RIBA在慢性丙型病毒性肝炎中的应用价值

采用含ＨＣＶ三种抗原（Ｃ_（２２），Ｃ_（３３），Ｃ_（１００））的重组免疫印迹试验（ＲＩＢＡ）检测了７１例抗－ＨＣＶ阳性（ＲＩＡ－２法）的慢性非甲非乙型肝炎患者血清并与ＨＣＶＲＮＡ结果进行了比较。ＲＩＢＡ阳性５６例，可疑８例，对应ＨＣＶＲＮＡ检出率分别为９４．６４％（５３／５６）和３７．５％（３／８），ＲＩＢＡ阴性的７例均未检出ＨＣＶ　ＲＮＡ。ＨＣＶＲＮＡ阳性病例中ＲＩＢＡ多为阳性，可疑仅占５．３６％（３／５６），无１例阴性。可见，ＲＩＢＡ阳性即表示ＨＣＶ感染且存在病毒血症，ＲＩＢＡ可作为第二代ＲＩＡ或ＥＬＩＳＡ查抗－ＨＣＶ的确证实验。     

丙型肝炎病毒1b DY株ns5a基因的克隆及其在大肠杆菌中的表达

目的克隆和表达丙型肝炎病毒（HCV）1b型地方株DY株ns5a基因。方法运用原核细胞基因工程技术。设计目的基因的特异引物，采用巢式PCR法，从含HCV1bDY株全长eDNA的质粒HCV17中扩增出约480bp的目的片段，将其插入克隆载体pMD18-Tvector中，再亚克隆到原核表达载体pET-28a中；经酶切、PCR及测序鉴定后转化入BL21菌株，在IPTG诱导下进行融合蛋白的表达；采用SDS-PAGE电泳及Western-blot检测NS5A蛋白的表达水平。结果成功构建了含有HCV1b DY株ns5a基因的重组体，并得以表达。结论成功构建和表达了HCV1b DY株ns5a基因，为进一步研究HCVns5a的基因型及探讨该基因编码的Ns5A蛋白的性质和生物学活性创造了条件。     

丙型肝炎病毒NS3-NS4基因的表达及鉴定

目的　构建丙型肝炎病毒 (HCV)非结构蛋白NS3 NS4全长基因重组质粒并诱导表达 ,鉴定NS3 NS4蛋白的抗原性。方法　应用PCR技术从pUC19/HCV中扩增目的基因 ,构建重组表达质粒 ,进行原核表达 ,用SDS PAGE、ELISA技术对表达产物的抗原性进行鉴定。结果　成功构建、表达了重组质粒pBV2 2 0 /NS3 NS4 ,用 5 0份质控标准血清对表达蛋白质进行抗原性检测 ,与第二代诊断试剂相比 ,总符合率为 96 %。结论　全长NS3 NS4蛋白是一个优势免疫原性区 ,应该是HCVEIA诊断试剂的有效成分。     

THE CHANGE OF SERUM TUMOR NECROSIS FACTOR-α LEVEL AND ITS CLINICAL SIGNIFICANCE DURING THE TREATMENT OF CHRONIC HEPATITIS B WITH LAMIVUDINE

Objective To evaluate the effect of lamivudine on immunity of chronic hepatitis B by observing the sequential changes of serum TNF-α and HBV-DNA level. Methods 31 CHB patients with elevated serum ALT/AST level and HBVDNA level higher than 106 copies/mL were treated with lamivudine (100mg/day) for one year. The sequential serum samples, which were taken at the 0, 3rd, 6th, 12th month after initiation of therapy, were used to detect serum level of TNF-α and quantity of HBV-DNA respectively. Results ① The serum TNF-α levels were higher than normal value before treatment in all patients; ② At In the 3rd month of treatment, The the serum HBV-DNA level began to decline and became negative in the 54.9% of all patients. At the end of treatment, HBV-DNA was negative in 48.4% of all patients; ③ The decrease of TNF-α level was later than HBVDNA level drop. TNF-α level began to decline after 6 months treatment. At the end of treatment, TNF-α level was lower than that at in 6th month, TNF-α level returned to normal in the 38.7% of all patients; ④ The TNF-α level decreased significantly after 6 months treatment in the patients with ALT>80IU/L at the beginning of treatment. But in the patients with ALT≤80IU/L, the TNF-α level decreased just after 12 months treatment; ⑤ TNF-α level fell obviously and early in patients whose HBVDNA became negative at in the 3rd month. Conclusion Lamivudine can suppress the replication of HBVDNA quickly, and decrease TNF-α level in the serum TNF-α level. It suggests that lamivudine can modulate immune response directly or indirectly. The changes of serum TNF-α level may be used to evaluate the clinical efficacy of lamivudine.     

