URI基因稳定表达对肝癌SMMC-7721细胞增殖能力的影响

目的:瞬转以及筛选出能够稳定表达URI(RPB5-mediating protein)基因的SMMC-7721细胞株,以其为模型研究URI基因对人肝癌SMMC-7721细胞增殖的影响.方法:首先,抽提URI的重组质粒并转染到SMMC-7721细胞中,在G418药物的筛选下选出能够稳定表达URI基因的细胞株,RT-PCR法和酶切检测该稳定细胞中URI基因的表达效率以确认URI是否稳定表达,MTT法和克隆形成实验检测URI基因对SMMC-7721细胞增殖的影响.结果:成功建立URI基因过表达的稳定细胞株.与SMMC-7721细胞对照组比较,其URI mRNA表达水平显著上调,能稳定表达URI细胞株的增殖,克隆形成率明显升高.结论:pFLAG-CMV-4-URI重组质粒能使URI在肝癌SMMC-7721细胞内稳定表达,过表达URI基因有可能帮助细胞通过G2/M期检验点来提高肝癌细胞SMMC-7721的增殖能力.     

乙型肝炎病毒X基因真核表达载体的构建及表达

目的:构建含有天然完整的乙型肝炎病毒(HBV)X基因序列的真核表达载体,观察其在肝癌细胞株中的表达。方法:设计并合成HBV X基因的引物,用PCR方法从含完整HBV全基因的HepG2细胞中扩增X基因序列,并将其连接到真核表达载体pVAX-1上,酶切、PCR鉴定;用Triton X-114去除质粒内毒素后,采用电穿孔法将重组质粒pVAX-HBV X和空质粒pVAX-1分别转染SMMC-7721细胞,RT-PCR法检测HBV X基因mRNA的表达,Western印迹鉴定HBV X蛋白(HBx)的表达。结果:酶切和PCR鉴定证实pVAX-HBV X载体中包含完整的HBVX基因片段,该重组质粒转染的SMMC-7721细胞中HBV X基因mRNA及HBx蛋白的表达稳定。结论:构建了HBV X基因的真核表达载体,为X基因及其编码蛋白的生物学功能的研究提供了可靠的基因材料。     

怀化医学高等专科学校医学形态学实验中心

目的:探讨抑癌基因p16对肝癌细胞生长的抑制作用及其机制。方法:将p16 cDNA亚克隆至pcDNA3.1真核表达载体上,并经脂质体介导转染至人肝癌细胞株SMMC-7721。用MTT法和Western blot分析转染细胞的生长情况。结果:成功构建重组表达质粒pcDNA3.1-p16,转染pcDNA3.1-p16的SMMC-7721细胞生长速度受到明显抑制;转染后有外源p16蛋白的表达,且伴随Bax上调,Bcl-2和cIAP2的下调。结论:重组pcDNA3.1-p16质粒能在人肝癌细胞SMMC-7721内表达,且能抑制SMMC-7721的生长,其机理与诱导肿瘤细胞凋亡相关。     

抑癌基因p16对人肝癌细胞生长抑制的机制研究

目的：探讨抑癌基因p16对肝癌细胞生长的抑制作用及其机制。方法：将p16cDNA亚克隆至pcDNA3．1真核表达载体上,并经脂质体介导转染至人肝癌细胞株SMMC-7721：用MTT法和Western blot分析转染细胞的生长情况。结果：成功构建重组表达质粒pcDNA3．1-p16,转染pcDNA3．1-p16的SMMC-7721细胞生长速度受到明显抑制;转染后有外源p16蛋白的表达,且伴随Bax上调,Bcl-2和cIAP2的下调。结论：重组pcDNA3．1-p16质粒能在人肝癌细胞SMMC-7721内表达,且能抑制SMMC-7721的生长,其机理与诱导肿瘤细胞凋亡相关。     

RNAi及DNA芯片分析肝癌细胞系中受DNMT3B调控的下游基因

为揭示DNA甲基转移酶3B（DNMT3B）在肝癌中是否参与了肿瘤的发生,应用Western blotting及细胞免疫化学方法分析DNMT3B蛋白在人的正常肝细胞株、肝癌癌旁细胞株及肝癌癌细胞株中的表达。构建了DNMT3B的RNAi稳定表达的重组载体,并转染人肝癌细胞株SMMC-7721中。以半定量RT-PCR及Western blotting分别鉴定DNM73B RNAi表达载体对内源性DNMT3B的抑制效率。用高通量的cDNA基因芯片分析了SMMC-7721中DNMT3B抑制后有影响的下游基因谱。结果显示,DNMT3B在肝癌细胞株中的表达水平明显高于肝癌癌旁和正常肝细胞株。DNMT3B的RNAi稳定表达重组载体转染SMMC-7721细胞株2个月后,观察到DNMT3B明显受到抑制。cDNA基因芯片分析发现,DNMT3B抑制后诱导26条基因表达下调,115条基因表达上调,包括一些发育相关基因以及肿瘤相关基因,如SNCG、NOTCH1、MBD3、WNT11、MAOA、FACL4等。提示DNMT3B的高表达可能与肝癌的发生有关,并以调控其他相关基因的表达而起作用,包括与发育相关的重要基因。     

