多种肝外组织内丙型肝炎病毒RNA和抗原表达的研究

我们分别采用原位杂交和链霉菌亲生物素蛋白过氧化物酶连接（ＳＰ）法检测了９例丙型肝炎患者肝外组织内丙型肝炎病毒（ＨＣＶ）ＲＮＡ和多种抗原。一、资料与方法９例均为１９８６～１９９４年收住院的重型肝炎死亡患者。男８例，女１例。年龄２５～４８岁。经血清学（抗ＨＣＶ）、肝组织内ＨＣＶＲＮＡ和抗原检测证实为ＨＣＶ感染者，均合并乙型肝炎病毒（ＨＢＶ）感染。患者肾、心、胰腺、肠（仅５例）、胆囊（仅１例）和肝等共４２份标本取自尸解组织存档石蜡块。原位杂交和ＳＰ方法及特异性确证实验设置参照文献［１，２］。主…     

肝病患者肝组织HCV抗原的检测及意义

目的 探讨肝病患者肝组织丙型肝炎病毒（HCV）抗原的表达及意义。方法 应用免疫组织化学方法对252例肝病患者石蜡包埋肝组织中HVC抗原进行了检测。结果 多克隆抗HCV检出HcAg阳性25例（9．92％）,单克隆抗HCV-NS3检出HCV—NS3蛋白阳性19例（7．54％）。19例HCV—NS3阳性肝组织均同时表现HcAg阳性,二者在肝细胞分布状况相似,不同病理类型肝炎患者肝组织中HCV抗原检出率无统计学意义。252例血清检出HCV标志物阳性71例,单独抗HCV阳性者无1例肝组织中检出抗原。结论 肝组织中抗原的检出和血清HCV标志具有良好对应性,但不同血清HCV标志模式抗原检出率有统计学意义。     

慢性丙型肝炎患者外周血单个核细胞中HCV RNA和NS5抗原的检测

目的 研究丙型肝炎病毒（ＨＣＶ）感染外周血个核细胞（ＰＢＭＣ）的情况及其意义。方法 运用非核素原位杂交法（ＮＩＳＨ）和抗生蛋白链菌素－生物素法（ＳＡＢＣ）分别检测２０例慢性丙型肝炎患者ＰＢＭＣ中的ＨＣＶ ＲＮＡ和ＮＳ５抗原。结果 ２０例患者中有８例（４０％）,ＰＢＭＣ中ＨＣＶ ＲＮＡ呈阳性,其中６例（３０％）ＰＢＭＣ中ＮＳ５抗原同时呈阳性。ＨＣＶＲＮＡ主要分布于胞浆中,而ＮＳ５抗原还可以出现在胞膜     

丙型肝炎核心抗原检测在丙型肝炎诊断中的意义

目的了解丙型肝炎核心抗原（HCV-cAg）检测方法的敏感性及特异性,确定具有临床意义的S/CO值,探讨其在丙型肝炎诊断中的意义。方法使用ELASA方法检测丙型肝炎核心抗原,RT-PCR检测HCV RNA定量,观察不同S/CO值所对应的HCV RNA定量之间的关系,以HCV RNA为诊断金标准,列四格表做诊断实验。结果 HCV-cAg抗原检测的敏感性为87.05%,特异性为76.67%,阳性预测值为96.53%,阴性预测值为44.23%。结论 （1）随着HCV-cAg的S/CO值逐渐增大,其与HCV RNA阳性符合率明显增高,随着HCV-cAg的S/CO值减小,其与HCV RNA阴性符合率明显增高;（2）S/CO值=2可以作为临床判断HCV感染病毒血症存在的一个标准;（3）本实验的敏感性和特异性较好,检测方法简单,可以作为丙型肝炎临床诊断的补充试验及筛查。     

