MiR-147a通过抑制细胞自噬促进HIF-1α蛋白的积累

目的:HIF-1α是由低氧诱导表达的一个重要的调节肿瘤生长、代谢的转录因子,它的降解除了通过泛素-蛋白酶体途径降解之外还与可以通过细胞自噬途径降解。通过研究miR-147a对细胞自噬的影响从而进一步研究miR-147a对HIF-1α降解的影响。方法:在HeLa细胞中过表达miR-147a,用Western blot和Q-PCR检测细胞自噬相关的标志物LC3B、P62、LAMP-2A的变化。再通过溶酶体-自噬泡共定位实验共聚焦显微镜观察自噬泡的数量以及共定位情况。最后通过加入自噬诱导剂(EBSS)和自噬抑制剂(Bafilomycin A1),用Western blot检测转染NC与miR-147a后HIF-1α蛋白的表达情况。结果:过表达miR-147a后自噬相关的标志物LC3B、P62表达量上升,LAMP-2A表达量下降,且溶酶体与自噬泡的共定位增多;加入自噬诱导剂和自噬抑制剂后HIF-1α蛋白的表达量增加。结果表明miR-147a可以抑制细胞自噬的巨自噬途径以及分子伴侣介导的自噬途径,积累HIF-1α蛋白。结论:miR-147a通过抑制细胞自噬从而减少HIF-1α蛋白的降解,但是miR-147a作用靶点的分子机制需要进一步研究。     

MicroRNA-18a通过HIF-1-alpha调控缺氧引起的人肺动脉平滑肌细胞增殖

目的：研究microRNA-18a (miR-18a) 对缺氧引起的人肺动脉平滑肌细胞(human pulmonary artery smooth muscle cells, hPASMCs)增殖的调控作用及其可能机制。方法：体外培养hPASMCs,分为未转染组、miR-18a 模拟物对照组、miR-18a 模拟物组、 miR-18a 抑制剂对照组、miR-18a 抑制剂组、siRNAcontrol组、siHIF-1-alpha组、miR-18a 抑制剂和siHIF-1-alpha共转染组。分别于常氧(21% O2)和低氧(3%O2)作用24小时。采用CCK-8 法检测细胞的增殖情况,萤光素酶报告基因系统验证缺氧诱导因子-1-alpha(HIF-1alpha)是 否为miR-18a 的靶基因,并通过western-blot 以及实时荧光定量PCR 技术检测相关蛋白和基因的表达。结果：缺氧可促进 hPASMCs 增殖,使miR-18a 表达减少;miR-18a 模拟物可抑制hPASMCs 增殖,而miR-18a 抑制剂可促进hPASMCs 增殖;抑制 miR-18a 可使HIF-1-alpha的表达上调。同时抑制miR-18a 和HIF-1-alpha,可使miR-18a 对hPASMCs 增殖调控的能力消失。结论：缺氧通 过抑制miR-18a,上调HIF-1-alpha的表达,促进hPASMCs增殖。     

HIF-1α/BNIP3信号通路对BCG诱导巨噬细胞自噬的影响

【背景】缺氧诱导因子1α(hypoxia-inducible factor 1-alpha,HIF-1α)是响应细胞低氧反应的关键因子,在红细胞生成、血管形成、能量代谢及调节宿主免疫代谢中发挥着重要作用。【目的】探讨HIF-1α/Bcl-2-腺病毒E1B相互作用蛋白3(Bcl-2-adenovirus E1B 19-kDa interacting protein 3,BNIP3)信号通路对牛分枝杆菌卡介苗(Bacillus Calmette-Guérin,BCG)诱导小鼠巨噬细胞RAW 264.7自噬的影响。【方法】构建HIF-1α的小干扰RNA (siHIF-1α),转染RAW 264.7细胞后,结合BCG感染,采用流式细胞仪检测细胞自噬率,用Western blotting或免疫荧光技术检测HIF-1α、BNIP3、LC3、Beclin 1、Rheb和mTOR的表达水平。【结果】BCG感染显著上调巨噬细胞中LC3和HIF-1α的表达,用siHIF-1α结合BCG感染后显著下调巨噬细胞中HIF-1α、BNIP3、LC3、Beclin 1和细胞自噬率水平,并促进Rheb和p-mTOR的表达。【结论】在BCG感染RAW 264.7细胞过程中,干扰HIF-1α表达抑制了HIF-1α/BNIP3信号通路,进而激活了mTOR途径,抑制BCG感染诱导的细胞自噬。     

