Distinctive Drug-resistant Mutation Profiles and Interpretations of HIV-1 Proviral DNA Revealed by Deep Sequencing in Reverse Transcriptase

Objective To investigate distinctive features in drug-resistant mutations （DRMs） and interpretations for reverse transcriptase inhibitors （RTIs） between proviral DNA and paired viral RNA in HIV-l-infected patients. Methods Forty-three HIV-l-infected individuals receiving first-line antiretroviral therapy were recruited to participate in a multicenter AIDS Cohort Study in Anhui and Henan Provinces in China in 2004. Drug resistance genotyping was performed by bulk sequencing and deep sequencing on the plasma and whole blood of 77 samples, respectively. Drug-resistance interpretation was compared between viral RNA and paired proviral DNA. Results Compared with bulk sequencing, deep sequencing could detect more DRMs and samples with DRMs in both viral RNA and proviral DNA. The mutations M1841 and M2301 were more prevalent in proviral DNA than in viral RNA （Fisher＇s exact test, P〈0.05）. Considering ＇majority resistant variants＇, 15 samples （19.48%） showed differences in drug resistance interpretation between viral RNA and proviral DNA, and 5 of these samples with different DRMs between proviral DNA and paired viral RNA showed a higher level of drug resistance to the first-line drugs. Considering ＇minority resistant variants＇, 22 samples （28.57%） were associated with a higher level of drug resistance to the tested RTIs for proviral DNA when compared with paired viral RNA. Conclusion Compared with viral RNA, the distinctive information of DRMs and drug resistance interpretations for proviral DNA could be obtained by deep sequencing, which could provide more detailed and precise information for drug resistance monitoring and the rational design of optimal antiretroviral therapy regimens.

Sequencing of PCR amplified HBV DNA pre-c and c regions in 2.2.15 cells and antiviral action by targeted antisense oligonucleotide directed against sequence

AIM To study the specific inhibition of HBV gene expression by liver-targeting antisense oligonucleotide (ASON) directed against pre-c and c regious in a sequence-specific manner.METHODS According to the result of direct sequencing of PCR amplified products, a 16-mer phosphorothioate analogue of the antisense oligonucleotide (PS-ASOn) directed against the HBV U5-like region was synthesized and then linked with one live-targeting ligand, the galactosylated poly-L-lysine. Their effect on the expression of HBV gene was observed using the 2.2.15 cells.RESULTS HBV DNA in the 2.2.15 cells was from HBV with surface antigen subtype ayw1 by sequencing so that antisense oligonucleotides could bind specifically to the target sequence through base piring. Under the same experimental conditions, the inhibitory rates of PS-ASON to HBsAg and HBeAg were 70% and 58% at a concentration of 10μmol/L, while by ligand-PS-ASON they were 96% and 82%, the amount of HBV DNA in cultured supernatant and cells was reduced significantly. An unrelated sequence oligonucleotide showed no effectiveness. All the oligonucleotides had no cytotoxicity.CONCLUSION Antisense oligonucleotides complexed by the liver-targeting ligand can be targeted to cells via asialoglycoprotein receptors, resulting in supecific inhibition of HBV gene expression and replication.