罗氏COBAS HCV RNA定量一代和二代检测试剂对556例丙型肝炎患者HCV RNA定量检测结果的比较

目的评估两代HCV核酸定量检测试剂的临床应用。方法收集南京市第二医院住院和门诊HCV感染者556例,采用罗氏COBAS HCV核酸定量一代和二代检测试剂检测其标本,其中HCV RNA阴性血浆93份;HCV RNA阳性血浆350份;血清血浆配对标本52份;干扰样本61份。将阳性血浆样本分为3组,低浓度(3次方及以下)61例、中浓度(4、5次方组)191例和高浓度89例(6次方及以上)。采用线性分析法,计算相关系数和回归方程,比较各组之间的关系。结果总体分析2种HCV RNA定量检测试剂的结果具有相关性(r=0.948,P0.05)。低浓度组的线性回归方程y=0.9809x+0.1359,r2=0.8047,P0.05;中浓度组的线性回归方程y=0.8130x+1.0727,r2=0.6956,P0.01;高浓度组的线性回归方程y=0.6746x+1.8914,r2=0.3088,P0.01。血清血浆有效对比检测结果的相关性良好(r2=0.9698,P0.05),线性回归方程y=0.9368x+0.4469。结论两代HCV RNA定量检测试剂符合程度较好,在低浓度组中具有良好的相关性,更适用于临床HCV RNA低浓度的检测。     

慢性丙型肝炎患者肝组织中病毒和Fas抗原表达及其与干扰素应答的关系

目的 探讨丙型肝炎患者肝组织中HCV Ns5和Fas抗原表达及其与肝组织损伤和干扰素治疗反应的关系.方法 应用a干扰素对一组慢性丙型肝炎患者进行24周治疗,停药后随访6个月,以血清丙氨酸转氨酶(ALT)恢复正常及血清HCV RNA持续阴转为完全应答标准,采用免疫组化方法检测组织中病毒和Fas抗原表达状况.结果 29例慢性丙型肝炎患者肝组织中,HCV NS5和Fas抗原呈阳性者均为19例(65.5%),两者在肝组织中的表达显著相关;单因素相关分析时,两者均与组织病变程度有关,但多因素分析表明只有HCV NS5抗原表达水平与组织炎症活动度呈正相关.治疗和随访结束时分别有17例(58.6%)和15例(51.7%)患者表现为近期和远期完全应答.治疗前Fas表达水平与干扰素应答有关,但未见HCV NS5水平与干扰素应答有明显关系;5例随访结束时重复肝穿,3例完全应答者病毒表达显著受抑,但Fas抗原无明显改变,2例无应答者病毒表达无明显改变甚或增强,而Fas表达均增强.结论 慢性丙型肝炎肝组织中HCV Ns5表达在慢性丙型肝炎发病过程中有重要意义;Fas抗原表达水平与组织中病毒表达相关;治疗前肝组织Fas表达状况与干扰素治疗反应有一定联系.     

肝病患者肝组织中HCV抗原检测及意义

应用免疫组织化学方法对一组肝病患者石蜡包埋肝组织中丙肝毒抗原进行了检测，２５２分肝组织中，采用多克隆抗体ＨＣＶ检出ＨＣＡｇ阳性２５份（９．９２％）采用单克隆抗ＨＣＶ－ＮＳ３检出ＨＣＶ－ＮＳ３蛋白阳性１９份（７．５４％），１９份ＨＣＶ－ＮＳ３阳性肝组织均同时表现ＨＣＡｇ阳性，二者在肝细胞分布状况相似，另有６例肝组织中检出ＨＣＡｇ阳性，阳性细胞周围多无坏死灶和炎性，不同病理类型肝炎患者肝组织中丙肝病毒     

