流感病毒感染诱导MDCK细胞调亡的研究

用荧光染色、ＤＮＡ凝胶电泳等方法检测了Ａ型流感病毒株Ａ１／京防８６－１和Ｂ型流感病毒株Ｂ／沪防９３－１诱导狗肾传代细胞（ＭＤＣＫｃｅｌｌｓ）的凋亡情况,并采用ＭＴＴ法和流式细胞仪比较了这２株病毒对ＭＤＣＫ细胞毒力和调亡诱导能力水平。结果显示：病毒感染６ｈ后,细胞ＤＮＡ发生断裂,病毒感染１２ｈ后,可见明显的染色质凝聚;在一定范围内,细胞调亡强度表现出明显的时间和剂量依赖关系;并且,Ａ型流感病毒株的毒     

流感病毒与细胞凋亡

流感病毒与细胞凋亡的相关研究近年来一直是流感研究的热点,流感病毒可使MDCK细胞,呼吸道细支气管细胞,淋巴细胞以及绒毛膜细胞和胎膜细胞发生凋亡,弄清楚流感病毒与细胞凋亡相互间的关系无疑对流感病毒的致病机制及抗感染免疫的研究有着积极的促进作用,本文仅对近几年来,流感病毒与MDCK细胞以及其它相关细胞的凋亡情况或关系的研究作一综述。     

流感病毒感染诱导细胞凋亡的研究进展

10年前,人们在流感病毒的研究中发现,这种病毒在体内和体外均可引起细胞凋亡.对流感病毒诱导细胞凋亡的研究不但有利于深入了解流感病毒的致病机制,而且为运用生物学方法治疗恶性肿瘤提供新的线索.本文对流感病毒感染与细胞凋亡关系的研究进展进行了综述.     

流感病毒诱导宿主细胞凋亡的研究进展

本文综述了流感病毒诱导细胞凋亡的研究进展,重点阐述在流感病毒诱导细胞凋亡过程中可能存在的信号转导途径,并探讨了ｂｃｌ－２基因对流感病毒诱导细胞凋亡的影响。     

黑色素抑制流感病毒诱导宿主细胞凋亡

The apoptosis induced by influenza virus in cultured MDCK cells was reported and the selective inhibitory effect of melamin on the apoptosis induced by influenza virus was investigated. The results showed that the DNA ladder could be first detected at 6 h post-infection (p.i.), accompanied by nuclear condensation and nuclear fragmentation could be easily detected at 12 h p.i. In addition, the apoptosis-induced activity of influenza virus A1/Jingfang 86-1 strain was more potent than that of B/Hufang 93-1 strain (P＜0.05). In the range of 20-125 μg/mL, melanin was found to significantly (P＜0.001) inhibit apoptosis induced by 64 hemagglutination unit influenza virus infection with a inhibitory rate comparable to that obtained by virazole and showed no cytotoxicity. The inital results suggested that the mechanism of melanin against the apoptosis induced by influenza virus was related to the blockage of viruses' adsorbtion to the host cells.     

黑色素抑制流感病毒诱导宿主细胞凋亡

报道了流感病毒体外诱导狗肾细胞系（ＭＤＣＫ）细胞凋亡的检测结果,对黑色素选择性抑制流感病毒诱导细胞凋亡的可能性进行了探讨,同时与临床上常用的抗病毒药物病毒唑的效果进行比较。结果显示：病毒感染６ｈ后,即可观测到宿主细胞核固缩现象、ＤＮＡ凝胶电泳出现特征性的梯状图谱,感染１２ｈ后,细胞核可见明显的裂解;并且流感病毒株Ａ１／京防８６１诱导细胞凋亡能力强于Ｂ沪防／９３－１;在２０～１２５μｇ／ｍＬ浓度范围内,黑色素可有效抑制６４个血凝单位（ＨＵ）的流感病毒感染诱导的细胞凋亡而无细胞毒性作用,其抑制效率类似病毒唑。初步研究结果表明：黑色素抗流感病毒诱导细胞凋亡机理与其阻断病毒吸附侵入宿主细胞有关     

MDCK细胞和MDCK-G1细胞感染H1N1流感病毒后病毒滴度和细胞转录组表达差异分析

目的通过分析细胞表达基因及其相关信号通路的差异等信息,探索MDCK和MDCK-G1细胞株在接种H1N1流感病毒后出现病毒滴度和细胞生长趋势差异的内在生物学特性等原因。方法通过Illumina测序平台对2株细胞进行转录组测序(RNA sequencing, RNA-seq)和测序结果生物信息学分析后,选择了17个差异基因进行实时定量聚合酶链反应(quantitative real-time polymerase chain reaction, qRT-PCR)验证。结果 MDCK和MDCK-G1细胞差异基因(differentially expressed genes, DEGs)|log_2(FoldChange)|0和padj≤0.05总数为2 786个。相比于MDCK细胞,MDCK-G1细胞有967个基因的表达显著上升,1 819个基因的表达显著下降。差异基因通过基因本体(Gene Ontology, GO)数据库富集功能注释和京都基因和基因组数据库(Kyoto Encyclopedia of Genes and Genomes, KEGG)进行通路分析发现,2株细胞在免疫调控和细胞生长周期调控上均存在差异,qRT-PCR验证的17个基因中有16个基因的表达趋势与转录组测序结果一致。结论 2株细胞的转录谱存在显著差异。     

转谷氨酰胺酶2抑制H1亚型流感病毒在MDCK细胞中的增殖

组织转谷酰胺酶(transglutaminase 2,TGM2)是一种普遍存在的多功能蛋白,与不同细胞的粘附和肿瘤形成有关.有证据表明,TGM2参与了宿主细胞与病毒间的相互作用,但是对于流感病毒在细胞内增殖的影响还未有报道.为了探究MDCK细胞中TGM2对H1N1亚型流感病毒增殖的影响,本研究构建了TGM2过表达和敲除...     