肠道病毒A71型亚单位蛋白对RD细胞自噬活化的影响

肠道病毒A71型（Enterovirus-A71, EV-A71）能够活化宿主细胞的自噬并依赖自噬促进其复制,然而EV-A71的亚单位蛋白对自噬的活化目前仍不清楚。为探讨EV-A71亚单位蛋白对人横纹肌肉瘤（Human rhabdomyosarcoma, RD）细胞自噬活化的影响,将EV-A71的亚单位蛋白重组真核质粒转染至RD细胞,采用抑制剂MK-2206阻断PI3K/Akt途径,共聚焦显微镜和免疫印迹检测自噬活化。过表达EV-A71亚单位蛋白的RD细胞中PI3K/Akt途径、p38、JNK和ERK途径均呈现不同程度活化,同时RD细胞呈现出绿色荧光表明自噬发生活化,特别是EV-A71的VP2和2A。EV-A71亚单位蛋白使LC3-II/LC3-I的转化水平提升,EV-A71亚单位蛋白（VP2、VP3、VP4、2A、2B和2C）显著提升p62的表达水平,EV-A71 VP1显著下调p62的表达水平但显著上调LAMP-1和LAMP-2的表达水平。阻断PI3K/Akt途径后,过表达EV-A71亚单位蛋白的RD细胞绿色荧光强度显著减弱、自噬被阻断,同时LC3-II/LC3-I的转化水平显著降...     

COPII囊泡衣被蛋白SEC24A在巨自噬通路中的功能研究

细胞自噬是一种重要且保守的细胞内降解过程,通过形成双层膜的自噬体包裹细胞内容物进行降解。内质网来源的COPII囊泡被认为是饥饿诱导的应激过程中自噬体的膜源。探究了COPII囊泡衣被蛋白SEC24A在巨自噬通路中的作用。利用siRNA干扰技术敲低SEC24A的表达,EBSS饥饿处理对照组和SEC24A敲低组HeLa细胞2 h诱导自噬发生,经Western blot和免疫荧光实验检测自噬底物蛋白p62和自噬标志蛋白LC3-II的蛋白水平变化,以确定SEC24A是否参与自噬。通过RFP-GFP-LC3串联荧光检测自噬体和自噬溶酶体的数目,利用蛋白酶K保护实验验证自噬缺陷发生在自噬体闭合之前或者之后,利用免疫荧光实验检测敲低SEC24A对自噬通路上ATG复合物的影响,以确定SEC24A调控自噬通路的位点。通过免疫共沉淀实验验证SEC24A与自噬相关蛋白ATG9A是否存在相互作用。蛋白检测实验发现,饥饿条件下与对照细胞相比,敲低SEC24A细胞内自噬底物蛋白p62积累,而标志蛋白LC3-II减少。RFP-GFP-LC3串联荧光实验显示,敲低SEC24A后自噬体及自噬溶酶体的数目均减少。蛋白酶K保护实验显示,SEC24A敲低细胞中受膜结构保护的p62和GFP-LC3均减少,提示SEC24A作用位点在自噬体闭合之前。免疫荧光实验显示,敲低SEC24A的表达后ATG14L、ATG16L1点状结构减少,而ATG9A点状结构的数量没有明显变化,提示SEC24A作用于ATG14L、ATG16L1上游。免疫共沉淀实验显示SEC24A与ATG9A存在相互作用。研究结果不仅有助于深化对自噬体形成过程和分子机制的了解,也为全面解读COPII囊泡及其衣被蛋白在自噬中的重要作用提供了信息。     

缺氧通过SIRT1调控结直肠癌细胞自噬

目的:探究缺氧调控结直肠癌细胞自噬的分子机制。方法:分别在常氧及缺氧(1%氧气浓度)条件下处理细胞,western blot检测细胞内沉默信息调节因子1(Silencing Information Regulator 1,SIRT1)及自噬相关标志分子的表达情况;慢病毒转染构建SIRT1稳定过表达或敲减细胞株,利用透射电镜观察细胞内自噬体形成的情况;使用m RFP-GFP-LC3双标腺病毒感染细胞,在激光扫描共聚焦显微镜下观察细胞自噬流的进展。结果:Western blot结果显示,缺氧条件下,HCT116及SW480细胞内SIRT1的表达水平随着缺氧时间的延长而降低,自噬特异性底物p62蛋白水平降低且LC3-I/II转换增加;与对照组相比,SIRT1过表达细胞内自噬特异性底物p62的表达水平升高而LC3-I/II转换受到抑制;相反在SIRT1敲减细胞内,p62的表达水平降低而LC3-I/II转换进一步促进。透射电镜结果发现SIRT1过表达后,细胞内自噬溶酶体形成减少、自噬体数量增多;激光共聚焦结果显示,SIRT1过表达细胞内绿色荧光淬灭减少、自噬体与自噬溶酶体的融合收到明显抑制,说明SIRT1通过抑制自噬溶酶体的形成,阻断自噬流的进展。结论:缺氧通过抑制SIRT1的表达促进结直肠癌的细胞自噬。     

