乙肝反义全基因真核载体的构建及在SMMC—7721细胞内的表达

目的 研究HBV反义全基因真核表达载体在抗HBV基因治疗的可能性,方法 采用基因重组技术,将HBV全基因片段反向插入真核细胞表达载体pBK-CMV克隆位点中,通过FuGENE^TM6转染试剂,将重组体转染人肝癌细胞系SMMC-7721细胞,结果 经RT-PCR表明,重组体导入的人肝癌细胞内有HBV反义RNA转录表达,且重组体导入对细胞生长无明显影响。结论 HBV全基因真核细胞表达载体在SMMC-7721细胞中能转录表达反义RNA,因而有可能利用反义技术和转基因方法进行HBV全基因反义RNA抗HBV研究。     

乙肝反义全基因真核载体的构建及在 SMMC－ 7721细胞内的表达

目的研究HBV反义全基因真核表达载体在抗HBV基因治疗的可能性。方法采用基因重组技术,将HBV全基因片段反向插入真核细胞表达载体pBK－CMV克隆位点中,通过FuGENETM6转染试剂,将重组体转染人肝癌细胞系SMMC－7721细胞。结果经RT－PCR表明,重组体导入的人肝癌细胞内有HBV反义RNA转录表达,且重组体导入对细胞生长无明显影响。结论HBV全基因真核细胞表达载体在SMMC－7721细胞中能转录表达反义RNA,因而有可能利用反义技术和转基因方法进行HBV全基因反义RNA抗HBV研究。     

乙肝反义全基因真核载体的构建及在SMMC-7721细胞内的表达

目的 研究HBV反义全基因真核表达载体在抗HBV基因治疗的可能性。方法 采用基因重组技术 ,将HBV全基因片段反向插入真核细胞表达载体pBK -CMV克隆位点中 ,通过FuGENETM6转染试剂 ,将重组体转染人肝癌细胞系SMMC -7721细胞。结果 经RT-PCR表明 ,重组体导入的人肝癌细胞内有HBV反义RNA转录表达 ,且重组体导入对细胞生长无明显影响。结论 HBV全基因真核细胞表达载体在SMMC -7721细胞中能转录表达反义RNA ,因而有可能利用反义技术和转基因方法进行HBV全基因反义RNA抗HBV研究。     

实时荧光定量RT-PCR技术检测siRNA对HBV复制的干扰效果

目的 运用实时荧光定量RT-PCR的方法,检测小RNA分子（small interfering RNAs,siRNA）对乙型肝炎病毒（hepatitis B virus,HBV）复制的抑制作用.方法 在HepG2.2.15细胞内导入筛选的三条特异抗HBV的siRNA分子,用实时荧光定量RT-PCR的方法,检测干扰后HBV的mRNA表达水平.结果 导入特异siRNA分子的细胞中HBV的mRNA表达量明显降低.结论 实时荧光定量RT-PCR技术能准确可靠的检测mRNA的表达水平,对siRNA分子干扰效果的评价有很好的应用价值.     

HBV核心蛋白拮抗α干扰素抗病毒活性的初步研究

目的 探讨HBV核心蛋白（HBc）对α干扰素（IFN-α）抗病毒活性可能存在的拮抗机制。 方法 以表达抗黏液病毒A蛋白(MxA)的重组质粒pcDNA3.1-Flag-MxA （野生型）转染肝胚瘤细胞株HepG2.2.15细胞,用ELISA和real-time PCR法分别检测转染后HepG2.2.15细胞上清HBsAg、HBeAg和细胞外HBV DNA。HBc核心蛋白表达质粒（pHBc-EGFP）转染HepG2细胞后,RT-PCR法分析IFN-α抗病毒蛋白MxA、双链RNA激活的蛋白激酶（PKR）和2′,5′-寡腺苷酸合成酶（2′,5′-OAS）等mRNA水平。结果 转染MxA重组质粒的HepG2.2.15细胞能够表达MxA,转染48 h时HBsAg、HBeAg比空白对照组显著减少（P＜0.05）,HBV DNA无显著性差异。转染pHBc-EGFP的HepG2细胞,经1 000 IU/ml IFN-α处理后,抗病毒蛋白PKR和2′,5′-OAS mRNA水平未见明显改变,而MxA-mRNA水平显著降低（P＜0.05）。 结论 HBV核心蛋白可能通过抑制抗病毒蛋白MxA的表达,而发挥对IFN-α的拮抗作用。     