Early hepatitis B viral DNA clearance predicts treatment response at week 96

AIM To investigate whether hepatitis viral DNA load at 24 wk of treatment predicts response at 96 wk in patients with chronic hepatitis B.METHODS A total of 172 hepatitis B envelope antigen(HBe Ag)-positive chronic hepatitis B patients who received initial treatment at 16 tertiary hospitals in Hunan Province, China were enrolled in this study. All patients received conventional doses of lamivudine and adefovir dipivoxil, telbivudine, entecavir dispersible tablets, or entecavir tablets for 96 wk. Patients who used other antiviral drugs or antitumor and immune regulation therapy were excluded. Patients were stratified into three groups according to their viral DNA load at 24 wk: < 10 IU/m L(group 1), 10-103 IU/m L(group 2), and > 103 IU/m L(group 3). Correlations of 24-wk DNA load with HBe Ag negative status and HBe Ag seroconversion at 96 wk were analyzed. Receiver operating characteristic curve analysis was used to test the predictive value of the HBV DNA load at 24 wk for long-term response.RESULTS The rates of conversion to HBe Ag negative status and HBe Ag seroconversion rates were 53.7% and 51.9%, respectively, in group 1; 35.21% and 32.39% in group 2; and 6.38% and 6.38% in group 3. The receiver operating characteristic curves for the three subgroups revealed that the lowest DNA load(< 10 IU/m L) was better correlated with response at 96 wk than a higher DNA load(10-103 IU/m L). Nested PCR was used for amplifying and sequencing viral DNA in patients with a viral DNA load > 200 IU/m L at 96 wk; resistance mutations involving different loci were present in 26 patients, and three of these patients had a viral DNA load 10-103 IU/m L at 96 wk. CONCLUSION Hepatitis B viral DNA load at 24 wk of antiviral treatment in patients with chronic hepatitis B is a predictor of the viral load and response rate at 96 wk.

SEN virus does not affect treatment response in hepatitis C virus coinfected patients but SEN virus response depends on SEN virus DNA concentration

AIM: To clarify the effect of SEN virus (SENV) infection on a combination therapy including interferon alfa (IFN-α) or pegylated-IFN with ribavirin in patients with chronic hepatitis and the effect of a combination therapy on SENV.METHODS: SENV DNA was determined by polymerase chain reaction in serum samples from 95 patients with chronic hepatitis C. Quantitative analysis was done for SENV H DNA.RESULTS: Twenty-one (22%) of 95 patients were positive for SENV DNA. There was no difference in clinical and biochemical parameters between patients with HCV infection alone and coinfected patients. The sustained response rate for HCV clearance after combination therapy did not differ between patients with SENV (52%) and without SENV(50%, n.s.). SENV DNA was undetectable in 76% of the initially SENV positive patients at the end of follow-up. SENV H response to combination therapy was significantly correlated with SENV DNA level (P=-0.05).CONCLUSION: SENV infection had no influence on the HCV sustained response rate to the combination therapy.Response rate of SENV to the combination therapy depends on SENV DNA level.

HBV C基因型有关的HBsAg阴性HBV DNA阳性患者S区突变对HBsAg的影响

目的通过构建HBV C基因型突变质粒研究HBsAg阴性HBV DNA阳性患者HBV S区突变对HBsAg水平的影响。方法收集2022年8月至2023年4月首都医科大学附属北京佑安医院107例HBsAg-/HBV DNA+患者血液样本,对成功提取扩增的HBV DNA S区进行测序,通过构建HBV C基因型突变质粒对HBV S区突变位点进行细胞功能验证,探讨OBI可能发生的分子机制。结果对成功提取扩增的68例患者进行测序,发现HBV S区存在大量突变,包括免疫逃逸突变(如sG145R、sK122R、sS114T、sT131P等)和跨膜结构域(transmembrane domain,TMD)突变(如sT5A、sG10D、sF20S等)。通过构建HBV C基因型突变质粒,进行细胞转染和细胞免疫荧光实验发现sG145R突变会明显降低HBsAg的表达,但是sK122R、sI26N、sQ29N、sR169H、sS114T、sT131P这6个突变位点并未影响细胞内外HBsAg的表达。结论通过测序发现HBsAg-/HBV DNA+患者HBV S区存在大量突变位点,通过构建sG145R、sK122R、sI26N、sQ29N、sS114T和ST131P等突变质粒发现sG145R突变会明显降低细胞内外HBsAg的表达,但是sK122R、sI26N、sQ29N、sR169H、sS114T、sT131P并未明显降低细胞内外HBsAg的表达。

HBV cccDNA in patients' sera as an indicator for HBV reactivation and an early signal of liver damage

