磷脂爬行酶1对干扰素抑制乙型肝炎病毒(HBV)作用的影响

研究磷脂爬行酶1(Phospholipid scramblase 1,PLSCR1)对干扰素抑制HBV作用的影响。设计合成PLSCR1特异性小干扰RNA(siRNA),以完全随机序列的阴性小干扰(NCsiRNA)作为对照,转染HepG2细胞,于转染48h后分别检测PLSCR1mRNA和蛋白水平表达量的变化,筛选出对PLSCR1具有沉默作用的siRNA;将HepG2细胞分为正常对照组和干扰素处理组,将1.3倍乙型肝炎病毒(HBV)全基因真核细胞表达载体HBV1.3质粒分别与PLSCR1siRNA或NCsiRNA共同转染HepG2细胞或干扰素处理的HepG2细胞,转染48h后检测各组细胞中PLSCR1mRNA表达量及培养液上清中HBsAg表达水平。PLSCR1特异性小干扰RNA siRNA911转染后能够显著抑制HepG2细胞中PLSCR1基因在mRNA和蛋白水平的表达;与HepG2细胞对照组比较,干扰素处理组细胞转染HBV1.3质粒、NCsiRNA+HBV1.3质粒后,细胞培养液中HBsAg表达水平均显著降低(P0.05);而PLSCR1siRNA与HBV1.3共转染IFN处理的HepG2细胞组与共转染HepG2细胞组相比较,细胞培养液中HBsAg的表达水平没有显著差异。提示抑制PLSCR1的siRNA可抑制干扰素的抗HBV活性,提示PLSCR1在干扰素抑制HBV复制中具有重要作用。     

乙型肝炎病毒对HepG2细胞侵袭的影响

目的研究HBV全长对HepG2细胞侵袭相关基因表达及活性的影响,探讨HBV在整体水平对HepG2细胞侵袭的影响。方法采用定量PCR分析HBV对HepG2细胞MMP2、9和TIMP1-4基因转录的影响;通过明胶酶谱及反相明胶酶谱检测MMP2、MMP9及TIMPs的活性;应用体外侵袭小室法检测细胞的侵袭能力。结果HBV的复制可以促进HepG2细胞MMP2、MMP9、TIMP1和TIMP3基因的转录,抑制TIMP4基因转录,增强HepG2细胞MMP2、MMP9的活性并增强细胞中TIMP1、TIMP3功能,HBV稳定复制的细胞具有更强的体外侵袭能力。结论HBV可影响HepG2细胞MMPs和TIMPs的基因转录、表达及功能,促进HepG2细胞的体外侵袭,这可能与HBV相关的HCC侵袭转移密切相关。     

MyD88抑制乙型肝炎病毒复制依赖活化NF-κB信号通路

目的 研究髓样细胞分化蛋白(MyD88)抗乙型肝炎病毒(HBV)效应的作用机制。方法 构建MyD88的截短突变体,获得核因子kappa B(NF-κB)超抑制剂IkBa-SR或者NF-κB信号通路激活剂IKKα/IKKβ的表达质粒,分别与HBV复制型质粒瞬时转染Huh7细胞,检测细胞上清液中HBeAg,HBsAg的表达以及胞质中HBV复制中间体DNA的含量,并以NF-κB依赖的荧光素酶报道系统检测它们活化NF-κB的程度。结果 MyD88全长蛋白和2个截短突变体M(1-151)、M(151-296)活化NF-κB的程度与其抑制HBV蛋白以及复制中间体DNA合成的能力相一致。与空载相比,表达NF-κB信号通路激活剂IKKα/IKKβ的质粒共同瞬转细胞后,转染MyD88和HBV表达质粒的细胞中NF-κB的通路明显活化,同时HBV core蛋白的合成显著降低;而NF-κB的超抑制剂IκBα-SR共同瞬转的细胞中core蛋白的表达量显著增加,检测细胞培养上清液中HBeAg和HBsAg及胞质中HBV复制中间体DNA的合成,得到相似结果。结论 NF-κB信号通路的活化在MyD88抑制HBV复制中发挥了关键作用     

应用微阵列技术筛选PS1TP1基因转染细胞差异表达基因

阐明乙型肝炎病毒(HBV)前-S1蛋白反式激活蛋白1(PS1TP1)的表达对于肝细胞的基因表达谱的影响.应用基因芯片技术对于pcDNA3.1(-)和pcDNA3.1(-)-PS1TP1分别转染的HepG2细胞的基因表达谱进行分析.以肝癌细胞系HepG2基因作为模板,应用聚合酶链反应(PCR)技术扩增PS1TP1基因片段,以常规的分子生物学技术构建表达载体pcDNA3.1(-)-PS1TP1.以脂质体技术转染肝母细胞瘤细胞系HepG2,提取总RNA,逆转录为cDNA,与转染空白表达载体pcDNA3.1(-)的HepG2细胞进行DNA芯片分析并比较.在4096个基因表达谱的筛选中,发现有8个基因表达水平显著上调,14个基因表达水平显著下调.PS1TP1基因的表达对于肝细胞基因表达谱有显著影响.DNA芯片技术是分析反式调节靶基因的有效技术途径.     

