干扰素α(IFN-α)治疗慢性乙型肝炎(CHB)引起骨髓抑制的特点和差别、对抗病毒疗效的影响和两类干预药物的效果。方法 252例CHB患者分别给予Peg-IFN-α或IFN-α治疗48周,比较IFN应用过程中Peg-IFN-α治疗组和IFN-α治疗组白细胞(WBC)、中性粒细胞(Neu)和血小板(PLT)的变化及差异;以IFN治疗24周内血细胞计数最低值将患者再分组,比较各亚组干扰素的疗效;比较口服升白细胞药物和重组人粒细胞集落刺激因子(rhG-CSF)对干扰素所致Neu减少的疗效。结果 Peg-IFN-α治疗组引起骨髓抑制较IFN-α治疗组常见,两组WBC、PLT减少比率、Neu≤0.75×109/L比率差异有统计学意义(P<0.05);3.6×109/L>WBC≥2.5×109/L亚组较WBC≥3.6×109/L亚组有更高的HBeAg和HBVDNA阴转率(P<0.05),1.5×109/L>Neu≥0.75×109/L亚组较Neu≥1.5×109/L亚组有较高的HBeAg阴转率(P<0.05);与口服升白细胞药物相比,rhG-CSF对IFN引起的Neu减少疗效快速而短暂,二者升白细胞治疗1周和1月时的Neu计数及有效率差异均有统计学意义(P<0.05)。结论 Peg-IFN-α引起骨髓抑制较IFN-α更为常见,白细胞下降幅度可一定程度预测IFN的疗效,rhG-CSF用于IFN治疗CHB引起的骨髓抑制的临床价值值得进一步探讨。     

消痛散胶囊治疗偏头痛50例

笔者近五年来运用自拟消痛散胶囊治疗偏头痛50例,取得了较为满意的疗效,现报告如下。