联合应用siRNA和拉米夫定抑制HBV复制和表达的实验研究

观察联合应用小干扰RNA和拉米夫定对HepG2.2.15细胞中HBV抗原表达和复制的抑制作用。构建并转染重组质粒psil-HBV到HepG2.2.15细胞中。转染后的细胞培养基中加入拉米夫定(0.05μm),分别于48、72、96 h收获细胞。用ELISA方法检测HBeAg和HBsAg;HBV DNA水平用实时定量PCR测定;用逆转录PCR检测HBV mRNA水平。96 h后联合应用小干扰RNA和拉米夫定组细胞培养上清中HBeAg和HBsAg抑制率分别为91.8%和82.4%(P<0.05);HBV mRNA表达水平明显降低。HepG2.2.15细胞中联合应用小干扰RNA和拉米夫定对HBV复制的抑制作用比单独应用siRNA或拉米夫定更有效。     

抑制核转录因子PPARα的反义寡核苷酸的抗乙型肝炎病毒活性研究

目的:研究靶向过氧化物酶体增殖活化受体α(PPARα)的寡核苷酸是否具有抑制乙型肝炎病毒(HBV)复制的活性作用。方法:反义寡核苷酸作用于能稳定表达HBV丹氏颗粒的HepG2.2.15细胞,ELISA检测细胞上清中HBV表面抗原(HBsAg)及e抗原(HBeAg)的分泌;实时荧光定量PCR考察反义寡核苷酸对HBV DNA复制水平的影响;通过反转录PCR和Western印迹考察反义寡核苷酸作用于细胞后靶基因及靶蛋白的差异表达情况。结果:抑制PPARα表达的反义序列PPARα-2可剂量依赖且特异性地抑制肝癌细胞中HBsAg和HBeAg的表达,且显著降低细胞中PPARαmRNA水平和蛋白水平。结论:通过筛选初步确定了基于PPARα设计的反义寡核苷酸有较好的抗HBV活性,同时也验证确定了PPARα可能成为抗HBV药物的新型作用靶点。     

pSUPER介导的RNAi抗乙肝病毒S基因的研究

目的 研究RNAi抗乙肝病毒的作用.方法 设计并合成一段针对HBV S基因的干扰序列及一段无关的序列,克隆进pSUPER载体中,分别构建pSUPEK-HBS、pSUPEK-nonsense载体,然后将pSUPER、pSUPEK-HBS、pSUPER-nonsense转染进HepG2.2.15细胞中,用ELISA方法对细胞上清中HBsAg、HBeAg进行检测.尾静脉注射HBV转基因小鼠,取血清检测.结果 pSUPER-HBS所表达的siRNA成功的抑制了HepG 2.2.15上清及HBV转基因鼠血清中的HBsAg、HBeAg,抑制率分别达到70%、51%以及60%、42%.结论 针对HBV S区的siRNA能明显抑制HBV的复制.     

化学发光Southern blot法检测HBV体外复制及其应用于药物对HBV的抑制分析

目的：建立化学发光Southern blot检测细胞内HBV DNA的方法,同时检测3种不同靶点抗乙肝药物的体外作用。方法：用地高辛标记HBV探针,优化杂交条件,检测来自HepG2及HepG2.2.15 HBV DNA复制中间体;利用建立的化学发光Southern blot检测HBV DNA的方法检测经拉米夫定、Bay41-4109、α-Galcer以不同药物浓度处理的HepG2.2.15HBV DNA复制中间体的水平。结果：（1）标记的HBV探针的检测灵敏度为0.1pg,杂交系统的检测灵敏度为1pg,可检测到HepG2.2.15细胞内的HBV DNA特异性信号;（2）以该法检测胞内HBV DNA可见3种药物都有明显的抑制作用,其半数有效量（IC50）分别为1.53μmol/L、0.41μmol/L、0.01μmol/L。结论：胞质HBV DNA的水平能准确地反映不同靶点抗HBV药物的抗病毒效果,建议在观察药物特别是中药抗病毒研究中采用。     