丙型肝炎病毒感染者肝组织中病毒抗原表达的免疫组化研究

为探讨丙型肝炎病毒（ＨＣＶ）在肝组织中的表达状况，应用免疫组化方法以多克隆人抗－ＨＣＶ和单克隆鼠抗－ＨＣＶ－ＮＳ＿３检测一组ＨＣＶ感染者石蜡包埋肝组织中ＨＣＶ抗原（Ａｇ）和ＨＣＶ－ＮＳ＿３，并对抗原检出与血清ＨＣＶ标志的关系进行分析。结果：ＨＣＶＡｇ和ＨＣＶ－ＮＳ＿３检出率分别为３８．３％（２３／６０）和３０．０％（１８／６０）；所有ＨＣＶ－ＮＳ＿２，阳性者均同时表现ＨＣＶＡｇ阳性；阳性颗粒定位于肝细胞胞浆；阳性细胞分布呈散在、弥漫状和簇状，周围多天坏死及炎性细胞浸润；不同血清ＨＣＶ标志模式间抗原检出率存在明显差别。研究结果对认识ＨＣＶ致病机理有一定意义。     

庚型肝炎病毒RNA、NS5抗原在肝组织中表达的研究

研究ＨＧＶ ＲＮＡ及ＨＧＶ抗原在肝组织中的表达。方法 对１７例ＨＧＶ血清标志阳性患者进行了肝组织ＨＧＶ ＲＮＡ的原位杂交及免疫组织化学的研究。结果 原位杂交ＨＧＶ ＲＮＡ阳性８例，阳性率４７．０５％，阳性信号均存在于肝细胞的胞浆中；免疫组化３例ＨＧＶ ＮＳ５抗原阳性，阳性率１７．６４％，均为胞浆型。     

FAS抗原与慢性丙型肝炎关系的研究

目的　通过观察慢性丙型肝炎 (CHC)患者肝组织及外周血单核细胞 (PBMC)FAS抗原表达对CHC病情的影响 ,进而探讨FAS系统及HCVRNA水平与CHC的关系及临床意义。方法　采用肝活检组织病理、免疫组织化学、PCR及血液生化等方法检测 41例CHC患者的肝组织和PBMC中FAS抗原、血清HCVRNA及肝功能 ,观察肝组织和PBMC中FAS抗原表达情况与血清HCVRNA水平及肝损伤的关系。结果　 ( 1)CHC患者PBMC的FAS阳性率为41.5 % ( 17/ 41) ,肝组织FAS阳性率为 65 .6% ( 2 1/ 3 2 )。 ( 2 )CHC患者PBMCFAS阳性组血清HCVRNA和ALT水平均显著高于FAS阴性组 (P <0 .0 5 )。结论　 ( 1)CHC患者肝组织和PBMC中FAS表达与肝组织炎症及血清病毒载量有关 ;( 2 )FAS系统可作为观察CHC病情的新指标 ,协助诊断与治疗。     

Correlation of serum HCV RNA and alanine aminotransferase levels in chronic hepatitis C patients during treatment with ribavirin

to evaluate the effect of ribavirin on serum hepatitis C virus (HCV) RNA and alanine aminotransferase (ALT) levels, 22 patients with chronic HCV infection were treated with oral ribavirin 1200 mg daily in three divided doses for 4 weeks. At the end of 4 weeks treatment, the serum ALT decreased in all but one patient and became normal in three individuals. The mean pretreatment serum ALT was reduced significantly from 193 ± 45 i.u./L to 95 ± 16 i.u./L after 4 weeks therapy (P= 0.009). However, 8 weeks after cessation of treatment, the serum ALT rose to a mean value of 154 ± 21 i.u./L. The mean pretreatment serum HCV RNA was not significantly decreased at the end of 4 weeks treatment (7.0 × 10⁵vs 4.1 × 10⁵ copies/mL, P > 0.05). However, serum HCV RNA levels were decreased in 12 and increased in 10 patients at the end of 4 weeks therapy. Eight weeks after cessation of therapy, the serum HCV RNA of 22 patients rose to a mean value of 4.9 ± 10⁵ copies/mL. Six patients who continued to have elevated serum ALT and positive HCV RNA after the initial 4 weeks treatment received oral ribavirin at the same dosage for an additional 24 weeks. The serum ALT again decreased in all six patients during therapy, but rose to pretreatment values by 8 weeks after cessation of the treatment. In addition, no significant changes were noted in the mean serum HCV RNA levels during and after 24 weeks of ribavirin therapy. Our results indicate that oral ribavirin only transiently lowered serum ALT values and did not efficiently suppress HCV synthesis in patients with chronic hepatitits C infection.     