AIM:To evaluate the covalently closed circle DNA (cccDNA) level of hepatitis B virus (HBV) in patients' liver and sera. METHODS:HBV DNA was isolated from patients' liver biopsies and sera.A sensitive real-time PCR method,which is capable of differentiation of HBV viral genomic DNA and cccDNA,was used to quantify the total HBV cccDNA.The total HBV viral DNA was quantitated by real-time PCR using a HBV diagnostic kit (PG Biotech,LTD,Shenzhen,China) described previously. RESULTS:For the first time,we measured the level of HBV DNA and cccDNA isolated from ten HBV patients' liver biopsies and sera.In the liver biopsies,cccDNA was detected from all the biopsy samples.The copy number of cccDNA ranged from from 0.03 to 173.1 per cell,the copy number of total HBV DNA ranged from 0.08 to 3 717 per cell.The ratio of total HBV DNA to cccDNA ranged from 1 to 3 406.In the sera, cccDNA was only detected from six samples whereas HBV viral DNA was detected from all ten samples.The ratio of cccDNA to total HBV DNA ranged from 0 to 1.77%.To further investigate the reason why cccDNA could only be detected in some patients' sera,we performed longitudinal studies.The cccDNA was detected from the patients' sera with HBV reactivation but not from the patients' sera without HBV reactivation.The level of cccDNA in the sera was correlated with ALT and viral load in the HBV reactivation patients. CONCLUSION:HBV cccDNA is actively transcribed and replicated in some patients' hepatoo/tes,which is reflected by a high ratio of HBV total DNA vs cccDNA.Detection of cccDNA in the liver biopsy will provide an end-point for the anti-HBV therapy.The occurrence of cccDNA in the sera is an early signal of liver damage,which may be another important clinical parameter.

HBV-DNA病毒载量在不同HBV感染产妇产后血清、唾液、乳汁中的变化及与肝功能的相关性研究

目的 观察不同HBV感染产妇的HBV-DNA病毒载量及其与肝功能的相关性。方法 102例HBV产妇根据HBV血清标志物分为三组,比较各组的HBV-DNA载量以及ALT、 AST水平,分析HBV-DNA载量与肝功能的相关性。结果 产妇血清、唾液、乳汁中的HBV-DNA载量,以及血清中ALT、 AST水平由高到低依次为A组、 B组、 C组(P <0.05)。不同HBV感染产妇的血清、唾液、乳汁HBV-DNA载量与ALT、 AST水平均呈正相关(P <0.05)。结论 随着HBV感染程度的加深,产妇血清、唾液、乳汁中的HBV-DNA病毒载量呈上升趋势,产后血清、唾液及乳汁中HBV-DNA病毒载量均与肝功能密切相关。

转录因子MYB转录调控MTFR2通过DNA损伤修复促进胃癌细胞化疗耐药性

目的探究v-myb禽成髓细胞病病毒癌基因同源物(MYB)转录调控线粒体裂变调节因子2(MTFR2)对胃癌(GC)细胞顺铂(DDP)耐药性的影响及分子作用机制。方法TCGA数据库分析GC中差异mRNA并预测上游调控分子,qRT-PCR检测MTFR2和MYB的表达,双荧光素酶和染色质免疫共沉淀(ChIP)实验验证MTFR2和MYB的调控关系,细胞计数盒8(CCK-8)检测细胞活力并计算IC_(50)值,流式细胞术检测细胞周期和细胞凋亡,彗星实验检测DNA损伤,蛋白质免疫印迹法检测DNA损伤相关蛋白(γ-H2AX、ATM、p-ATM)的表达。结果MTFR2在GC组织和细胞中显著高表达,敲低MTFR2能够降低细胞增殖,阻滞S期,诱导细胞凋亡,促进DNA损伤和DDP敏感性。生信预测MTFR2存在上游转录因子MYB,MYB在GC组织和细胞中的表达显著上调,双荧光素酶和ChIP验证了MTFR2启动子区域与MYB的结合关系。回复实验发现进一步过表达MTFR2能够逆转敲低MYB对GC细胞增殖和DDP耐药性的抑制作用。结论MYB上调MTFR2的表达通过DNA损伤途径促进GC细胞增殖和DDP耐药,表明靶向MYB/MTFR2调控轴可能是克服GC DDP耐药性的潜在途径。

