脂质体干扰素的体外抗乙型肝炎病毒效应

本文采用２．２．１５细胞进行脂质体干扰素与干扰素的体外抗乙型肝炎病毒实验，用ＥＬＩＳＡ法测ＨＢｅＡｇ，用反向血凝法及ＥＬＩＳＡ法测ＨＢｓＡｇ，用斑 交法测ＨＢＶ ＤＮＡ，结果证实，脂质体ＩＦＮ体外对ＨＢｅＡｇ的抑制率较ＩＦＮ高，能较好地抑制ＨＢＶ ＤＮＡ，但与ＩＦＮ一样，对ＨＢｓＡｇ无抑制作用。     

特异性乙型肝炎病毒X基因反义核酸体外抗病毒作用

为了观察互补于乙型肝炎病毒（ＨＢＶ）Ｘ基因的三段硫代反义核酸（ＡＳＯＮ）体外抗病毒作用，采用ＥＬＩＳＡ和ＰＡＰ－ＥＬＩＳＡ法检测ＡＳＯＮ作用前后２，２，１５细胞上清中乙型肝炎病毒表面抗原、ｅ抗原及Ｘ抗原（ＨＢｓＡｇ、ＨＢｅＡｇ、ＨＢｘＡｇ）含量变化及细胞原位杂交检测细胞内ＨＢＶＤＮＡ含量变化。结果表明，三段ＡＳＯＮ均可抑制ＨＢｓＡｇ、ＨＢｅＡｇ和ＨＢｘＡｇ的表达，其抑制率分别为８０．６５％、６２．７６％和７８．０７％；细胞内ＨＢＶＤＮＡ也明显减少。据此认为，ＨＢｘＡｇ表达量下降可能系ＡＳＯＮ序列特异性封闭作用所致，而ＨＢ－ｓＡｇ和ＨＢｅＡｇ表达量以及ＨＢＶＤＮＡ含量降低，可能是通过ＨＢｘＡｇ对ＨＢＶＤＮＡ启动子的反式激活功能降低而实现的。     

HBV感染者血清HBeAg/IC和HBV DNA间的关系

资料与方法：ＨＢＶ感染者１１７例,正常对照２０例,研究对象排除其它肝炎病毒感染,半年内未经抗病毒及免疫治疗。采用ＥＬＩＳＡ及Ｎａｓｔｅｄ－ＰＣＲ方法分别检测ＨＢＶ感染者血清中ＨＢｅＡｇ／免疫复合物（ＩＣ）和ＨＢＶ ＤＮＡ。 结果：１１７例ＨＢＶ感染者血清中共检出ＨＢｅＡｇ／ＩＣ阳性２３例,ＨＢＶＤＮＡ阳性 ５５例, ＨＢｅＡｇ／ＩＣ阳性者中 ＨＢＶＤＮＡ阳性率为９１．３％（２１／２３例）,而ＨＢｅＡｇ／ＩＣ阴性的９４例中ＨＢＶ ＤＮＡ阳性３４例,阳性率为３６．２％,两组差异有非常显著性,ｘ２＝８．１５…     

乙型肝炎病毒前C／C基因区硫代和脂肪链—硫代反义寡核苷酸的体外抗…

为了研究修饰的反义寡核苷酸（ＡＳＯＮ）的抗乙型肝炎病毒（ＨＢＶ）作用。以２．２．１５细胞为靶细胞，在ＨＢＶ前Ｃ／Ｃ基因区设计合成了１６聚硫代和脂肪链－硫代两种修饰的ＡＳＯＮ，用酶联免疫吸附和斑点杂交技术分别检测作用细胞的乙型肝炎病毒表面抗原（ＨＢｓＡｇ）和ｅ抗原（ＨＢｅＡｇ）以及ＨＢＶ ＤＮＡ的分泌情况。结果显示，ＡＳＯＮ在浓度为１０μｍｏｌ／Ｌ时能特异性抑制细胞９２％￣９５％ＨＢｓＡｇ和８４％￣     

反义乙肝病毒S基因真核载体构建及体外抗乙肝病毒作用

目的：探讨反义ＲＮＡ抗乙肝病毒（ＨＢＶ）作用。方法：构建了正，反义ＨＢＶＳ基因重组ＥＢ病毒载体ｐＭＥＰ４ｓ，ｐＭＥＰ４Ｓａｓ，ＤＮＡ－磷酸钙共沉淀法将重组载体ＤＮＡ转染２．２．１５细胞，潮霉素筛选１个月得到抗性细胞克隆，ＥＬＩＳＡ，斑点杂交法分别检测抗性细胞上清ＨＢｓＡｇ，ＨＢｅＡｇＨＢＶＤＮＡ水平，结果：转染后１，２月，反义载体ＨＢｓＡｇ，ＨＢｅＡｇ的抑制率分别达７５％，５１．６％，７０％，４６     