应用微阵列技术筛选PS1TP1基因转染细胞差异表达基因

阐明乙型肝炎病毒(HBV)前S1蛋白反式激活蛋白1(PS1TP1)的表达对于肝细胞的基因表达谱的影响。应用基因芯片技术对于pcDNA3.1()和pcDNA3.1()PS1TP1分别转染的HepG2细胞的基因表达谱进行分析。以肝癌细胞系HepG2基因作为模板,应用聚合酶链反应(PCR)技术扩增PS1TP1基因片段,以常规的分子生物学技术构建表达载体pcDNA3.1()PS1TP1。以脂质体技术转染肝母细胞瘤细胞系HepG2,提取总RNA,逆转录为cDNA,与转染空白表达载体pcDNA3.1()的HepG2细胞进行DNA芯片分析并比较。在4096个基因表达谱的筛选中,发现有8个基因表达水平显著上调,14个基因表达水平显著下调。PS1TP1基因的表达对于肝细胞基因表达谱有显著影响。DNA芯片技术是分析反式调节靶基因的有效技术途径。     

HBV PreS2 S/IFN-α融合基因真核表达载体的构建及其表达

构建含HBVPrdS2 S和IFN α融合基因的真核表达载体pcDNA3.1.S2S/IFN α并在真核细胞中进行表达。应用重叠延伸剪切技术 (splicingbyoverlappingextension ,简称SOE)经两次PCR获得嵌合基因片段S2S/IFN α ,回收后直接克隆到 pcDNA3.1V5 /HisTOPOTA克隆载体 ,得到真核重组载体pcDNA3.1.S2S/IFN α。然后用脂质体法转染VeroE6细胞。对重组载体进行了限制性酶切及PCR鉴定 ,证明连接正确 ;经间接免疫荧光检测证实该重组载体能在真核细胞中表达插入的外源性基因编码的融合蛋白。真核表达载体pcDNA3.1.S2S/IFN α的成功构建及在VeroE6细胞中的有效表达 ,为进一步探讨HBV感染的特异性免疫治疗提供了实验依据     

HBV影响XRN2基因在HepG2-H7细胞中表达的机制研究

目的利用稳定表达HBV的HepG2-H7细胞,研究HBV对XRN2基因表达的调控,并对其作用机制进行初步探讨。方法用RT—PCR和Real-time PCR的方法检测HepG2细胞及稳定表达HBV的HepG2-H7细胞中XRN2在mRNA水平的表达差异。构建XRN2启动子的萤火虫荧光素酶报告质粒,分别转染HepG2细胞及HepG2-H7细胞,检测HBV对XRN2启动子的影响。将XRN2启动子质粒与HBV4种蛋白的真核表达质粒共转染HepG2细胞,寻找对启动子影响较大的HBV蛋白。结果RT—PCR和Real-time PCR的结果显示XRN2在HepG2-H7细胞中的表达较HepG2细胞有所下降。荧光素酶活性分析显示HBV能抑制XRN2启动子的活性,且HBx和HBp蛋白在这一过程中起主要作用。结论HBV蛋白可以通过抑制XRN2启动子活性调节其在HepG2-H7细胞中的表达。     

利用基因捕获技术建立稳定表达HBV的细胞模型

pGEM-HBV1.3质粒经HindIII限制性内切酶消化,将HBV1.3全长DNA切下,与同样经HindIII限制性内切酶降解过的PU21连接,得到PU21-HBV重组质粒。将该重组质粒采用电击转染方法导入HepG2细胞中,G418筛选阳性克隆并以X-gal染色,RT-PCR、Southern blot等方法验证HBV DNA的插入和表达。 PU21-HBV重组质粒经测序证明HBV1.3全长DNA正确与PU21载体连接,该重组质粒转染HepG2细胞后经G418筛选,得到一系列阳性克隆, Southern blot证实HepG2细胞基因组中含HBV DNA,RT-PCR结果表明HBV DNA在HepG2细胞中有功能基因的转录。HBV1.3已被整合在HepG2细胞染色体中并能稳定表达其RNA。稳定的HBV表达细胞模型构建成功。HBV表达细胞模型的建立,为进一步研究相关基因对HBV的转录、复制、转录后调节以及HBV各种蛋白的表达机理研究提供实验材料。     