氧化苦参碱对HepG2.2.15细胞中乙型肝炎病毒DNA表达量的影响

目的 :观察氧化苦参碱对 Hep G2 .2 .15细胞中乙型肝炎病毒 (HBV ) DNA表达量的影响 ,进一步探讨其抗病毒机制。方法 :用不同浓度的氧化苦参碱作用 Hep G2 .2 .15细胞 ,留取培养上清后 ,采用 PCR- EL ISA技术定量上清中 HBV DNA,并比较其抑制率。结果 :氧化苦参碱能直接抑制 Hep G2 .2 .15细胞内 HBV DNA的复制 ,且随着药物浓度升高抑制作用逐渐增强。保持药物在培养上清中的浓度是保证抑制率的关键。 结论 :氧化苦参碱可以在 DNA复制水平直接抑制乙型肝炎病毒的合成。

乙型肝炎病毒外膜大蛋白与HBV DNA的关系研究

目的:探讨乙型肝炎患者血清中乙型肝炎病毒外膜大蛋白(HBV-LP)与HBV DNA的关系。方法:采用酶联免疫吸附试验(ELISA)和实时荧光定量PCR法,检测320例乙型肝炎患者血清中的HBV-LP水平及HBV DNA载量。结果:HBV-LP水平与HBV DNA拷贝数变化相一致,两者呈正相关(r=0.949);不同模式的HBeAg血清中HBV-LP与HBV DNA阳性率,差异均无统计学意义(P>0.05)。结论:血清HBV-LP水平能反映乙型肝炎患者体内HBV复制程度,是判断HBV复制的血清学指标。

化学发光法与乙肝病毒DNA荧光定量在乙肝检测中的应用价值

目的:明确化学发光法(CLIA)、乙肝病毒DNA荧光定量技术在乙肝检测中的价值。方法:选取285例乙肝患者和280例肝硬化患者为检测对象,分别对样本进行CLIA、乙肝病毒DNA荧光定量分析,统计相关检测数据。结果:乙肝组HBsAg、HBsAb、HBeAg、HBeAb、HBcAb水平均明显低于肝硬化组(P<0.05),HBV-DNA水平明显高于肝硬化组,随着HBV-DNA水平上升,乙肝患者的肝功能指标均有不同程度提高(P<0.05)。经Pearson相关性分析,HBV-DNA和HBsAg、HBsAb、HBeAg、HBeAb、HBcAb均为负相关(t=-0.876、-0.956、-0.974、-0.922、-0.981)。结论:CILA、乙肝病毒DNA荧光定量检测技术在乙肝检测中都有一定价值,两者之间为负相关,可为乙肝诊断、预后评估提供参考信息。

Serum concentration of sFas and sFasL in healthy HBsAg carriers,chronic viral hepatitis B and C patients

AIM:To estimate the amount of apoptosis among healthy HBsAg carriers,patients with chronic HBV infection treated wibh lamivudine and patients with chronic HCV infection treated with interferon alpha and ribavirin.Activity of apoptosis was evaluated by serum sFas/sFasL concentration measurement. Moreover dependence between apoptosis and HBV-DNA or HCV-RNA levels was studied. METHODS:Eighty-six persons were included into study:34 healthy HBsAg carders,33 patients with chronic HBV infecl^on and 19 patients with chronic HCV infection.Serum levels of sFas/sFasL were measured by ELISA assay.HBV-DNA and HCV-RNA were measured by RT-PCR assay.Levels of sFas/sFasL were determined before and 2 and 12 wk after therapy in patients with chronic hepatitis B and C infection. HBV-DNA or HCV-RNA was detected before treatment and 6 mo after treatment. RESULTS:Twenty-four (71%) healthy HBsAg carders showed HBV-DNA over 10~5/mL,which was comparable to the patients with chronic hepatitis B.independently from HBV-DNA levels, the concentration of sFas among healthy HBsAg carders was comparable to healthy persons.Among patients with chronic hepatitis B and C,the concentration of sFas was significantly higher in comparison to healthy HBsAg carriers and healthy persons.In chronic hepatitis B patients the concentration of sFas was decreased during lamivudine treatment.Among chronic hepatitis C patients the concentration of sFas was increased during IFN alpha and ribavirin treatment,sFasL was not detected in control group.Furbhermore sFasL occurred more frequently in chronic hepatitis C patients in comparison to chronic hepatitis B patients. CONCLUSION:There are no correlations between apoptosis and HBV-DNA levels.However ther is an association between apoptosis and activity of inflammation in patients with chronic HBV infection.Apoptosis can be increased in patients with chronic hepatitis C by effective treatment which may be a result of apoptosis stimulation by IFN-α.