反义寡核苷酸抗HBV作用的双峰现象

ＨＢＶ基因组含３－２ｋｂＤＮＡ,有至少４个ＯＲＦ,其中Ｓ区包含Ｓ基因,ｐｒｅＳ１基因和ｐｒｅＳ２基因,编码形成大、中、小３种蛋白,构成ＨＢＶ的包膜蛋白（ＨＢｓＡｇ,ｐｒｅＳ１Ａｇ,ｐｒｅＳ２Ａｇ）．其中ＨＢｓＡｇ在体内引起的免疫病理反应是ＨＢＶ感染中的重要致病机制,ｐｒｅＳ２Ａｇ较ＨＢｓＡｇ有更强的抗原性,并具有反式激活作用,在ＨＢＶ感染及肝癌发生中关系密切．因而如何从基因水平抑制ＨＢｓＡｇ和ｐｒｅＳ２Ａｇ的表达,对抗ＨＢＶ治疗有重要意义．我们设计了针对Ｓ基因翻译起始区和ｐｒｅＳ２基因翻译起…     

感染乙型肝炎病毒MT2淋巴细胞表达Fas

目的：观测Ｔ淋巴细胞－ＭＴ２细胞对乙型肝炎病毒感染的敏感性，了解病毒感染对淋巴细胞表达Ｆａｓ的影响。方法：体外培养ＭＴ２细胞，用ＨＢＶ ＤＮＡ阳性血清直接感染，用酶联免疫和斑点杂交，ＰＣＲ法检测感染后的细胞病毒复制与表达状况，用酶联免疫法检测了Ｆａｓ抗原表达。结果：ＭＴ２细胞于阳性血清感染后第八天可测到ＨＢｅＡｇ，ＨＢｓＡｇ和ＨＢＶＤＮＡ同时出现Ｆａｓ表达信号，结论：ＭＴ２细胞对ＨＢＶ易感，ＨＢＶ     

海藻硫酸多糖抗乙型肝炎病毒的实验研究

１．材料与方法：海藻硫酸多糖（ＳＰＳ）是自制的一种天然硫酸酯多糖。在ＨＢＶ基因转染的人肝癌细胞系 ２．２．１５细胞中，观察ＳＰＳ在对ＨＢｅＡｇ和ＨＢｓＡｇ的抑制作用，以齐多夫定和灵芝多糖作为对照药物。选用鸭肝炎动物模型，以阿昔洛韦为阳性对照，观察ＳＰＳ对ＤＨＢＶ感染鸭血清ＤＨＢＶ ＤＮＡ水平的抑制作用。 ２．结果：ＳＰＳ在 ２．２．１５细胞培养中对ＨＢｓＡｇ和ＨＢｅＡｇ的分泌有明显抑制作用，随着ＳＰＳ浓度的增高，对ＨＢｅＡｇ和ＨＢｓＡｇ表达的抑制作用增强，最高抑制率分别为７０．１％±４．２％（ＩＣ５…     

硫代磷酸反义寡核苷酸对乙型肝炎病毒抗原表达的体外抑制作用

目的 研究硫代磷酸反义寡核苷酸（Ｓ－ＡＳＯＮ）的抗乙型肝炎病毒（ＨＢＶ）作用。方法 作者以２．２．１５细胞作为研究对象，在ＨＢＶ基因Ｓ区和Ｃ区的翻译起始位点设计合成了２段１６聚Ｓ－ＡＳＯＮｓ，采用酶联免疫吸附试验检测了培养细胞上清中表面抗原（ＨＢｓＡｇ）和ｅ抗原（ＨＢｅＡｇ）的分泌情况。结果 当Ｓ－ＡＳＯＮ浓度为每天２μｍｏｌ／Ｌ时，对ＨＢｓＡｇ和ＨＢｅＡｇ的抑制率可分别达到８８％和７５％，而无关     

乙肝病毒S基因区反义寡核苷酸对表面抗原表达的抑制作用

为了研究硫代反义寡核苷酸的抗乙型肝炎病毒作用，以２．２．１５细胞为靶细胞，在ＨＢＶ Ｓ基因起始区设计了１段ＡＳＯＮ，用酶联免疫吸附法检测了作用细胞的ＨＢＶ的分泌情况。结果显示，ＡＳＯＮ在每天给予２μｍｏｌ／Ｌ的浓度情况下抑制８８％的ＨＢｓＡｇ产生，无关序列的寡核苷酸无明显抑制作用，同时未见ＡＳＯＮ的细胞毒作用。     