Fas靶向RNA干扰逆转录病毒载体的构建及生物活性鉴定

本研究构建Fas靶向RNA干扰逆转录病毒载体,为探讨抑制Fas表达在再生障碍性贫血等疾病治疗中的意义、以及为促进RNA干扰技术的广泛开展奠定基础.用PCR方法获得小鼠U6＋27启动子盒及产生siRNA的相应DNA模板,然后连同增强绿色荧光蛋白（EGFP）基因克隆入商业化逆转录病毒载体pLXSN,以获得逆转录病毒载体穿梭质粒pLXSN/EGFP-siFas.包装、滴定重组逆转录病毒载体,并感染P815细胞,检测绿色荧光蛋白表达情况及对细胞Fas表达的抑制情况.结果表明：构建的逆转录病毒载体pLXSN/EGFP-siFas能够有效被包装并感染靶细胞,在靶细胞内可同时表达siRNA、绿色荧光蛋白以及新霉素抗性.结论：成功构建了抑制小鼠Fas表达的RNA干扰逆转录病毒载体.     

靶向C区基因的M1GS RNA核酶胞内抗乙型肝炎病毒的实验研究

目的探讨靶向作用于乙型肝炎病毒C区基因的M1GS RNA核酶胞内抗乙型肝炎病毒的作用。方法设计并应用PCR技术合成M1RNA核酶，应用pEGFP-C1载体构建M1GSRNA核酶的真核表达质粒，应用脂质体Lipofectamine TM 2000转染HepG2．2．15细胞，以ELISA法检测细胞培养液中病毒抗原，以反转录-聚合酶链反应法（RT-PCR）检测细胞内病毒mRNA，以荧光定量PCR法检测分泌入培养液的HBVDNA含量，用MTT法检测核酶对细胞增殖活性的影响。结果质粒载体表达的M1GS RNA核酶能明显抑制细胞培养液中HBeAg的表达及病毒mRNA的表达，抑制率分别为31．58％，32．5％。但M1GS RNA核酶对培养液中的HBV DNA含量无明显影响，亦不影响HepG2．2．15细胞的增殖活性。结论M1GS RNA核酶能特异性抑制HepG2．2．15细胞内HBV C区基因表达，是一种很有潜力的抗HBV基因治疗方法。     

单纯疱疹病毒Ⅱ型潜伏相关转录体真核表达载体的构建及表达

背景：单纯疱疹病毒Ⅱ型潜伏相关转录体开放读码框1真核表达载体的构建可以为研究单纯疱疹病毒Ⅱ型潜伏相关转录体开放读码框1在病毒潜伏复发中的功能奠定基础。目的：构建含存单纯疱疹病毒Ⅱ型潜伏相关转录体开放读码框1的真核表达载体,并通过转染非洲绿猴肾细胞（Vero）,验证载体的体外表达情况。方法：根据单纯疱疹病毒Ⅱ型潜伏相关转录体开放读码框1基因序列设计一对引物,以单纯疱疹病毒Ⅱ型333标准株基因组为模板PCR法扩增出开放读码框1基因,克隆至真核表达载体上,并进行酶切鉴定以及测序;利用X-fect转染试剂盒将重组质粒pEGFP-C2/潜伏相关转录体开放读码框1转染vero细胞,RT-PCR检测其mRNA水平的表达,并用荧光倒置显微镜观察融合蛋白的表达。结果与结论：开放读码框1基因的体外扩增目的片段为741bp,所构建的真核表达载体pEGFP-C2/潜伏相关转录体开放读码框1经双酶切鉴定,与预期大小一致,测序结果与NCBI收录的开放读码框1基因序列一致;重组质粒pEGFP-C2/潜伏相关转录体开放读码框1转染vero细胞后,RT-PCR验证有目的基因的转录,荧光显微镜观察到融合蛋白在转染的Vero细胞中表达。表明成功构建pEGFP-C2/潜伏相关转录体开放读码框1真核表达载体,并且实现其在非洲绿猴肾细胞（Vero）中的表达。     

bcr/abl反义RNA片段对Ph~ 人白血病细胞系致瘤性和存活的影响(英文)