丙型肝炎病毒C区截短型基因原核表达载体的构建及在大肠杆菌中的表达

目的建立稳定表达丙型肝炎病毒(HCV)核心蛋白的原核表达系统,获得高产量的纯化核心蛋白。方法应用多聚酶链反应(PCR),以HCV-H株全长cDNA序列为模板,扩增获得C区羧基端部分缺失的基因片段,克隆入原核表达载体pBVIL1,构建原核表达载体pBVIL1-Ct,转化HB101宿主菌,通过温度诱导表达截短型C蛋白。结果扩增得到目的基因长度为462bp,构建pBVIL1-Ct表达载体,在HB101宿主菌中通过温度诱导获得稳定表达,表达蛋白占菌体总蛋白含量的24%。Western-Blot及ELISA检测证实表达产物可与HCV患者阳性血清发生特异性结合反应。结论羧基端部分缺失的HCV C区基因片段可在大肠杆菌中稳定表达并具有良好的反应原性。     

白细胞介素-10启动区基因多态性与慢性丙型肝炎的相关性

目的　探讨白细胞介素 -10(IL- 10)启动区- 592 位点的基因多态性与慢性丙型肝炎的发生、发展以及转归的关系。方法　采用聚合酶链反应- 限制性片段长度多态性分析(PCR- RFLP)方法检测 85 例慢性丙型肝炎患者和 75 名健康对照者 IL- 10基因启动区域 592位点的基因型。结果　IL -10 基因启动区 592 位点基因型在慢性丙型肝炎患者与健康对照之间的分布无显著性差异(P> 0. 05); IL- 10 基因启动区 592 位点基因型在血清 HCV RNA> 105拷贝/mL组和HCV RNA≤105拷贝/mL组之间的分布无显著性差异(P>0.05);IL- 10 基因启动区 592 位点基因型在慢性丙型肝炎组和肝硬化组之间的分布无显著性差异(P>0.05); IL- 10 基因启动区 592 位点基因型在 ALT≥80U/L组中A/A基因型出现的比率高于ALT<80U/L组,两组之间分布存在显著性差异(P<0.01)。结论　IL- 10基因启动区域 592位点的基因多态性与慢性丙型肝炎和肝硬化患者的病毒血症水平无显著相关性;IL- 10 启动区域 592位点的基因多态性可能与慢性丙型肝炎患者的肝脏炎症反应有关。     

干扰素治疗乙型肝炎对照研究

采用精制人白细胞干扰素治疗乙型肝炎进行对照研究，结果表明，治疗组ＨＢｓＡｇ清除率（１０．５２％，４／３９），ＨＢＶＤＮＡ转阴率（４１．９３％，１３／３１）、ＨＢｅＡｇ转阴率（４５．９４％、１７／３７）明显高于对照组（３．６０％，９．９０％１６．００％），Ｐ＜０．０５，证实干扰素具有清除乙肝病毒，抑制其复制活性作用。     

HIGN LEVEL EXPRESSION OF HEPATITIS C VIRUS CORE GENE IN E.COLI

Hepatitis C virus (HCV) is the major agentfor the posttransfusinal and diffusive non- A,non-B hepatitis that could transform to chronic hepati-tis,and also has close relation to liver cancer andcirrhosis[1] .HCV is a single positive- stranded RNA viruswhose genome(about9.4kilobases) contains asingle uninterrupted open reading frame (ORF )which encodes a polyprotein precursor of 30 1 0～30 1 1 amino acid residues.The genome of HCV issimilar to flavivirus and pestivirus.In vitrostudieshav…     

Screening of hepatocyte proteins binding to NS5ABP37 protein by yeast-two hybrid system