乙型肝炎病毒(HBV)反基因与HBV的结合及对HBV复制的影响

目的 研究三螺旋形成寡核苷酸与HBV基因的结合情况及其对HBV复制的影响。方法 全盛一段能与HBV核心启动子位点（1734-1753nt)形成三螺旋的寡核苷酸（TF020）并标记上生物素分别用脂质体-TF020及裸TF020转染HepG2.2.15细胞,用链酶亲和素-生物素法（SABC）检测其转染效率及其与HepG2.2.15细胞中HBVDNA的结合情况;并采用ELISA、半定量逆转录（RT）-PCR及荧光定量PCR法分别检测经寡核苷酸处理的HepG2.2.15细胞及空白对照细胞上清HBsAg、HBeAg、HBV DNA及细胞中HBV RNA的水平。结果 脂质体-TF020转染HepG2.2.15细胞的效率可达80%,而裸TFO20转染率最高为40%;TF020主要定位于细胞核及胞浆中,TF020处理组细胞上清中HBsAg、HBeAg含量分别较空白对照组降低37.0%及78.2%,HBV DNA水平较空白对照组明显降低;而细胞中2.4kb/2.1kb RNA、3.5kb/3.4kbRNA亦分别降低31.6%及70.2Z%。结论 TF020通过脂质体包裹后可以很好地进入细胞并与HBV DNA结合;TF020能够有效地抑制HBV复制,发挥抗病毒作用。     

Inhibition of hepatitis B virus replication by pokeweed antiviral protein in vitro

AIM： To explore the inhibitory effects of pokeweed antiviral protein seed （PAP-S） and PAP encoded by a eukaryotic expression plasmid on hepatitis B virus （HBV） replication in vitro. METHODS： HepG2 2.2.15 cells in cultured medium were treated with different concentrations of PAP-S. HBsAg, HBeAg and HBV DNA in supernatants were determined by ELISA and fluorescent quantitative PCR respectively. MTT method was used to assay for cytotoxicity. HepG2 were cotransfected with various amounts of PAP encoded by a eukaryotic expression plasmid and replication competent wild-type HBV 1.3 fold overlength plasmid. On d 3 after transfection, HBsAg and HBeAg were determined by using ELISA. Levels of HBV core-associated DNA and RNA were detected by using Southern and Northern blot, respectively. RESULTS： The inhibitory effects of PAP-S on HBsAg, HBeAg and HBV DNA were gradually enhanced with the increase of PAP concentration. When the concentration of PAP-S was 10 μg/mL, the inhibition rates of HBsAg, HBeAg and HBV DNA were 20.9%, 30.2% and 50%, respectively. After transfection of 1.0 μg and 2.0 μg plasmid pXF3H-PAP, the levels of HBV nucleocapside- associated DNA were reduced by 38.0% and 74.0% respectively, the levels of HBsAg in the media by 76.8% and 99.7% respectively, and the levels of HBeAg by 72.7% and 99.3% respectively as compared with controls. Transfection with 2 μg plasmid pXF3H-PAP reduced the levels of HBV nucleocapside-associated RNA by 69.0%.CONCLUSION： Both PAP-S and PAP encoded by a eukaryotic expression plasmid could effectively inhibit HBV replication and antigen expression in vitro, and the inhibitory effects were dose-dependent.     

Inhibitory effect of antisense oligodeoxynucleotides complementary to HBVon HepG2.2.15 cell line

AIM To explore the therapeutic potential of antisense oligodeoxynucleotides on hepatcellular cacinoma(HCC).METHODS Four antisense phosphorothioated oligodeoxynucleotides (asON), complementary to differentsites of HBV, were synthesized and assayed for their anti-HBV activity in HepG22. 2.15 cells with ELISA.The most effective asON was chosen for the following study: FACSCAN, TRAP and immuno-staining wereused respectively for checking apoptosis, telomerase activity and expression of oncogene p21ras and p62C-myc inHepG2.2.15 cells after treated by asON.RESULTS The oligomer directed against the initiator of pre-S2 was the most effective one with aninhibitory rate of 66% on HBsAg and 91% on HBeAg (P<0.02). Two inhibitory peaks (bimodal)appeared. Telomerase activity as well as the expression of p21fas and p62C-myc decreased drastically 3 days afterasON-HBpreS2 treatment. Meanwhile, apoptosis appeared in the experiments.CONCLUSION The inhibitory effects of as-preS2 on the HBV gene expression and the reversion of somemalignant behaviour in HepG2.2.15 cells were the significant, effective therapy against HBV infection andhepatocellular carcinoma.     