FOXQ1在肝癌中的临床意义及其对SMMC-7721细胞体外血管形成的影响

探讨叉头框蛋白Q1(forkhead box Q1, FOXQ1)基因在肝癌中的临床意义及对肝癌细胞体外血管生成作用.利用 qRT-PCR法及Western印迹法,检测24例肝癌、癌旁组织、正常肝细胞L02及肝癌细胞SMMC-7721中FOXQ1的mRNA和蛋白质的表达;利用免疫组织化学法检测68例肝癌及癌旁组织中FOXQ1的蛋白质表达.合成shRNA-FOXQ1及shRNA-NC慢病毒,转染到SMMC-7721细胞.用体外血管生成实验检测转染shRNA-FOXQ1的肝癌细胞血管生成能力. 用qRT-PCR和Western印迹法检测细胞间FOXQ1、VEGF基因和蛋白质的表达.结果显示,癌组织和SMMC-7721细胞中FOXQ1 mRNA和蛋白质的表达均高于癌旁组织和正常肝细胞(P<0.05),FOXQ1蛋白的表达与TNM分期、肿瘤分化程度、肿瘤数目、肿瘤大小等参数差异显著(P<0.05).shRNA-FOXQ1组血管生成能力明显低于shRNA-NC组和空白组(P<0.05),FOXQ1、VEGF基因和蛋白质的表达也明显低于shRNA-NC组和空白组(P<0.05).研究结果证实,FOXQ1在肝癌中高表达,如果沉默FOXQ1的表达可抑制肝癌细胞血管生成,与肝癌的临床病理特征密切相关.     

RNA干扰抑制nm23-M1基因表达对骨髓瘤SP2/0细胞增殖的影响

建立RNA干扰 (RNA interference, RNAi)抑制nm23-M1基因表达的骨髓瘤SP2/0细胞株, 初步探讨nm23-M1基因对小鼠骨髓瘤细胞增殖的影响.针对 nm23-M1 mRNA序列设计3个小干扰RNA (small interfering RNA, siRNA)序列, 分别构建表达这3个序列及阴性对照序列的pGenesil-1重组质粒, 再转染小鼠骨髓瘤SP2/0细胞并经G418抗性筛选稳定表达细胞株.采用半定量RT-PCR及Western 印迹检测3个重组质粒对nm23-M1 mRNA及蛋白质表达的抑制效果, 然后用MTT法观察抑制nm23-M1表达对SP2/0细胞增殖的影响.结果显示, 设计的3条siRNA不同程度地特异抑制骨髓瘤SP2/0细胞nm23-M1 mRNA及蛋白质的表达, 其中siRNA-2抑制作用最强, 其对nm23-M1 mRNA和蛋白质的抑制率分别为74.4%和62.1%; 且siRNA-2抑制nm23-M1基因表达后SP2/0 细胞增殖受到明显抑制 (P < 0.05).本研究成功构建了pGenesil-1-nm23-M1 siRNA重组质粒, 筛选出稳定抑制nm23-M1基因表达的SP2/0细胞株, 提示抑制nm23-M1基因表达有抑制骨髓瘤细胞增殖的作用; 为进一步研究nm23基因的生物学功能及临床应用打下了基础.     

在表达HCV Core蛋白的肝癌细胞中建立Notch1低表达稳定转染细胞株

目的筛选并包装Notch1基因shRNA低表达的慢病毒,感染稳定表达丙型肝炎病毒(hepatitis C virus,HCV)核心蛋白(core)的肝癌细胞,在此基础上建立低表达Notch1基因的SMMC7721-Core稳定转染细胞株。方法设计针对Notch1基因mRNA的干扰序列,并将其连接入载体质粒,转染HEK293T细胞,构建带有Notch1基因的慢病毒表达载体,感染稳定表达HCV Core蛋白的人肝癌细胞株SMMC7721-Core,采用定量聚合酶链反应(quantitative PCR,qPCR)和免疫印迹法(Western blot)检测干扰效果。结果构建了带有干扰Notch1基因的慢病毒表达载体,感染SMMC7721-Core细胞后筛选获得稳转细胞株,qPCR测得Notch1 mRNA低表达,Western blot检测到Notch1蛋白低表达。结论成功构建了Notch1低表达SMMC7721-Core人肝癌稳定转染细胞株,为进一步开展对HCV Core蛋白诱导肝癌过程中Notch1所起作用的研究提供了依据。     

人线粒体转录终止因子3基因RNA干扰表达载体的构建筛选与干扰效果评价

目的:构建人线粒体转录终止因子3(MTERF3)基因的短发夹RNA(shRNA)干扰表达载体,并在mRNA和蛋白质水平对其干扰效率进行验证,以检测和筛选出干扰效率最优的shRNA表达载体。方法:根据人MTERF3基因全长cDNA序列,利用Oligoengine在线软件设计4个shRNA序列,合成4条互补寡核苷酸链,退火成双链后克隆至psiU6.1载体,得到4个重组干扰质粒psi-MTERF3-1～psi-MTERF3-4,并通过PCR和DNA测序对其进行鉴定;将重组干扰质粒利用脂质体介导瞬时转染He La细胞,转染48 h后采用real time RT-PCR检测4个干扰质粒对MTERF3 mRNA表达水平的影响,采用Western印迹检测其对MTERF3蛋白表达水平的情况,并筛选出最有效的shRNA干扰质粒。结果:PCR鉴定和DNA测序鉴定证实重组质粒中已插入目的DNA序列;real time RT-PCR和Western印迹结果表明4个重组载体均可以显著降低人MTERF3基因mRNA和蛋白质的表达水平(P0.05),其中以pSi-MTERF3-4的干扰效率最优,对MTERF3 mRNA表达的抑制率为70.1%,对MTERF3蛋白表达的抑制率为72.2%。结论:在人宫颈癌He La细胞系筛选出有效干扰MTERF3基因表达的shRNA序列,构建了针对MTERF3基因的RNA干扰真核表达载体且能够有效抑制目的基因的表达,为进一步研究人MTERF3在宫颈癌发生发展中的作用机制提供了实验基础。     

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.     

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     

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.