构建了表达2、3和4个相同241bp b3/a2型bcr/abl融合基因的反义RNA片段的重组质粒,酶切电泳和以241bp bcr/abl基因片段为探针进行的菌落原位杂交证实各目的片段已插入逆转录病毒表达载体,分别命名为pDAB2、pDAB3和pDAB4。用脂质体介导的方法将pDAB3导入Ph~ 人白血病细胞系(K562和BV173细胞),48小时后用G418进行抗性筛选,观察了对靶细胞增殖的影响。结果表明,外源质粒在靶细胞内表达的反义RNA片段,使细胞致瘤性消失及诱发凋亡。探讨了可能的作用机理。     

维持性透析患者严重的感染症之一:病毒性肝炎

乙型肝炎及丙型肝炎的病原学一、乙型肝炎病毒 (HBV)为DNA病毒 ,其直径为 4 2～ 4 7nm的球型颗粒 (Dane颗粒 )。HBV由两层外壳及内核组成。外壳有许多小球状颗粒 ,含表面抗原 (HbsAg)。内核由环状双股DNA、DNA多聚酶、核心抗原 (HbcAg)和e抗原 (HbeAg)。HBV双链DNA的一条为短链 ,为正链 ,含 2 80 0对核苷酸 ,该链不完全闭合 ,有一缺口 ,不完整 ,不含开放读码区 ,不能编码蛋白。双链DNA中另一条为长链 ,为负链 ,含 32 0 0对核苷酸 ,有四个开放读码区 ,编码全部病毒蛋白。这四个开放读码区分别是S基因区、C基因区、P基因区和X基…     

核酸扩增技术在献血者血液HBV DNA、HCV RNA及HIV-1 RNA筛查中的应用研究

目的探讨在我国无偿献血者的血液筛查中引进核酸扩增技术(NAT)的必要性,了解献血者血清学病毒标志物检测阴性、NAT检测阳性的感染状况。方法应用Roche PCR、PCR-微流芯片、实时荧光PCR方法对深圳市131 174人(次)血清学检测病毒标志物阴性的献血者进行HBV DNA、HCV RNA和HIV-1 RNA检测,对NAT阳性献血者追踪检测并做定量分析。结果HBV DNA阳性22例,阳性率为1/5 962,其中15例为抗-HBc阳性,阳性率为1/8 745;HCV RNA阳性1例,阳性率1/131 174;HIV-1 RNA未检出阳性。对14名HBV DNA阳性者的追踪发现,8人发生了血清转换现象。结论采用高灵敏度的NAT筛查献血者血液中的HBV和HCV,有助于提高输血及血液安全。     

Detection of hepatitis B virus DNA in serum with nucleic acid probes labelled with 32P, biotin, alkaline phosphatase or sulphone.

To facilitate the clinical application of dot-blot hybridization for assaying hepatitis B virus (HBV) DNA, we compared the ability of nucleic acid probes labelled with 32P or with various non-radioactive markers to detect HBV DNA in patient serum. Cloned HBV DNA was hybridized with (1) 32P-labelled HBV DNA cloned in M13, (2) the 32P-labelled HBV RNA probe included in the HepProbe kit, (3) an alkaline phosphatase-labelled synthetic oligonucleotide of HBV, (4) biotin-labelled HBV DNA, and (5) sulphonated HBV DNA. Detection was either by autoradiography or an enzymatic colour reaction. The lowest level of detection of cloned HBV DNA was achieved with the 32P-labelled HBV RNA probe (0.3 pg HBV DNA, corresponding to 3 x 10(4) genomes in 50 microliters), followed by the 32P-labelled DNA probe (0.3-2 pg), sulphonated DNA (1-2 pg), biotin-labelled DNA (4 pg), and an alkaline phosphatase-labelled synthetic oligonucleotide (30 pg). Subsequently, sera from 159 patients with various constellations of HBsAg, HBeAg, and anti-HBe were tested with the most sensitive radioactive method (HepProbe) and the corresponding nonradioactive method (sulphonation). The overall concordance rate was 71% (r = 0.42). Compared with HepProbe results, sulphonation showed a sensitivity of 80% and a specificity of 67%. We conclude that radiolabelling (in particular 32P-labelling of HBV RNA) still allows the most sensitive and reliable detection of HBV DNA in patient serum using conventional dot-blot hybridization.     