脂多糖通过mTOR途径抑制肝细胞脂质自噬

本研究旨在探讨脂多糖(lipopolysaccharide, LPS)对肝细胞脂质自噬的影响及其机制。体外培养人肝癌细胞株HepG2,用0.1 mmol/L软脂酸(palmitic acid, PA)负荷,分为对照(0μg/mL LPS)组、LPS (10μg/mL)组、LPS+DMSO组、LPS+雷帕霉素(rapamycin, RAPA, 10μmol/L)组。油红O染色观察HepG2细胞内脂质积聚情况;自噬双标腺病毒mRFP-GFP-LC3转染细胞后激光共聚焦显微镜观察细胞自噬流;通过氟硼二吡咯BODIPY 493/503荧光染料和溶酶体标记物溶酶体关联膜蛋白1 (lysosomal associated membrane protein 1, LAMP1)进行脂滴和溶酶体的共定位,反映细胞内脂质自噬水平;Western blot检测哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)、p-mTOR、核糖体S6激酶1 (ribosome protein subunit 6 kinase 1,S6K1)、p-S6K1、LC3II/I、P62蛋白表达。结果显示,与对照组相比,LPS组细胞油红O染色红染脂滴增加,自噬体增加,自噬溶酶体明显降低,LAMP1/BODIPY共定位率降低(P 0.05),p-mTOR/mTOR、p-S6K1/S6K1和LC3II/LC3I比值升高,P62蛋白表达增加(P 0.05)。加入RAPA干预后,与LPS+DMSO组相比,自噬体减少,自噬溶酶体明显增加,LAMP1/BODIPY共定位率升高(P 0.05),肝细胞油红O染色红染脂滴减少(P 0.001)。综上,LPS通过激活mTOR通路抑制HepG2细胞脂质自噬,从而加重细胞内脂质积聚。     

293T细胞中SQSTM1/p62-GFP的表达及其与LC3在诱导自噬状态下的定位分布

为探究SQSTM1/p62蛋白在细胞发生自噬时的定位,以人肺腺癌A549细胞的cDNA为模板,PCR扩增SQSTM1基因(编码p62蛋白)并将其插入pEGFP-N1真核表达质粒.将重组质粒转染进入人胚肾293T细胞中表达p62绿色荧光融合蛋白(GFP-p62),利用Earle's盐平衡溶液饥饿诱导细胞自噬,Western blotting和激光共聚焦显微镜检测GFP-p62的表达及其与自噬相关蛋白LC3在自噬细胞中的定位.结果发现,重组载体pEGFP-N1-p62转染293T细胞后,293T细胞能高效表达GFP-p62融合蛋白,内源性p62所占细胞内总p62比例很低.饥饿诱导后细胞自噬体形成,LC3-Ⅱ/LC3-Ⅰ的比例明显增高,p62蛋白表达下调.同时还发现,GFP-p62在自噬细胞中仅定位于大多数自噬体且与LC3共定位,小部分自噬体及自噬体以外区域没有明显GFP-p62分布.本研究建立了一种通过p62-GFP与LC3的共定位检测人胚肾293T细胞自噬时p62蛋白和LC3聚集体的有效方法,.提示了使用内源性p62低表达细胞作为研究p62和细胞自噬的优势,也将有助于开展关于p62在细胞中定位与其生物学功能关系的研究工作.     

维生素D对烟曲霉感染的肺巨噬细胞 自噬机制的初步研究

目的探究维生素D在鼠肺细胞感染烟曲霉后对细胞自噬的影响。方法用一定量的烟曲霉活化孢子感染细胞后,一组细胞加入维生素D(维生素D组),一组细胞加入生理盐水,感染一定时间后用溶酶体探针检测自噬相关分子的表达;收集各组细胞并裂解细胞,离心取上清,用Western-blot法检测上清液中的LC3BII、Dectin-1及ROS的表达水平。结果活化的孢子感染肺巨噬细胞后,维生素D组自噬体与溶酶体共定位减少、吞噬孢子的速率在减少以及ROS水平降低,其对应的胞内Dectin-1、ROS、LC3BII减少且差异有统计学意义。结论烟曲霉感染肺巨噬细胞后,维生素D可通过减弱细胞自噬体与溶酶体的融合并下调自噬信号通路蛋白的表达以达到抵抗烟曲霉感染的作用。     

阿司咪唑对宫颈癌细胞HeLa自噬的影响

该研究利用蛋白质免疫印迹法(Western blot)、稳定表达m Cherry-EGFP-LC3B(microtubuleassociated proteins 1A/1B light chain 3B,LC3)自噬双标荧光细胞、mt-Keima荧光探针活细胞扫描技术在人宫颈癌细胞He La中检测了多种自噬相关标志物,探究了阿司咪唑(astemizole,AST)对He La细胞自噬的影响。结果表明,阿司咪唑在He La细胞中引起自噬标志蛋白质LC3-II与SQSTM1/p62的显著累积,并存在剂量和时间依赖性效应;阿司咪唑导致He La细胞存活率显著降低;用自噬抑制剂Bafilomycin A1(Baf-A1)阻断自噬流(autophagic flux)后,再加入阿司咪唑不能促进LC3-II进一步累积;阿司咪唑处理细胞后自噬体与自噬溶酶体的数量均显著增加;此外,阿司咪唑显著抑制了线粒体氧化磷酸化解偶联剂羰基氰化物间氯苯腙(carbonyl cyanide 3-chlorophenylhydrazone,CCCP)导致的线粒体标志蛋白质TOMM20(translocase of outer mitochondrial membrane 20)的降解,其线粒体自噬水平显著低于对照组。以上结果证明了阿司咪唑有抑制自噬流的作用,导致损伤的线粒体不能被顺利降解,并且提示该作用可能是通过抑制溶酶体功能实现的。     

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).