Objective To investigate the biological function of NS5ABP37 and to look for proteins interacting with NS5ABP37 protein in hepatocytes. Methods We constructed bait plasmid expressing NS5ABP37 protein of hepatitis C virus (HCV) by cloning the gene of NS5ABP37 protein into pGBKT7, then the recombinant plasmid DNA was transformed into yeast AH109 (α type). The transformed yeast AH109 was mated with yeast Y187 (α type) containing liver cDNA library plasmid in 2×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-α-gal for selection and screening. After extracting and sequencing of plasmids from positive (blue) colonies, we made a sequence analysis by bioinformatics. Results We screened twenty-five proteins binding to NS5ABP37, including Homo sapiens cyclin Ⅰ (CCNI) gene, Homo sapiens matrix metallopeptidase 25 (MMP25) and Homo sapiens talin 1. Conclusion The yeast-two hybrid system is an effective method for identifying hepatocyte proteins interacting with NS5ABP37 of HCV. And the biological function of NS5ABP37 may be associated with glycometabolism, lipid metabolism and apoptosis.     

Induction of apoptosis and inhibition of proliferation of C6 glioma cells in vitro by tamoxifen

Objective To investigate the anti-tumor effect and mechanism of tamoxifen on rat C6 glioma cells. Methods C6 cells were cultured in Dulbecco＇s modified Eagle＇s medium （DMEM） with 3% fetal calf serum （FCS）, and treated with tamoxifen of different concentrations, i.e. group A （1.25 μmol/L）, group B （2.50 μmol/L）, group C （5. 00 μmol/L）, group D （10. 00 μmol/L）, group E （20. 00 μmol/L） and control group （0. 00 μmol/L）. Morphological changes, MTT assay and 5-bromo-2＇-deoxyuriding labeling ratio were assessed. Apoptosis was observed by flow cytometry. Results C6 cells treated with different doses of tamoxifen for 24, 48, and 72 hours became irregular in shape, while cells treated with vehicle grew normally. MTT assay showed that tamoxifen did not suppress C6 cell growth until 72 hours after treatment. Seventy-two hours after treatment, there were significant differences in cell viable rate between group A versus groups C, D and E; so did group B versus group D as well as group E （P〈 0.05 ）. BrdU incorporation assay indicated significant difference of BrdU labbled index （BrdU LI） among groups A, C, E and control group 48 hoers after treatment （P〈0.05）. And the BrdU LI decreased with the increased concentration of tamoxifen. Flow cytometry （FCM） showed significant difference between treated group and control group at 24, 48, and 72 hours after treatment （P〈0.05）. Conclusion Tamoxifen significantly suppresses the growth of C6 glioma cells in a time- and dose-dependent manner. The mechanism of tamoxifen suppressing C6 glioma cells may be inhibiting proliferation and inducing apoptosis. Therefore, tamoxifen can be a candidate as a chemotherapy agent for glioma.     

NIDDM患者红细胞免疫功能和C_3与CIC含量关系的研究

应用改良的红细胞酵母花环法测定了50例非胰岛素依赖型糖尿病(NIDDM)患者红细胞免疫功能,并与40例正常人的测定结果进行比较,结果表明:NIDDM患者红细胞免疫功能低于正常人(P<0.01)。本文还对红细胞免疫功能下降在NIDDM中的发病机理进行了初步探讨,并对红细胞免疫功能与循环免疫复合物(CIC)及补体C_3的关系也作了初步研究。     

丙型肝炎病原学检测的临床应用效果评价

目的　了解我国丙型肝炎病毒感染的病原学诊断现状 ,探讨合理有效的临床应用模式。方法　用酶联免疫吸附试验 (ELISA)检测抗HCV抗体 ,用荧光定量反转录聚合酶链式反应 (FQRT PCR)技术定量检测HCV RNA ,用速率法检测ALT水平。结果　 188份抗HCV IgG阳性标本中 10 3份HCV RNA阳性 (大于 10 3 拷贝·mL-1) ,阳性率 5 4 .7% ;10 7份HCV RNA阳性标本中 ,有 4份抗HCV IgG阴性 (3.7% ) ,二者一致率 6 9.8% ,不一致率 30 .2 % ;HCV RNA载量与ALT水平无明显相关性。结论　用ELISA检测抗HCV IgG筛查丙肝病人会有 3.7%的病毒血症者漏检 ,且有 4 5 .2 %的假阳性 (无传染性 ) ;用抗HCV IgG阳性加ALT升高作为诊断丙型肝炎现症患者价值有待探讨 ;抗HCV IgG的出现与否与HCV RNA载量无关。     

