乙型肝炎病毒X蛋白上调环加氧酶2表达促进肝癌细胞增殖的实验研究

乙肝病毒X蛋白（Hepatitis B virus X protein,HBx）高表达与乙肝相关肝癌的发病密切相关,但HBx发挥促癌作用的机制并不清楚。环加氧酶2（Cyclooxygenase,COX2）具有促进肝癌细胞增殖的功能,乙肝相关肝癌中COX2的表达增加且与HBx呈正相关,提示上调COX2可能是HBx促进肝癌细胞增殖的分子机制。为了阐明HBx是否通过上调COX2促进肝癌细胞增殖,本实验培养了肝癌HepG2细胞并分为对照组、转染pcDNA3.1-HBx质粒的HBx组、转染NC siRNA的si-NC组、转染NC siRNA及pcDNA3.1-HBx质粒的si-NC+HBx组、转染COX2 siRNA及pcDNA3.1-HBx质粒的si-COX2+HBx组。检测细胞增殖活力OD_490nm的水平,COX2、B淋巴细胞瘤2基因（BCL2）、生存素（Survivin）的表达量,前列腺素E2（英文名,PGE2）的含量。实验结果显示,HBx组的OD_490nm水平,细胞中COX2、BCL2、Survivin的表达量,培养基中PGE2的含量均高于对照...     

乙肝病毒X 蛋白通过激活NF-κB 信号通路上调ABCG2 的表达

目的:研究乙肝病毒X蛋白(HBx)通过核因子-κB(NF-κB)信号通路对半转运蛋白(ABCG2)的调节作用。方法:用特异性的NF-κB信号通路阻断剂PDTC阻断NF-κB信号通路,荧光双标激光扫描共聚焦显微镜观察L02细胞系转染HBx基因前后及PDTC加入前后NF-κB信号通路的激活、失活情况,同时用Real-time PCR和Western Blot技术检测转染前后及PDTC加入前后ABCG2在mRNA及蛋白水平的表达变化。结果:以L02细胞为参照,转染HBx基因后的L02-HBx细胞NF-κB信号通路被激活,ABCG2 mRNA和蛋白水平分别增加3.62±0.15和4.61±0.73倍,差异有统计学意义(P<0.05);PDTC作用24h后L02/HBx细胞NF-κB信号通路阻断,ABCG2 mRNA和蛋白表达分别为2.15±0.32倍和2.37±0.55倍,与未加入PDTC作用的L02-HBx细胞相比均有统计学意义(P<0.05)。结论:NF-κB信号通路是HBx上调ABCG2表达的途径之一。     

乙肝病毒X蛋白通过NF-κB通路调节肝癌细胞迁移、侵袭的研究

慢性乙肝病毒感染是亚洲国家肝癌的常见病因,乙肝病毒所表达的乙肝病毒X蛋白(Hepatitis B virus X protein,HBx)是肝癌发生发展的重要推动因子。核因子-κB(Nuclear factorκB,NF-κB)是模式识别受体下游重要的转录因子,肝细胞生长因子(Hepatocyte growth factor,HGF)/c-Met途径能够促进NF-κB活化,并通过活化的NF-κB来调节肝癌细胞的迁移、侵袭等生物学环节。但HBx是否直接通过NF-κB通路来调节肝癌细胞的迁移、侵袭尚未明确。为探究HBx通过NF-κB通路调节肝癌细胞迁移、侵袭的作用,本研究采用培养肝癌HepG2细胞并分组,空白对照组用不含药物及质粒的DMEM处理,空白质粒组转染1.2μg的pcDNA3.1空白质粒,HBx质粒组转染不同浓度的HBx表达质粒pcDNA3.1-HBx,HBx+PDTC组转染1.2μg的HBx表达质粒pcDNA3.1-HBx并用含有50μmol/L PDTC的DMEM进行处理;皮下注射HepG2细胞建立移植瘤小鼠模型,称量移植瘤的质量。检测细胞迁移及侵袭活力、细胞及移植瘤中NF-κB通路分子、迁移基因、侵袭基因的表达。结果显示,与空白对照组、空白质粒组比较,HBx质粒组细胞中NF-κB、HGF、c-Met、N-cadherin、Vimentin、MMP2、MMP9的蛋白表达水平及相对愈合面积、侵袭数目均明显增多(P0.05);与HBx质粒组比较,HBx+PDTC组细胞中NF-κB、HGF、c-Met、N-cadherin、Vimentin、MMP2、MMP9的蛋白表达水平及相对愈合面积、侵袭数目均明显减少(P0.05);与空白对照组、空白质粒组比较,HBx质粒组移植瘤的质量及移植瘤中NF-κB、HGF、c-Met的蛋白表达水平明显增加(P0.05)。本研究得出结论,HBx能够促进肝癌细胞的迁移、侵袭,且该作用与激活NF-κB通路有关。     