Evolution of HBV Viral Load during Clinical and Biological Follow-Up of Chronic Hepatitis B Patients at the Saint Camille Hospital in Ouagadougou

Hepatitis B virus (HBV) infection is a major public health problem worldwide. The aim of this study was to document the dynamics of HBV viral load during the follow-up of chronic hepatitis B patients at the Saint Camille Hospital in Ouagadougou (HOSCO) from 2017 to 2021. This descriptive retrospective study was carried out in the Hepato-Gastro-Enterology Department of HOSCO and focused on patients who were undergoing treatment for chronic viral hepatitis B. A total of 260 cases of chronic hepatitis B were included in the study. The most affected age group was 21 to 30 years, accounting for 48.08% of the cases. Lifestyle factors included alcohol consumption (3.08%) and tobacco use (2.69%). Major risk factors for transmission included lack of vaccination (98.46%), family history of HBV infection (68.00%) and engagement in high-risk activities (28.00%). Patients requiring treatment were prescribed Tenofovir 300 mg tablets. FibroScan® showed the presence of stage F3-F4 fibrosis (2.14%) and S3 steatosis (13.33%). After one year of follow-up, 6.92% of patients achieved an undetectable viral load with normalized transaminase levels. The majority of other patients had a detectable viral load but below 20,000 IU/mL. The prevalence of viral hepatitis B remains significant worldwide. Although effective and well-monitored treatment can lead to undetectable viremia, prevention remains the most effective strategy for successful management of this disease.

Pathogenic and pathological characteristic of new type gosling viral enteritis first observed in China

AIM: To study the purifying method and characteristics of new gosling viral enteritis virus (NGVEV),the etiological agent of new gosling viral enteritis (NGVE) which was first recognized in China, as well as the pathomorphological development in goslings infected artificially with NGVEV. METHODS: (1)NGVEV virions were purified by the procedure of treatment with chloroform and ammonium sulfate precipitation, dialysis to remove the sulfate radical and ammonium ion and separation by gel filtration chromatography, and SDS-PAGE. (2)Forty-2-day-old White Sichuan goslings were orally administered with NGVEV and 24 hr later 2 birds were randomly selected and killed at 24hr intervals until death occurred. Specimens(duodenum, ileum, liver, heart, kidney, spleen, lung, proventriculus, pancreas, esophagus, and the intestinal embolus) were taken until all birds in this group died and were sectioned and stained with hemotoxylin and eosin and studied by light microscope. RESULTS: NGVEV shared the typical characteristics of Adenovirus and which structural proteins consisted of 15 polypeptides. Necrosis and sloughing of the epithelial cells covering the villus tips of the duodenum were first observed in goslings 2 days postinfection artificially with NGVEV. With the progress of infection, this lesion rapidly occurred in the epithelium at the base of the villus and with infiltration of the inflammatory cells, the jejunum tended to be involved. With the intensification of mucosa necrosis and inflammatory exudation of the small intestine, fibrinonecrotic enteritis was further developed and embolus composed of either intestinal contents wrapped by pseudo-membrane or of the mixture of fibrous exudate and necrotic intestinal mucosa were observed in the middle-lower part of the small intestine. This structure occluded the intestinal tract and made the intestine dilated in appearance. The intestinal glandular cells underwent degeneration, necrosis and might be found sloughed into the lumen. Hemorrhage and hyperemia could be observed on the lung and kidney. Epithelial cells of the renal tubular underwent degeneration. In some cases, granular degeneration and fatty degeneration could be found in the liver and in some cases at a later stage of this disease the epithelial cells of trachea and proventriculus might be found sloughed. In some cases at an early stage of this disease, cardiac hyperemia and hemorrhage could be observed. Esophagus, pancreas and brain were found normal. Analyses and comparisons between the pathologic lesions of NGVE and Gosling Plague (GP) were available in this paper as well. CONCLUSION: (1)NGVEV is adenovirus. (2)Pathological characteristic could be as the data for NGVE diagnosis.