复方六月雪体外抗HBV的实验研究

目的观察复方六月雪(CLYX)体外抗乙型肝炎病毒(HBV)的作用。方法在最大无毒浓度(TC0)基础上观察不同浓度药物作用于HepG2.2.15细胞,分别在第4天和8天收集细胞培养上清液,采用实时荧光定量PCR法检测上清液HBV DNA的含量,采用ELISA法测定上清液HBsAg和HBeAg的滴度。结果无毒浓度的复方六月雪在HepG2.2.15细胞培养中可有效地抑制细胞HBV DNA的复制及HBsAg和HBeAg的分泌。结论CLYX在体外有显著的抗HBV的作用。     

Inhibition of hepatitis B virus gene expression and replication by artificial microRNA

AIM： To investigate the inhibitory effects of hepatitis B virus （HBV） replication and expression by transfecting artificial microRNA （amiRNA） into HepG2.2.15 cells. METHODS： Three amiRNA-HBV plasmids were constructed and transfected into HepG2.2.15 cells. HBV antigen secretion was detected in the cells with transient and stable transfection by time-resolved fluoroimmunoassays （TRFIA）. HBV DNA replication was examined by ？ uorescence quantitative PCR, and the level of HBV S mRNA was measured by semi- quantitative RT-PCR. RESULTS： The efficiency of transient transfection of the vectors into 2.2.15 cells was 55%-60%. All the vectors had significant inhibition effects on HBsAg and HBeAg at 72 h and 96 h after transfection （P 〈 0.01 for all）. The secretion of HBsAg and HBeAg into the supernatant was inhibited by 49.8% ± 4.7% and 39.9% ± 6.7%, respectively, at 72 h in amiRNA- HBV-S608 plasmid transfection group. The copy of HBV DNA within culture supernatant was also significantly decreased at 72 h and 96 h after transfection （P 〈0.01 for all）. In the cells with stable transfection, the secretion of HBsAg and HBeAg into the supernatant was significantly inhibited in all three transfection groups （P 〈 0.01 for all, vs negative control）. The copies of HBV DNA were inhibited by 33.4% ± 3.0%, 60.8% ± 2.3% and 70.1% ± 3.3%, respectively. CONCLUSION： In HepG2.2.15 cells, HBV replication and expression could be inhibited by artif icial microRNA targeting the HBV S coding region. Vector-based artificial microRNA could be a promising therapeutic approach for chronic HBV infection.     

长片段RNA抑制HepG2.2.15细胞中HBV基因的表达和复制

目的 评估长的反义RNA干扰片段在培养细胞株中对HBV复制的抑制效应.方法将HBV基因组S区的全部核苷酸序列插入至pTARGETTM载体中,并将重组载体转染入HepG2.2.15细胞中.用酶联免疫吸附法检测HBsAg与HBeAg水平,用荧光定量PCR法检测HBVDNA水平.对数据采用多个独立样本Kruskal-Wallis检验与两两比较的Mann-Whitney U检验.结果 经过处理后,HepG2.2.15细胞上清液中HBsAg表达量(A值)在HBS2组(携带长片段反义RNA)为0.621±0.027,在HBS4组(携带正义RNA)为3.399±0.018,对照组为2.232±0.187;HBeAg表达量(A值)在HBS2组、HBS4组和对照组分别为0.749±0.019、1.548±0.025和1.570±0.044; HBV DNA水平(×104拷贝/ml)在HBS2组、HBS4组、对照组分别为1.597±0.082、3.381±0.297和3.610±0.063.与对照组相比,HBS2组HBsAg、HBeAg和HBV DNA表达量均降低,统计量Z值均为-2.309,P值均＜0.05; HBS4组HBsAg表达量增高(Z=-2.309,P＜0.05),而HBeAg和HBV DNA表达量无明显差异,统计量Z值分别为-0.866、-1.155,P值均＞0.05.结论 长片段反义RNA能抑制HBV基因的表达和病毒复制.