免疫筛查阴性献血者血样病毒核酸检测的研究

目的了解二次酶联免疫筛查献血者血样漏检的原因。方法将二次酶联免疫筛查阴性的献血者血样在加样仪上实现血液样本汇集,用全自动核酸提取仪提取样本核酸,以核酸扩增检测仪做HBV、HCV和HIV自动扩增检测。对HBsAg阴性、HBVDNA阳性献血者用核酸筛查试剂定量检测HBV,并每隔2周对其跟踪采血,做HBV两对半免疫检测和HBsAgV3的确认试验。结果16320份二次酶联免疫筛查阴性的合格献血者血样中,8份HBVDNA阳性(漏检率0.49‰),未发现HCV和HIV1RNA阳性。8份HBVDNA阳性献血者乙肝两对半免疫检测HBsAg、HBsAb和HBeAg均为阴性,HBcAb均为阳性,3份HBeAb为阳性。6例HBsAg阴性HBVDNA阳性献血者血样的病毒滴度在(76～1490)copies/ml,2例病毒滴度过低,未定量检测到病毒。跟踪6名HBVDNA阳性献血者,1例18周时HBsAg确认试验阳性,其余5例仍为阴性。结论现行的二次酶联免疫技术的血液筛查存在HBV漏检,原因可能是隐匿性乙型肝炎病毒感染。应重视血液筛查工作中HBV的漏检及输血传播,并在现有的血液筛查模式中或增加HBcAb检测,或增加病毒核酸筛查。     

反义锁核酸体外抗乙肝病毒的实验研究

目的探讨锁核苷酸(LNA)修饰的反义寡核苷酸抑制乙型肝炎病毒(HBV)表达的效果及其抗HBV作用的特点。方法针对HBV S基因同一靶位分别合成三段不同化学修饰的反义寡核苷酸:锁核酸修饰、未修饰和全硫代修饰,并以无关序列为对照直接作用于HepG22.2.15细胞,ELISA法动态检测细胞上清液中HBsAg含量;实时荧光定量PCR法(FQ-PCR)检测细胞上清中HBV DNA含量;MTT法监测细胞活性。结果针对HBV S基因的反义LNA能显著抑制HepG22.2.15细胞表达HBsAg(P<0.05)且第3天出现抑制高峰,抑制作用随时间延长逐渐减弱。反义锁核酸组对HBsAg及细胞内、细胞外HBV DNA均有较强抑制作用,最高抑制率分别为51.19%、56.73%、86.9%,与其他实验组相比差异有统计学意义(P<0.05),与对照组比较差异有统计学意义(P<0.01);LNA在20μmol/L浓度范围内对细胞代谢无影响。结论针对S基因的反义LNA体外能发挥高效、特异、低毒的抗HBV作用,为乙型肝炎的基因治疗注入了新的思路。     

Inhibition of hepadnaviral replication by polyethylenimine-based intravenous delivery of antisense phosphodiester oligodeoxynucleotides to the liver.

Antisense oligodeoxynucleotides (ODNs) appear as attractive anti-hepatitis B virus (HBV) agents. We investigated in vivo, in the duck HBV (DHBV) infection model, whether linear polyethylenimine (lPEI)-based intravenous delivery of the natural antisense phosphodiester ODNs (O-ODNs) can prevent their degradation and allow viral replication inhibition in the liver. DHBV-infected Pekin ducklings were injected with antisense O-ODNs covering the initiation codon of the DHBV large envelope protein, either in free form (O-ODN-AS2) or coupled to lPEI (lPEI/O-ODN-AS2). Following optimization of lPEI/O-ODN complex formulation, complete O-ODN condensation into a homogenous population of small (20-60 nm) spherical particles was achieved. Flow cytometry analysis showed that lPEI-mediated transfer allowed the intrahepatic delivery of lPEI/O-ODN-AS2 to increase three-fold as compared with the O-ODN-AS2. Following 9-day therapy the intrahepatic levels of both DHBV DNA and RNA were significantly decreased in the lPEI/O-ODN-AS2-treated group as compared with the O-ODN-AS2-treated, control lPEI/O-ODN-treated, and untreated controls. In addition, inhibition of intrahepatic viral replication by lPEI/O-ODN-AS2 was not associated with toxicity and was comparable with that induced by the phosphorothioate S-ODN-AS2 at a five-fold higher dose. Taken together, our results demonstrate that phosphodiester antisense lPEI/O-ODN complexes specifically inhibit hepadnaviral replication. Therefore we provide here the first in vivo evidence that intravenous treatment with antisense phosphodiester ODNs coupled to lPEI can selectively block a viral disease-causing gene in the liver.     