干扰素γ增强GH3细胞中人生长激素基因表达机制

为了研究干扰素γ(IFN γ)对大鼠垂体GH3细胞中人生长激素 (hGH)基因启动子活性的影响及其可能的作用机制 ,采用荧光素酶报告基因方法 ,将含hGH基因启动子 (- 4 84～ 2bp)和荧光素酶报告基因的表达质粒pGL3 4 84 Luc单独转染或与垂体特异性核转录因子Pit 1蛋白表达质粒 (pcDNA Pit 1 cDNA)或Pit 1反义寡核苷酸 (Pit 1OND)共转染于大鼠垂体GH3细胞中 ,观察加入IFN γ及细胞内信号转导途径的抑制剂后GH3细胞中荧光素酶表达的变化 ,反映其对hGH启动子活性的影响 ;将含不同长度hGH基因启动子序列的荧光素酶表达质粒pGL3 3 80 Luc(- 3 80～ 2bp)、pGL3 2 5 0 Luc(- 2 5 0～ 2bp)、pGL3 1 3 2 Luc(- 1 3 2～ 2bp)和 pGL3 6 6 Luc(- 6 6～2bp)分别转染GH3细胞 ,观察它们对IFN γ的反应 ,以寻找IFN γ影响hGH基因启动子活性的关键序列。结果表明 ,IFN γ (1 0 5u/L ,1 0 6u/L)均能促进大鼠垂体GH3细胞中荧光素酶的表达 ,最高达对照组的 1 3 1 % (P <0 .0 0 1 ) ;在胞内信号转导抑制剂中 ,只有丝裂原活化蛋白激酶 (MAPK)信号转导途径抑制剂PD980 5 9(4 0 μmol/L) ,能完全阻断IFN γ的促进作用 ;Pit 1蛋白过表达和表达被抑制对IFN γ的促进作用没有影响 ;含不同长度hGH基因启动子序列质粒中 ,只有 pGL3 3 8     

病毒感染相关基因微阵列的制备及其在HBV感染应答基因筛选中的应用

为筛选乙型肝炎(乙肝)病毒(HBV)感染应答基因,探讨HBV感染分子机理,采用生物信息学分析、筛选宿主细胞中与乙肝病毒、丙型肝炎(丙肝)病毒、流行性感冒(流感)病毒等感染密切相关的基因,设计并合成寡核苷酸探针,制备了含231种病毒感染相关基因的寡核苷酸微阵列.利用此微阵列比较HepG2细胞、HepG2.2.15细胞之间的基因表达谱差异,筛选乙肝病毒感染候选应答基因,从分子水平对乙肝病毒感染作用机理进行初步研究.制备的病毒感染相关基因表达谱微阵列的监测结果显示,阳性对照和看家基因探针出现较强信号,空白点样液和阴性对照探针未出信号,大部分基因探针信号强度在可分析范围内,上矩阵和下矩阵反映的基因表达情况一致,证明微阵列的特异性、敏感性、重复性都较好.HepG2.2.15与HepG2细胞基因表达谱比较结果显示,28个宿主基因在HepG2.2.15细胞中高表达,包括ASGR1、AFP、Fibronectin、APOC等基因;4个基因低表达,包括RRM1、ICSBP等基因.初步筛选获得HBV感染候选应答基因.此结果表明,制备的微阵列敏感性、特异性、重复性好,可为研究病毒宿主相互作用关系提供技术平台,应用此微阵列筛选获得的HBV候选应答基因可为揭示HBV感染的分子致病机理提供新的信息,为抗HBV药物研究提供潜在的作用靶点.     

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.     

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     

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.     

鸡传染性法氏囊病病毒研究进展

传染性法氏囊病(Infection bursal disease, IBD)是由鸡传染性法氏囊病毒(Infectious bursal disease virus, IBDV)引起的鸡和火鸡的一种高度接触性传染病,给世界各国的禽养殖业带来了巨大损失.自IBDV发现至今新的变异株不断出现,分子结构的改变导致病毒致病力的改变及宿主对疫苗应答的改变,使得传统的疫苗已不能控制其流行,因此各国学者对其基因组结构和功能进行了广泛深入的研究,并积极研制新型有效的疫苗以达到防治的目的.     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).