乙肝病毒X蛋白通过激活NF-κB信号通路上调ABCG2的表达

目的：研究乙肝病毒X蛋白（HBx）通过核因子-κB（NF-κB）信号通路对半转运蛋白（ABCG2）的调节作用。方法：用特异性的NF-κB信号通路阻断剂PDTC阻断NF-κB信号通路,荧光双标激光扫描共聚焦显微镜观察L02细胞系转染HBx基因前后及PDTC加入前后NF-κB信号通路的激活、失活情况,同时用Real-time PCR和Western Blot技术检测转染前后及PDTC加入前后ABCG2在mRNA及蛋白水平的表达变化。结果：以L02细胞为参照,转染HBx基因后的L02-HBx细胞NF-κB信号通路被激活,ABCG2 mRNA和蛋白水平分别增加3.62±0.15和4.61±0.73倍,差异有统计学意义（P〈0.05）;PDTC作用24h后L02/HBx细胞NF-κB信号通路阻断,ABCG2 mRNA和蛋白表达分别为2.15±0.32倍和2.37±0.55倍,与未加入PDTC作用的L02-HBx细胞相比均有统计学意义（P〈0.05）。结论：NF-κB信号通路是HBx上调ABCG2表达的途径之一。     

MiR-199a-5p对肝星状细胞活化增殖的影响

miR-199a-5p是miRNAs家族的一员.为探讨对人肝星状细胞活化增殖和迁移的影响,为临床肝纤维化治疗提供新的思路,本研究构建miR-199a-5p过表达载体、合成miR-199a-5p反义寡聚核苷酸(anti-sense oligonucleotide,ASO)经脂质体转染LX-2细胞.采用CCK-8法和Transwell分别检测细胞增殖和迁移能力;集落形成实验检测LX-2细胞的集落形成能力;实时荧光定量PCR技术检测细胞中转染miR-199a-5p后纤维化相关基因α-SMA和Collagen Ⅰ的mRNA表达水平;Western blot检测各组细胞中α-SMA表达水平.结果 表明miR-199a-5p可以促进LX-2细胞增殖(P＜0.01)、迁移(P＜0.01)和集落形成(P＜0.01);而ASO-199a-5p-5p组细胞增殖(P＜0.01)、迁移能力(P＜0.01)和集落形成能力(P＜0.01)受到抑制.RT-qPCR结果显示miR-199a-5p在受TGF-β1刺激后的LX-2细胞中的miRNA表达水平高于未受刺激组的(P＜0.05),转染miR-199a组细胞中α-SMA的mRNA(P＜0.01)和蛋白(P＜0.05)表达水平升高.以上结论表明miR-199a-5p过表达能促进肝星状细胞活化、增殖.     

HBV X基因的表达及在真核细胞中对内质网压力的作用

运用PCR技术获得HBx基因,分别克隆到原核表达载体pET-his和真核表达载体pcDNA3.1(-)上.重组质粒pET-his-HBx转化大肠杆菌BL21(DE3)后, IPTG诱导表达,利用Ni柱纯化后的蛋白免疫家兔,获得特异性的抗-HBx兔抗血清.重组质粒pcDNA3.1(-)-HBx分别转染HepG2和Hep3B细胞系后,经RT-PCR和Western blot检测,证明HBx可以在这两种细胞系中表达.通过报告基因的表达研究了HBx对XBP1和GRP78启动子的激活活性,结果表明瞬时转染HBx的细胞系中,XBP1和GRP78启动子介导的荧光素酶活性比相应的对照细胞增加了3～7倍.通过RT-PCR分析证明,转染了HBx的细胞中XBP1 mRNA发生了剪切.因此,可以初步推断HBx在HepG2和Hep3B细胞中的表达可以引起内质网压力反应,为进一步阐明HBx表达对内质网的影响和肝脏病原发生机制奠定了基础.     

HBV X基因的表达及在真核细胞中对内质网压力的作用

运用PCR技术获得HBx基因,分别克隆到原核表达载体pET-his和真核表达载体pcDNA3.1(-)上。重组质粒pET-his-HBx转化大肠杆菌BL21(DE3)后,IPTG诱导表达,利用Ni柱纯化后的蛋白免疫家兔,获得特异性的抗-HBx兔抗血清。重组质粒pcDNA3.1(-)-HBx分别转染HepG2和Hep3B细胞系后,经RT-PCR和Westernblot检测,证明HBx可以在这两种细胞系中表达。通过报告基因的表达研究了HBx对XBP1和GRP78启动子的激活活性,结果表明瞬时转染HBx的细胞系中,XBP1和GRP78启动子介导的荧光素酶活性比相应的对照细胞增加了3～7倍。通过RT-PCR分析证明,转染了HBx的细胞中XBP1mRNA发生了剪切。因此,可以初步推断HBx在HepG2和Hep3B细胞中的表达可以引起内质网压力反应,为进一步阐明HBx表达对内质网的影响和肝脏病原发生机制奠定了基础。     