乙型肝炎表面抗原/抗体同时存在的血清学模式与病毒血症的关系

目的分析乙型肝炎病毒（HBV）感染者乙肝表面抗原（HBsAg）与乙肝表面抗体（HBsAb）同时阳性的模式,与HBV DNA的关系,以及其产生的原因及临床意义。方法采用化学发光微粒子免疫分析法（CMIA）检测样本的HBV血清标志物,从中选出HBsAg和HBsAb同时阳性的样本,采用荧光定量聚合酶链反应（FQ-PCR）检测其HBV DNA定量值。结果在选取的HBsAg和HBsAb同时阳性的37例样本中,出现3种HBV模式：（1）HBsAg、HBsAb、HBeAb、HBcAb阳性,阳性率46%,HBV DNA阳性率22%;（2）HBsAg、HBsAb、HBeAb、HBcAb阳性,阳性率30%,HBV DNA阳性率30%;（3）HBsAg、HBsAb、HBeAb、HBcAb阳性,阳性率24%,HBV DNA阳性率5%。37例样本中,有21例血清HBV DNA检测阳性,阳性率为57%。结论 HBsAg和HBsAb同时阳性并不代表疾病真正好转,部分感染者仍存在病毒血症,应当引起重视。     

不同血清学标志血清中HBV DNA和HCV RNA的分析研究

应用聚合酶链反应（ＰＣＲ）技术检测４８１例乙肝血清学五项指标至少一项阳性血清中ＨＢＶＤＮＡ和ＨＣＶＲＮＡ，结果显示ＨＢＶＤＮＡ的检出率为９１．５％，ＨＣＶＲＮＡ的检出率为１５．４％，并提示（１）当乙肝患者血清中抗ＨＢｅ阳性甚至抗ＨＢｓ阳性时，仍有相当部分的ＨＢＶ存在且仍在复制；（２）ＨＢＶ和ＨＣＶ感染可相互增加其易感性；ＨＢｓＡｇ阳性与否与抗ＨＣＶ可能有一定关系；（３）ＰＣＲ检测ＨＢＶＤＮＡ、ＨＣＶＲＮＡ具有较高的敏感性。     

Quantitative analysis of hepatitis D virus RNA and hepatitis B surface antigen serum levels in chronic delta hepatitis improves treatment monitoring

BACKGROUND/AIMS: Treatment of chronic delta hepatitis is long and difficult and better monitoring is needed. METHODS: In this study, hepatitis delta virus (HDV) RNA, hepatitis B surface antigen (HBsAg) and hepatitis B virus (HBV) DNA were retrospectively quantified in 53 patients with hepatitis B e antigen (HBeAg)-negative chronic hepatitis delta. Twenty-one had received 28 courses of 3-5 MU interferon-alpha2b (IFN-alpha2b) thrice weekly for a median of 12.6 months (interquartile range [IQR]: 7.3-31.6), five had received eight courses of 100 mg lamivudine (LAM) daily for 23.6 months (IQR: 8.4-61.5) and 27 were untreated. The controls were 54 untreated, randomly selected, HBeAg-negative chronic hepatitis B patients without delta infection. Quantification of serum HDV RNA, HBsAg and HBV DNA were performed at baseline, during and at the end of treatment and end of follow up. RESULTS: Untreated patients had significantly higher median HBsAg levels than controls (5,872 vs 3,501 IU/ml; P = 0.046), but lower median HBV DNA levels (2.933 vs 6.459 log10 copies/ml; P < 0.001). Median baseline HDV RNA (6.374 log10 copies/ml) was similar in IFN-alpha2b-treated, LAM-treated and untreated patients. At the end of treatment, IFN-alpha2b significantly suppressed in paired measurements HDV RNA (P = 0.012) and HBsAg (P = 0.043), but LAM was inefficient. In IFN-alpha2b-treated patients, HDV RNA became undetectable in five patients within a median of 30 months (IQR: 8-90), followed by a slower decrease in HBsAg. CONCLUSIONS: In untreated chronic delta hepatitis, suppressed HBV replication is associated with significantly increased HBsAg serum levels. IFN-alpha2b significantly suppresses both HDV RNA and HBsAg, but LAM has no effect. Long-term IFN-alpha2b treatment (IQR: 1.5-5.0 years) appears necessary for undetectable serum HDV RNA and further treatment is required for HBsAg loss. Monitoring of HDV RNA and HBsAg serum levels in patients with chronic delta hepatitis provides insight during treatment.     