半乳糖凝集素-3结合蛋白作为正向调节因子参与IFN-γ介导的抗HBV作用

摘要 目的：研究半乳糖凝集素-3结合蛋白（LGALS3BP）在IFN-?酌介导的抗HBV中的作用。方法：采用qRT-PCR及Western Blot分别检测HepG2细胞瞬转过表达质粒及干扰质粒后LGALS3BP转录水平和翻译水平的变化及检测IFN-γ对HepG2细胞内源性LGALS3BP转录水平和翻译水平的影响；采用CCK-8评价LGALS3BP表达量的变化对HepG2细胞增殖的影响及确定IFN-γ处理HepG2和HepG2.215的最适浓度；采用ELISA检测当LGALSEBP表达量发生变化时，IFN-γ刺激后对细胞外分泌的HBsAg和HBeAg的影响；采用q-PCR和RT-PCR分别检测细胞内HBV核衣壳中DNA和细胞内HBV RNA水平。结果：40 ng/mL的IFN-γ处理HepG2细胞48小时，LGALS3BP mRNA表达量与对照组相比提高2.87倍，但LGALS3BP表达量的变化对细胞增殖无显著作用；当过表达LGALS3BP时，在IFN-γ介导的抗HBV过程中，与对照组相比，HepG2.215细胞分泌的HBsAg、HBeAg以及细胞内HBV DNA和RNA的相对下降幅度分别为46%、67%、59%、49%；与对照组相比，转染pCH9-3091的HepG2细胞分泌的HBsAg、HBeAg以及细胞内HBV DNA和RNA的相对下降幅度分别为67%、53%、60%、29%；而当干扰LGALS3BP时，在IFN-γ介导的抗HBV过程中，与对照组相比，HepG2.215细胞分泌的HBsAg、HBeAg以及细胞内HBV DNA和RNA的相对上升幅度分别为46%、67%、59%、49%；与对照组相比，转染pCH9-3091的HepG2细胞分泌的HBsAg、HBeAg以及细胞内HBV DNA和RNA的相对上升幅度分别为67%、77%、67%、45%。结论：LGALS3BP作为正向调节因子参与IFN-?酌介导的抗HBV过程。     

靶向HBs基因的shRNA表达载体的构建及其抑制HepG2.2.15表达HBVsAg的作用

探索用PGenesil-1(Pg)构建的靶向乙型肝炎病毒表面抗原(HBsAg)基因的shRNA表达载体PGenesil-1-HBs(简称Pgs),对体外培养HepG2.2.15细胞中的HBV基因及其抗原表达的抑制作用.设计、合成靶向HBV S区的3对DNA序列,分别插入PGenesil-1中构建3个siRNA表达载体Pgs1、Pgs2、Pgs3,经限制性内切酶,DNA序列测定等技术鉴定确认.筛选并确定最佳细胞接种量及重组质粒转染量后,分别或按不同组合转染HepG-2.2.15细胞,48 h后采用半定量RT-PCR检测HBVsmRNA转录水平,免疫细胞化学技术检测HBsAg表达水平,MEIA分别检测细胞裂解液和培养上清中HBsAg和HBeAg的表达水平.结果表明,HBV真核表达载体Pgs1、Pgs2、Pgs3均能不同程度地抑制HepG2.2.15细胞中的HBsAg、HBeAg合成和HBs-mRNA转录.成功构建的HBV真核表达载体Pgs1、Pgs2、Pgs3,其中PgS3能显著抑制HBsAg表达(P＜0.01).多种表达载体联合对抗原表达的抑制作用效率不同.     

牡蛎多糖体外对HepG2.2.15细胞分泌HBsAg、HBeAg的影响

目的 探讨牡蛎多糖体外抗乙型肝炎病毒(HBV)的作用.方法 采用HepG2.2.15细胞为体外细胞模型,给予不同浓度牡蛎多糖进行混合培养,作用9d后收集上清液,用ELISA法测定上清液中HBsAg、HBeAg的水平,观察药物对HepG2.2.15细胞分泌病毒杭原的影响,同时以MTT法检测药物在体外对HepG2.2.15细胞生长的抑制作用.结果 牡蛎多糖浓度在0.1～1 000 μg/mL时,对细胞无明显的毒性作用;牡蛎多糖可明显抑制HBsAg、HBeAg的分泌,其半数有效浓度( IC50)分别为362.2、558.6 μg/mL,治疗指数(TI)为＞27.6和＞17.9.结论 牡蛎多糖体外具有一定的抗HBV作用.     