10-23 DNA enzyme对乙型肝炎病毒C基因体外转录产物的切割活性

目的:探讨10-23 DNA enzym e对乙型肝炎病毒C基因体外转录产物的特异切割活性。方法:设计并合成3种能针对乙型肝炎病毒C基因开放阅读框A1816UG的特异性脱氧核酶,分别命名为D rzBC-7、D rzBC-8和D rzBC-9。应用体外转录的方法获得相应的底物RNA,观察D rzBC-7、D rzBC-8和D rzBC-9对其的体外切割效应。以D rzBC-9为例,观察不同浓度的M gC l2对体外切割效率的影响,根据L inew eaver Burk作图法,计算相关的酶动力学参数Km、K cat和K cat/Km。结果:通过体外转录获得用于切割反应的底物RNA,其大小为300 nt。在特定的切割条件下,D rzBC-7、D rzBC-8和D rzBC-9均能对相应的底物RNA进行有效的切割,切割产物分别为109 nt和191 n。t以D rzBC-9为例,在切割反应体系中缺乏M gC l2时,未见有切割反应。M gC l2浓度在150 mm o.lL-1时达到最高切割效率,再提高M gC l2的浓度,切割效率未见明显增大。酶动力学参数Km、K cat和K cat/Km分别为1.4×10-9m o.lL-1,1.6 m in-1和1.1×109m o.lL-1.m in-1。结论:针对乙型肝炎病毒C基因的10-23 DNA enzym e在体外对相应的转录产物有特异切割作用。

Distribution of HBV DNA and HBsAg in the liver of patients with liver cirrhosis and its significance

Hepatitis B virus(HBV)DNA and HBsAg were detected in 51 human paraf- fin-embedded sections of liver cirrhosis by the double labelling technique of in situ hybfidiza- tion and PAP method.The results showed that the positive sections were 21(41.2％)for HBV DNA,43(84.3％)for HBsAg and 19(37.3％)for both HBV DNA and HBsAg.HBV DNA-positive grains were localized predominantly in the cytoplasm of hepatic cells or in both nucleus and cytoplasm,a few only in the nucleus or the inner side of the cell membrane.In active cirrhosis and inactive cirrhosis,the positive rates of HBV DNA were 52.8％ and 13.3％,respectively,and for both HBV DNA and HBsAg,they were 47.2％ and 13.3％ restively.The positive rate is higher in active cirrhosis than in inactive cirrhosis(P<0.05).The results further indicated that the infection of HBV and the existence and persistent action of HBV DNA in the liver tissues are one of the important factors of the development of liver cirrhosis.

前S1抗原和HBV-DNA与HBV血清标志物之间的相关性探讨

目的探讨前S1抗原和HBV-DNA与HBV血清标志物之间的关系。方法对1158例慢性乙型肝炎患者用酶联免疫吸附试验(ELISA)检测HBV血清标志物和PreS1,聚合酶链反应(PCR)方法检测HBV-DNA。结果1158例慢性乙型肝炎患者中HBV-DNA总阳性率68.9%,PreS1总阳性率54.8%,有显著性差异(χ2=53.24,P<0.005)。HBV-DNA、PreS1在HBeAg(+)组的阳性率均明显高于HBeAg(-)组(HBV-DNA:χ2=226.24,P<0.005;PreS1:χ2=49.64,P<0.005)。PreS1和HBeAg与HBV-DNA的总符合率分别为56.9%和63.3%,PreS1的敏感性高于HBeAg,但特异性则相反。随着HBV-DNA拷贝数的升高,PreS1和HBeAg的阳性率亦随之升高,HBV-DNA含量低时PreS1的阳性率明显高于HBeAg。结论联合检测HBV血清标志物、PreS1,并动态监测HBV-DNA的含量,在观察慢性乙肝患者HBV复制、疗效时具有重要的临床应用价值。PreS1可作为HBeAg阴性、无条件开展HBV-DNA检测时的补充指标。