Inhibition of bone resorption in vitro by antisense RNA and DNA molecules targeted against carbonic anhydrase II or two subunits of vacuolar H(+)-ATPase.

The bone resorbing cells, osteoclasts, express high levels of carbonic anhydrase II (CA II) and vacuolar H(+)-ATPase (V-ATPase) during bone resorption. We have used antisense RNA and DNA molecules targeted against CA II, and against 16- and 60-kD subunits of vacuolar H(+)-ATPase (V-ATPase), to block the expression of these proteins in vitro. Osteoclastic bone resorption was studied in two in vitro culture systems: release of 45Calcium from prelabeled newborn mouse calvaria cultures, and resorption pit assays performed with rat osteoclasts cultured on bovine bone slices. Both antisense RNA and DNA against CA II and the V-ATPase were used to compare their specificities as regards inhibiting bone resorption in vitro. The antisense molecules inhibited the synthesis of these proteins by decreasing the amounts of mRNA in the cells in a highly specific manner. In osteoclast cultures treated with the 16-kD V-ATPase antisense RNA, acidification of an unknown population of intracellular vesicles was highly stimulated. The acidification of these vesicles was not sensitive to amiloride or bafilomycin A1. This suggests the existence of a back-up system for acidification of intracellular vesicles, when the expression of the V-ATPase is blocked. Our results further indicate that blocking the expression of CA II and V-ATPase with antisense RNA or DNA leads to decreased bone resorption.     

Survivin反义核酸促进紫杉醇诱导HL-60细胞凋亡

目的　探讨Survivin反义核酸对紫杉醇诱导白血病细胞系HL 6 0细胞凋亡的影响。方法　将已构建成功的Survivin反义核酸真核表达载体pcDNA3 SVVas电转染HL 6 0细胞 ,并筛选转染成功的阳性克隆 ;锥虫蓝拒染法观察Survivin反义核酸与紫杉醇联合应用对HL 6 0细胞增殖的影响 ;细胞计数和MTT实验测定转染细胞对紫杉醇敏感性 ;琼脂糖凝胶电泳分析细胞凋亡DNA断裂情况 ;核染色检测凋亡细胞的细胞核变化。结果　转染Survivin反义核酸的HL 6 0SVVas细胞增殖明显受抑 ,与HL 6 0neo细胞、HL 6 0细胞进行比较 ,差异具有显著性 (P <0 .0 5 ) ;MTT实验显示 ,紫杉醇对HL 6 0SVVas、HL 6 0neo、HL 6 0细胞的IC50 值分别为 (14 .4± 1.87)ng ml,(31.9± 6 .38)ng ml和 (32 .0± 6 5 2 )ng ml。统计学分析证明差异具有显著性 (P <0 .0 1) ;经琼脂糖凝胶电泳 ,HL 6 0SVVas细胞可见到DNA梯形条带 ,而HL 6 0neo、HL 6 0细胞未见到 ;与HL 6 0neo、HL 6 0细胞相比 ,HL 6 0SVVas的细胞核呈致密浓染。结论　Survivin反义核酸可促进紫杉醇诱导HL 6 0细胞凋亡。