Abstract：

Objective To evaluate the inhibitory effects of long antisense RNA on HBV replication in HepG2.2.15 cells. Methods The coding region of HBV S gene was cloned into pTARGET vector in sense and antisense orientations and the recombinant plasmids were transfected into HepG2.2.15 cells which were divided into HBS2 (antisense RNA) group, HBS4 (sense RNA) group and control group. HBsAg and HBeAg in the culture supernant were detected by ELISA. The HBV DNA in the supernant was quantified by real-time PCR. Results After treatment, the levels of HBsAg in HepG2.2.15 cell supernatants of three groups were 0.621 ± 0.027, 3.399 ± 0.018 and 2.232 ± 0.187 respectively; the levels of HBeAg were 0.749 ± 0.019,1.548 ± 0.025 and 1.570 ± 0.044 respectively and the levels of HBV DNA were 1.597 ± 0.082, 3.381 ± 0.297 and 3.610 ± 0.063 respectively. The expressions of HBsAg and HBeAg and the HBV DNA level in HBS2 group were remarkably reduced as compared to the control (Z = -2.309, P ＜ 0.05); whereas the sense plasmid transfection (HBS4) did not affect HBeAg (Z= -0.866) and HBV DNA (Z = -1.155) levels in the culture supernant but slightly increased the HBsAg level (Z = -2.309). Conclusion Antisense RNA might be a useful tool to repress HBV replication.     

靶向S区和C区基因的M1GS RNA核酶共同抑制HBV的表达

目的:研究靶向HBVS区和C区基因的M1GSRNA核酶共同作用对HBV基因表达的影响.方法:选择HBVayw亚型S区基因294nt和C区基因2333nt为切割位点,以含有编码M1RNA的DNA序列的质粒pTK117为模板,通过PCR扩增得到M1GSRNA核酶的DNA模板,并将其克隆至真核表达载体pEGFP-C1得到重组质粒pEGFP-GSS和pEGFP-GSC.将2个重组质粒共转染HepG2.2.15细胞,转染后ELISA法测细胞培养液中的HBsAg和HBeAg,RT-PCR检测HBVmRNA.结果:成功构建了分别靶向HBVS区基因和C区基因的真核表达载体.共转染HepG2.2.15细胞后,HBsAg和HBeAg的表达分别被抑制了33.2%和39.1%,HBVCmRNA和SmRNA分别被抑制了32.5%和29.7%,而HepG2.2.15细胞的增殖无明显变化.结论:靶向HBVS区和C区基因的M1GSRNA核酶共同作用可特异性抑制HBVS区和C区基因的表达.     

反义锁核酸与拉米夫定在HepG2.2.15细胞中抗HBV作用的比较

目的:应用反义锁核酸与拉米夫定作用HepG2.2.15细胞,对他们抗乙型肝炎病毒(hepatitis B virus,HBV)效果进行比较.方法:设计针对HBV翻译起始区S基因mRNA的反义寡核苷酸,并进行锁核酸修饰,以阳离子脂质体介导反义锁核酸转染HepG2.2.15细胞;拉米夫定组直接作用HepG2.2.15细胞;分别于用药后第2、4、6、8、10天收集细胞培养上清液.用ELISA法和FQ-PCR法检测收集上清液HBsAg、HBeAg和HBVDNA的含量.MTT法分别检测反义锁核酸与拉米夫定对细胞存活率的影响.结果:拉米夫定对HBVDNA具有明显抑制作用,最高可达46.52%,但对HBsAg、HBeAg影响较小;反义锁核酸对HBsAg、HBeAg及HBVDNA均有较强抑制作用,对HBsAg、HBeAg和HBVDNA的最高抑制率分别达67.69%、59.71%和62.96%(P<0.05),且抑制随时间呈增高趋势.反义锁核酸与拉米夫定对细胞代谢均无明显影响.结论:反义锁核酸抗HBV作用机制与拉米夫定不同,反义锁核酸抗HBV作用明显优于拉米夫定.     

青蒿琥酯对乙型肝炎病毒复制及肝癌细胞株HepG2.2.15凋亡的影响

目的 探讨青蒿琥酯在体外对乙型肝炎病毒复制及肝癌细胞株HepG2.2.15凋亡的影响.方法 将不同浓度青蒿琥酯作用于转染乙型肝炎病毒全基因组DNA的肝癌细胞株HepG2.2.15,收集48 h上清,采用酶联免疫吸附实验检测上清中乙型肝炎病毒表面抗原(HBsAg)和e抗原(HBeAg),采用荧光定量PCR法检测HBV-DNA,流式细胞术检测细胞凋亡.结果 青蒿琥酯对HBV复制具有抑制作用,随着浓度增加,对HBsAg和HBeAg的抑制率逐渐上升,细胞内HBV-DNA 复制水平下降;青蒿琥酯可诱导肝癌细胞早期凋亡及导致细胞死亡,随浓度增加,HepG2.2.15细胞早期凋亡率及死亡率均增加.结论 青蒿琥酯对HepG2.2.15细胞HBsAg和HBeAg的分泌及HBV-DNA复制具有抑制作用,并具有诱导HepG2.2.15细胞凋亡的作用.