慢性乙型肝炎患者ALT、HBV DNA及血清肝纤维化标志物与肝纤维化程度的关系

目的探讨ALT、HBV DNA以及血清纤维化标志物透明质酸(HA)、层粘连蛋白(LN)、Ⅲ型前胶原肽(PⅢP)、Ⅳ型胶原(CⅣ)与慢性乙型肝炎肝纤维化程度的关系。方法检测281例慢性乙型肝炎患者血清中ALT、HBV DNA和纤维化标志物(HA、LN、PⅢP及CⅣ)的水平,并行肝活检检测肝组织病理纤维化分期。结果 HBeAg阴性慢性乙型肝炎患者HBV DNA水平较低、纤维化程度较高。HBeAg阳性患者纤维化程度与HBV DNA呈负相关(r=-0.251,P<0.001),S≥3组水平最低。慢性乙型肝炎患者纤维化程度与PⅢP水平呈正相关,其水平随着纤维化程度的加重而明显升高。结论 PⅢP水平可能作为评估慢性乙型肝炎患者肝纤维化程度的血清学指标,血清HBV DNA与肝脏纤维化严重程度的关系仍需进一步深入探讨。

CD137L在HBsAg DNA疫苗诱导小鼠细胞免疫应答中的佐剂效应

目的:研究共刺激分子CD137L重组质粒对HBsAg DNA疫苗诱导小鼠细胞免疫应答和回忆反应的佐剂作用。方法:将小鼠CD137L真核表达载体(pcD137L)体外转染NIH3T3细胞,RT-PCR、流式细胞仪和免疫荧光法分别检测CD137L mRNA和蛋白的表达;pcD137L与HBsAg DNA疫苗(pcDS)通过肌肉注射联合免疫BALB/c小鼠,LDH释放法测定体外脾细胞特异性CTL杀伤活性;流式细胞仪分析小鼠脾脏CD8+记忆T细胞数量;流式细胞仪检测CD8+T细胞及淋巴细胞中IFN-γ和IL-4的表达水平。结果:重组质粒pcD137L在NIH3T3细胞中获得有效表达。与pcDS单独免疫组比较:免疫后1周,pcDS+pcD137L免疫组能诱导更强的HBsAg特异性CTL杀伤活性(P<0.05);免疫后1周和12周,pcDS+pcD137L免疫组CD8+T细胞CD44high和CD127表达水平均显著增高(P<0.05或P<0.01);免疫后1周、12周和13周(即再次给予pcDS刺激后1周),pcDS+pcD137L免疫组分别明显上调CD8+T细胞和淋巴细胞中IFN-γ产生细胞的百分比,差异有显著意义(均P<0.05)。结论:共刺激分子CD137L能显著增强HBsAg DNA疫苗诱导的Tc1(I型)细胞免疫、特异性CTL应答及回忆反应,是诱导HBV特异性T细胞应答的有效佐剂。

乙型肝炎e抗原阴、阳性慢性乙型肝炎患者细胞免疫状态与乙型肝炎病毒DNA水平相关分析

目的探讨乙型肝炎e抗原(HBeAg)阴性及HBeAg阳性慢性乙型肝炎患者在细胞免疫状态及乙型肝炎病毒DNA(HBV DNA)水平之间的相关性。方法分别应用流式细胞仪、荧光定量聚合酶链反应(PCR)方法对HBeAg阴、阳性慢性乙型肝炎患者T淋巴细胞亚群及HBV DNA进行检测。结果HBeAg阴性慢性乙型肝炎患者的HBV DNA水平与CD8+T淋巴细胞比例之间存在正相关,而与CD4/CD8比值之间存在负相关,r值分别为0.567,-0.601,均P<0.01;与总T、CD4+T淋巴细胞比例无相关关系。HBeAg阳性组患者的HBV DNA水平与总T、CD4+T、CD8+T淋巴细胞水平相关关系。结论HBeAg阴性慢性乙型肝炎患者HBV DNA水平与机体细胞免疫密切相关。