栀子苷通过TGF-β1/Smad信号通路抑制肝纤维化和肝星状细胞活化

本研究旨在观察栀子苷对肝纤维化和肝星状细胞活化的影响,并探讨其可能机制。人肝星状细胞LX-2用5 ng/mL转化生长因子-β1 (transforming growth factor-β1, TGF-β1)处理,并用不同浓度(0, 1, 2.5, 5, 10, 20, 40, 60, 80, 100μmol/L)的栀子苷联合培养,MTT法进行细胞活力测定,LX-2分别经5 ng/mL TGF-β1、TGF-β1+栀子苷(20μmol/L)处理后,用qPCR和Western blot分别检测I型胶原蛋白(collagen I)、纤连蛋白(fibronectin)、α平滑肌肌动蛋白(α-smooth muscle actin, α-SMA)、p-Smad2和p-Smad3基因和蛋白的表达。BALB/c小鼠用CCl₄ (25%, 1 mL/kg)诱导肝纤维化,设立对照组、CCl₄组和CCl₄+栀子苷组(40 mg/kg),4周后检测肝功能及血清肝纤维化指标,用HE和Masson染色进行组织学观察,免疫组织化学分析组织α-SMA...     

藏红花酸对TGF-β1刺激的人肝星状细胞LX-2信号转导通路的影响

目的:研究藏红花酸(crocetin)对转化生长因子-β1(TGF-β1)刺激的人肝星状细胞LX-2信号转导通路的影响。方法:体外培养LX-2细胞,随机设立空白对照组,模型组,藏红花酸低剂量组,藏红花酸中剂量组,藏红花酸高剂量组,用含10%血清的1640培养液培养48 h,MTT法测各组细胞增殖,蛋白免疫印迹测定各组细胞α-SMA蛋白的表达,实时荧光定量PCR法测各组细胞Smad2、Smad3、Smad7mRNA的表达。结果:与对照组比较,TGF-β1刺激后人肝星状细胞LX-2增殖作用明显,且上调α-SMA蛋白、Smad2mRNA、Smad3mRNA表达,下调Smad7mRNA表达(P0.01)与模型组比较,藏红花酸组均能抑制LX-2细胞增殖,并呈剂量依赖性,高、中剂量组作用明显,能够明显下调α-SMA蛋白、Smad2mRNA、Smad3mRNA表达,上调Smad7mRNA表达,差异具有统计学意义(P0.05)。结论:藏红花酸对TGF-β1刺激的LX-2细胞中Smad7mRNA具有上调作用,对Smad2、Smad3mRNA具有下调作用,其抗肝纤维化作用可能与抑制TGF-β1/Smads信号转导通路有关。     

肝细胞癌微环境肌成纤维细胞的分布、表型分析及其意义

目的探讨肝细胞癌(hepatocellular carcinoma,HCC)微环境中肌成纤维细胞(myofibroblast,MF)的分布、活化表型及其临床意义。方法应用免疫组织化学法检测9例正常肝组织、10例肝硬化组织、62例肝细胞癌及对应癌旁组织中各种(肌)成纤维细胞的CD34、细胞活化标志物α-平滑肌肌动蛋白α-smooth muscle actin,α-SMA),细胞骨架系统的中间丝蛋白成分包括神经胶质酸性蛋白(glial fibrillary acidic protein,GFAP)、结蛋白(desmin)、巢蛋白(nestin)和波形蛋白(vimentin)的表达,分析它们的分布、表型及其与临床病理学参数的关系。结果 HCC中至少分布有活化型肝星状细胞(activated hepatic stellate cell,a HSC)、间隔区肌成纤维细胞(septal myofibroblast septal,SMF)、肝门区肌成纤维细胞(portal myofibroblast,PMF)和间隔/实质交界区肌成纤维细胞(interface between parenchyma and septum myofibroblast,IMF)4种。与正常肝脏及癌旁组织相比,HCC组织中a HSC、PMF和IMF的α-SMA表达、a HSC和IMF的vimentin表达、IMF的nestin表达均明显升高,且均与Edmondson分级(IMF中vimentin的表达除外)及肿瘤转移正相关;而α-SMA在a HSC、IMF中及nestin在IMF中的高表达与癌组织的TNM分期正相关;此外,a HSC中α-SMA的高表达还与肝细胞癌肝硬化正相关。结论 HCC微环境中存在多种异质型肌成纤维细胞,其中a HSC、PMF和IMF的活化在一定程度上促进HCC发生发展及转移。     

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.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.