丙型肝炎病毒非结构蛋白3全长基因的克隆和表达

目的克隆和表达丙型肝炎病毒(HCV)非结构基因NS3,为进一步研究其基因功能奠定基础。方法设计特异性引物,以HCV全长cDNA质粒为模板进行PCR扩增,获得全长NS3基因。将其插入克隆载体pMD18-Tvector中,经酶切、PCR及测序鉴定后,与表达载体pET32a重组,后转入BL21菌株并诱导表达。采用SDS-PAGE电泳及Western-blot检测NS3基因的蛋白表达水平。结果含有NS3的重组体构建成功并在大肠杆菌中表达。结论运用原核细胞基因工程技术成功构建和表达了HCV非结构基因NS3蛋白,为尽一步研究HCVNS3基因功能奠定了基础。     

A STUDY ON THE ALTERATION OF FREE RADICALS、PROSTAGLANDINS AND THEIR RELATIONSHIP IN CHILDREN EITH ACUTE GLOMERULONEPHRITIS

ＡＳＴＵＤＹＯＮＴＨＥＡＬＴＥＲＡＴＩＯＮＯＦＦＲＥＥＲＡＤＩＣＡＬＳ、ＰＲＯＳＴＡＧＬＡＮＤＩＮＳＡＮＤＴＨＥＩＲＲＥＬＡＴＩＯＮＳＨＩＰＩＮＣＨＩＬＤＲＥＮＥＩＴＨＡＣＵＴＥ　ＧＬＯＭＥＲＵＬＯＮＥＰＨＲＩＴＩＳＹａｎＸｉａｏｃａｉ；，ＷｅｉＪｕ...     

多发性骨髓瘤患者超氧化物歧化酶和脂质过氧化物的改变及其意义

用邻苯三酚自氧化法和改良硫代巴比妥酸（ＴＢＡ）荧光微量法测定了多发性骨髓瘤（ＭＭ）患者初发期、缓解期和复发期红细胞超氧化物歧化酶（ＳＯＤ）活性和血浆脂质过氧化物（ＬＰＯ）含量。结果表明：ＭＭ初发和复发病例红细胞ＳＯＤ活性明显降低，血浆中ＬＰＯ含量明显增高，较正常对照均有明显差异。缓解期患者的ＳＯＤ活性和ＬＰＯ含量大致恢复正常水平。ＭＭ红细胞ＳＯＤ活力与血浆ＬＰＯ含量呈负相关（ｒ＝－０．４２５，Ｐ＜０．０５）），与血红蛋白浓度呈正相关（ｒ＝０．７２２，Ｐ＜０．００１），与骨髓中浆细胞百分数呈负相关（ｒ＝－０．５２６，Ｐ＜０．０５）。以上结果提示自由基损伤作用参与了ＭＭ的发生、发展和转归过程，且与病情的严重程度有关。     

经颈静脉肝内门体分流术治疗肝硬化门脉高压症对机体液递物质的影响及疗效

目的探讨经颈静脉肝内门体分流术(TIPS)治疗肝硬化门脉高压症对机体液递物质的影响及其临床疗效评价。方法肝硬化门脉高压症患者40例,分为TIPS组和内科组,每组20例。另选健康成人20例,作为正常对照组。应用放免法及生化分析法测定各组各时段血清一氧化氮(NO)、白细胞介素-6(IL-6)及血浆丙二醛(MDA)的水平,观察临床表现及肝功能的变化。结果TIPS术后所有患者出血停止,一般状况改善明显,TIPS术后1 d血清丙氨酸氨基转移酶(ALT)较术前升高,且差异有显著性(P<0.01),术后7 d有所降低,与术前相比无显著性差异。两治疗组40例患者血清NOI、L-6及血浆MDA水平治疗前后均高于健康正常人(P<0.01)。TIPS组术后NO水平与内科组治疗一周后相比明显升高(P<0.01)。TIPS组术后NO水平较术前明显升高(P<0.01)。结论TIPS治疗肝硬化门静脉高压症近期疗效显著,术后肝功能无明显损害;术后血清NO水平升高,其机制可能与TIPS引起门脉及全身血流动力学变化有关;MDA无明显变化,表明肝脏总的血流灌注减少不明显。     