干扰素联合病毒唑对慢性丙肝患者正负链HCV RNA的影响

目的探讨干扰素联合病毒唑对正、负链丙型肝炎病毒 RNA(HCV RNA)的影响.方法 23例患者,依据 HCV RNA 及抗-HCV 阳性,肝功能反复不正常,病程持续1年以上,其他肝炎病毒标志阴性,被诊断为慢性丙型肝炎(CHC).其中,13例接受联合抗病毒治疗,即α-干扰素300万 U,皮下注射,每周3次,治疗3个月;和病毒唑1g,于干扰素治疗的第1个月,同时静脉滴注,1次/d,治疗1个月.10例单用干扰素治疗3个月(用法同上).作为对照.其血清及周围血单核细胞(PBMCs)中正、负链 HCV RNA 用套式反转录—聚合酶链反应技术检测.结果联合抗病毒组中,9例(69.23%)异常的 ALT 降至正常,5例于治疗停止后第24周复发,4例为完全应答者;而单用干扰素组,分别为6例(60%)、4例(66.67%)和2例(33.335).两组间无明显差异(P>0.05).治疗结束时,联合抗病毒组的血清正链阳性率由92.31%降至38.46%(P<0.05),PBMCs 的负链阳性率由76.92%降至38.46%(P<0.05);而单用干扰素组,分别为100%降至50%(P<0.05)和90%降至40%(P<0.05).但组间无差异(P>0.05).复发见于治疗前后 PBMCs 中正链 HCVRNA 持续阳性的患者.结论病毒唑似不能增加干扰素的疗效.血清 HCV RNA 的阴转并不意味着 HCV 的完全清除,因而用它判断疗效及病情转归有一定的局限性.因此,同时检测血清及 PBMCs 中正、负链 HCV RNA,具有重要的临床意义.     

Seven-years follow-up on trial of Interferon alpha in patients with HCV RNA positive chronic hepatitis C

AIM To study the long-term efficiency of therapy with Interferon alpha (IFN-a) in patients with HCVRNA positive chronic hepatitis C.METHODS Ten patients were enrolled in the study, whose age 31 -62 years (mean 53 years), course 6- 72months (mean 24 months), of whom, 6 patients with mild CHC, 4 moderate CHC. All patients receivedIFN-a 3 MU three times weekly for six to twelve months, and then followed up for seven years after the endof treatment. The results of hepatic functions and HCV RNA at the end of treatment and follow-up period inall patients were observed.RESULTS ( At the end of treatment, clinical symptoms recovered obviously in all patients, virologicalresponse (defined as HCV RNA loss) occurred in 5 of 7 (71.4%) patients (<60 years old) and in 1 of 3(33.3%) patients ( >60 years old). At the end of follow-up, the rates of HCV RNA loss were 42.9% (3/7)and 33.3% (1/3), respectively, in these group. Virological sustained response (defined as HCV RNA loss atthe end of treatment and follow-up) occurred in 3 of 6 (50%) patients (6 - 12 month-course) and in 1 of 4(25%) patients (> 12 month-course). A sustained HCV RNA response was observed in 2 of 7 (28.6%)patients with IFN-a therapy for 6m and in 2 of 3 (66.7%) patients with IFN-a therapy for more than 6 m. Ofall patients, 4 patients with sustained HCV RNA response were mild CHC, 4 patients with sustained HCVRNA positive were mild CHC (2 patients), moderate CHC (2 patients), respectively; other 2 patients withHCV RNA loss at the end of treatment but recurred at the end of follow-up, were moderate CHC. ②Biochemically sustained response (defined as ALT normalization at the end of treatment and follow-up) wasobserved in 5 out of 10 (50%) patients, and these 5 patients were mild CHC, of whom, 4 patients with HCVRNA sustained negative, 1 patient with HCV RNA loss and then recurred again. Two patients with ALTnormalization at the end of follow-up were one mild CHC, one moderate CHC, respectively. Other 3patients with no response were moderate CHC, of whom, 2 patients with HCV RNA sustained positive, 1patient with HCV RNA loss then recurred, and in these 3 patients, the lower limits of ALT were more than121 U/L- 148 U/L. ③ Of 10 patients, 3 moderate CHC patients were far from satisfactory to IFN-αtherapy, of whom, 2 coinfected with HBV, t with post-hepatitis cirrhosis.CONCLUSION The CHC patients with younger age, shorted course, and lighter liver changes in biopsy(mild CHC) have better response to IFN-α therapy, and the efficiency of therapy with IFN-α for 12 m aremore satisfactory than those for 6 m. The patients with coinfected HCV and HBV have a response to IFN-αtherapy worse than the others.     