多聚嘧啶序列结合蛋白（PTB）与HBV转录后调节元件（HPRE）结合抑制HBV表面抗原的基因表达

采用RT-PCR验证PTB与HPRE在体外的特异性结合。用HepG2.2.15细胞系、HBs-HPRE瞬时转染Hela细胞探讨PTB对HBV基因表达的影响。结果显示PTB能与HPRE特异性地结合。功能性研究证明PTB可以抑制HepG2.2.15细胞的HBsAg表达量,并呈浓度依赖性。由HPRE引起的HBsAg表达的增加也能被PTB所抑制。实验数据证明PTB通过与HPRE相互作用抑制HBsAg的基因表达。     

反义前S/S基因转移表达抗乙型肝炎病毒的研究

为了观察反义基因转录表达抗乙型肝炎病毒(HBV)的作用,将HBV ayw前S/S基因(preS/S)片段反向插入逆转录病毒载体质粒,构建preS/S反义基因重组体,经转染PA317细胞后,获得重组逆转录病毒颗粒.用重组病毒转导2.2.15细胞后,第3天即可见细胞培养上清HBV表面抗原(HBsAg)和e抗原(HBeAg)表达量减少,到转导后第5天,细胞培养上清中的HBsAg和HBeAg表达量降到最低水平,HBsAg抑制率为71%,HBeAg抑制率为27%,抑制作用至少可持续到转导后第11天.空载及正向插入基因的重组逆转录病毒转导对2.2.15细胞内HBV抗原表达无抑制作用.     

人工microRNA抗乙型肝炎病毒效果初探

目的:设计靶向乙型肝炎病毒(HBV)基因保守区的人工microRNA(amiRNA),考察其对HBV基因表达的抑制作用。方法:比对HBV全基因组现有序列,选择保守区设计amiRNA,定向克隆到pcDNA6.2-GW/EmGFP-miR载体,将amiRNA载体与HBV复制载体pHBV1.31共转染HepG2细胞,72 h后收取细胞上清,ELISA检测HBV表面抗原(HBsAg)及e抗原(HBeAg)的含量,荧光定量PCR检测HBV DNA含量。结果:amiRNA可显著抑制细胞上清HBsAg、HBeAg和HBV DNA的水平。结论:amiRNA作为防治HBV感染的潜在有效手段之一值得进一步深入研究。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

Synthesis and antimicrobial studies of hydrazone derivatives of 4-[3-(2,4-difluorophenyl)-4-formyl-1H-pyrazol-1-yl]benzoic acid and 4-[3-(3,4-difluorophenyl)-4-formyl-1H-pyrazol-1-yl]benzoic acid

Microbial resistance to antibiotics is an unresolved global concern, which needs urgent and coordinated action. One of the guidelines of the Centers for Disease Control and Preventions (CDC) to combat antibiotic resistance is the development of new antibiotics to treat drug-resistant bacteria. In our effort to find new antibiotics, we report the synthesis and antimicrobial studies of 30 new pyrazole derivatives. These novel molecules have been synthesized by using readily available starting materials and benign reaction conditions. Some of these molecules have shown activity with MIC values as low as 0.78?µg/mL against four bacterial strains; Staphylococcus aureus, methicillin-resistant S. aureus, Bacillus subtilis, and Acinetobacter baumannii. Furthermore, active molecules are non-toxic to mammalian cell line.

     

Novel compounds with potent CDK9 inhibitory activity for the treatment of myeloma

Cyclin-dependent kinases (CDKs) and Polo-like kinases (PLKs) play key role in the regulation of the cell cycle. The aim of our study was originally the further development of our recently discovered polo-like kinase 1 (PLK1) inhibitors. A series of new 2,4-disubstituted pyrimidine derivatives were synthesized around the original hit, but their PLK1 inhibitory activity was very poor. However the novel compounds showed nanomolar CDK9 inhibitory activity and very good antiproliferative effect on multiple myeloma cell lines (RPMI-8226).