STUDY ON THE IMMUNE FUNCTION OF ERYTHROCYTE IN PATIENT WITH NIDDM

ＳＴＵＤＹＯＮＴＨＥＩＭＭＵＮＥＦＵＮＣＴＩＯＮＯＦＥＲＹＴＨＲＯＣＹＴＥＩＮＰＡＴＩＥＮＴＷＩＴＨＮＩＤＤＭＸｕＪｉｎｇ（徐静）（ＤｅｐａｒｔｍｅｎｔｏｆＥｎｄｏｃｒｉｎｅ，ＳｅｃｏｎｄＡｆｆｉｌｉａｔｅｄＨｏｓｐｉｔａｌ，Ｘｉ＇ａｎＭｅｄｉｃａｌ...     

Methylation status of the interferon-gamma gene promoter in chronic hepatitis B

Objective To evaluate the methylation status at CpG site -55 in the interferon-gamma (IFN-7) gene promoter and its effect on IFN-7 expression in chronic hepatitis B. Method The authors recruited 30 patients with UBeAg-positive chronic hepatitis B (CHB), 30 HBeAg-negative CHB patients, and 30 healthy blood donors. Pyrosequeneing was used to determine the methylation status at CpG site -55 in the IFN-γ gene promoter following bisulfite treatment of DNA in peripheral blood mononuclear cells (PBMCs). The expression of IFN-γ was analyzed by real-time RT-PCR and ELISA. HBV DNA in PBMCs was detected by nested PCR. Results The methylation level at CpG site -55 in the IFN-γ gene promoter was significantly increased, resulting in subsequent down-regulation of the expression of this cytoldne in CHB. The methylation level at CpG site -55 was significantly higher in HBeAg-positive patients than in HBeAg-negative ones (P<0.01) and was also significantly higher in PBMCs from HBV DNA-positive patients than from HBV DNA-negative ones (P<0.01) ; the methylation level at CpG site -55 was positively correlated with the amount of HBV DNA in serum (P<0.01). Oonclusion IFN-γ gene expression appears to be regulated by methylation of the IFN-γ gene promoter in CHB; the methylation level at CpG site -55 is associated with HBV infection.     

多抗甲素对淋巴细胞免疫功能的影响

应用3H－TdR及14C－UR参入淋巴细胞的双标记方法，在体外培养条件下，研究多抗甲素对人淋巴细胞DNA及RNA合成功能的影响。结果表明，在多抗甲素的浓度为0．1～10mg／L时，可显著增强淋巴细胞DNA及RNA的合成功能；当多抗甲素的浓度增至50～100mg／L时，对淋巴细胞合成DNA及RNA的能力呈现抑制作用，3H及14C标记物的参入计数减少。多抗甲素对淋巴细胞DNA及RNA合成功能的影响呈现双相作用。     

人白细胞干扰素联合复方水飞蓟蜜丸治疗乙型慢性肝炎的疗效观察

本文报告人白细胞干扰素联合复方水飞蓟蜜丸治疗乙型慢性肝炎15例,采用小剂量(干扰素5万μ/日)长疗程(3～6个月)疗法,乙型肝炎血清标志物在15例中有10例出现有效指标,有效率达66.6%;对照组有效者5例,无效者10例,有效率33.3%,二者相比,有一定差异,但因病例少,差异不显著(P>0.05)。干扰素治疗前后曾检测血中干扰素含量,治疗前血中均测不出,治疗后3～7个月,血中干扰素量可达5～40μ/ml,停药后干扰素含量下降。用药后血中干扰素量明显增高者,疗效好,本文说明抗病毒药联合保肝药治疗乙型肝炎有一定作用,但仍需进一步观察。