慢性丙型肝炎病毒感染者外周血淋巴细胞增殖反应

目的　观察慢性丙型肝炎患者外周血淋巴细胞对丙型肝炎病毒（ＨＣＶ） 抗原刺激的增殖反应．方法　外周血单个核细胞（ＰＢＭＣ） 与ＨＣＶ 抗原ｃ２２ 、ｃ３３ 、ｃ１００ － ３ 、ＮＳ５ 和植物血凝素（ＰＨＡ） 分别共同孵育,加入胸腺嘧啶核苷（３ ＨＴｄＲ） ,然后收集细胞于液闪仪测定每分钟脉冲数（ｃｐｍ） ．结果　根据对不同ＨＣＶ 抗原的淋巴细胞增殖反应发现,以ｃ２２免疫原性最强,ｃ１００ － ３ 次之：淋巴细胞激活与ＨＣＶ 基因型关系不大;健康对照和慢性乙型肝炎患者对各ＨＣＶ 抗原未能显示有效的淋巴细胞增殖反应;与健康对照比较,慢性丙型肝炎和乙型肝炎患者对ＰＨＡ 刺激的淋巴细胞增殖反应降低．结论　ＨＣＶ 抗原ｃ２２ 免疫原性最强,丙型肝炎患者对ＨＣＶ 抗原的淋巴细胞增殖反应系特异性;慢性丙型肝炎和乙型肝炎患者存在抑制的细胞免疫应答．     

Genetic and biochemical diversity in the HCV NS5B RNA polymerase in the context of interferon α plus ribavirin therapy

Summary. The hepatitis C virus (HCV) RNA polymerase (RdRp) may be a target of the drug ribavirin, and it is an object of drug development. Independent isolates of any HCV subtype differ genetically by approximately 10%, but the effects of this variation on enzymatic activity and drug sensitivity are poorly understood. We proposed that nucleotide use profiles (G/U ratio) among subtype 1b RdRps may reflect their use of ribavirin. Here, we characterized how subtype 1b genetic variation affects RNA polymerase activity and evaluated the G/U ratio as a surrogate for ribavirin use during pegylated interferon α and ribavirin therapy. Genetic and biochemical variation in the RdRp was compared between responders who would be largely sensitive to ribavirin and relapsers who would be mostly resistant. There were no consistent genetic differences between responder and relapser RdRps. RNA polymerization, RNA binding and primer usage varied widely among the RdRps, but these parameters did not differ significantly between the response groups. The G/U ratio among a set of subtype 1a RdRps increased rather than decreased following failed therapy, as would be expected if it reflected ribavirin use. Finally, RdRp activity was significantly associated with ALT levels. These data indicate that (i) current genetic approaches cannot predict RNA polymerase behaviour, (ii) the G/U ratio is not a surrogate for ribavirin use, (iii) RdRp activity may contribute to liver disease by modulating viral mRNA and antigen levels, and (iv) drug candidates should be tested against multiple patient‐derived enzymes to ensure widespread efficacy even within a viral subtype.