H1N1亚型流感病毒诱导外周血单个核细胞凋亡研究

A型流感病毒能诱导淋巴细胞、单核巨噬细胞的凋亡,为进一步探讨淋巴细胞和单核巨噬细胞在凋亡中可能存在的相互作用,用H1N1亚型流感病毒诱导人外周血淋巴细胞和单核巨噬细胞的凋亡．结果显示,前48 h,H1N1流感病毒能诱导淋巴细胞和单核巨噬细胞的凋亡,但在培养48 h后,流感病毒对单核巨噬细胞表现为凋亡抑制作用,同时流感病毒对淋巴细胞吸附不同时间后,荧光染色和流式细胞术检测凋亡未见明显差异,说明细胞凋亡与病毒吸附时间长短并无相关性．检测p53抑制剂Pifithrin-α(PFT-α)加入前后淋巴细胞和单核巨噬细胞的凋亡情况,结果显示,淋巴细胞和单核巨噬细胞的凋亡均被抑制, 提示通过p53诱导的凋亡可能是流感病毒诱导细胞凋亡的一条重要途径．     

MDCK细胞的悬浮驯化及初步应用

目的：驯化MDCK细胞使之适应悬浮培养,以之作为生产流感病毒疫苗的介质,为以细胞代替鸡胚制备流感病毒疫苗提供保证。方法：通过直接法和间接法分别驯化MDCK细胞,放大培养能悬浮生长的MDCK细胞,并以之作为介质培养流感病毒。结果：获得了能悬浮培养的MDCK细胞,其在悬浮条件下生长良好;以之作为载体培养流感病毒,可获得较高产量的病毒。结论：建立了悬浮培养的MDCK细胞,以之作为载体培养流感病毒可获得较高病毒滴度,为细胞培养生产流感病毒疫苗奠定了基础。     

Different progress of MDCK cell death after infection by two different influenza virus isolates

The effect of influenza strains A (H₃N₂) and B, isolated during the seasons of 1994 and 1995 in the Czech Republic, on MDCK cells was studied. Various concentrations of virus and conditions of nutrition were used during the cell culture. The virus replication and consequently fragmentation of genomic DNA together with cytotoxicity were investigated in the absence and presence of 10 per cent calf serum. Virus replication, regardless of type A or B, caused earlier DNA fragmentation in comparison to non-infected cells in tissue culture. The results showed that the influenza B strain had a greater cytotoxic effect on MDCK cells than influenza A. A higher infection dose of influenza A virus accelerated the onset of apoptosis; conversely, a higher infection dose of influenza B virus delayed the onset of apoptosis. The absence of serum enhanced the progress of influenza-induced apoptosis in conditions in vitro. © 1997 John Wiley & Sons, Ltd.     

人精液对RAJI细胞中EPSTEIN-BARR病毒早期抗原的诱导

在大戟科、瑞香科、豆科和其它科植物中,发现有些植物含有能诱导人伯基特淋巴瘤细胞——Raji细胞中EB病毒早期抗原的物质,并已证明其中有的是很强的促癌物质,如12-0-十四烷酰巴豆醇-13乙酸酯(TPA)、桐油提纯的HHPA、芫花和黄芫花等。某些人的精液也含有能激活EB病毒的物质。本文报告中国人的精液,以及人精液与从宫颈癌病人分离的细菌培养液,对EB病毒早期抗原的诱导作用和协同诱导作用。     

MECHANISMS OF THE INDUCTION OF APOPTOSIS IN HUMAN HEPATOMA CELLS BY TUMOUR NECROSIS FACTOR-α

Tumour necrosis factor alpha (TNF-α) at 20 ng/ml induced apoptosis in human hepatoma cellsin vitro . The effect of TNF-α-induced apoptosis was exacerbated by the hypoxanthine-xanthine oxidase (HX/XO) system and cycloheximide (CHX), but alleviated by superoxide dismutase (SOD), suggesting that TNF-α-induced apoptosis may be due to oxidative stress, and independent of protein synthesis. TNF-α elevated free Ca²⁺concentration, triggered lipid peroxidation and decreased the expression of bcl-2 protein. The findings suggest that TNF-α-induced apoptosis may be involved in stimulating Ca²⁺-dependent endonuclease activity and increasing membrane lipid peroxidation. Bcl-2 may play a pivotal role in serving as a Ca²⁺regulator or antioxidant, preventing lipid peroxidation in the process.     

单克隆抗体对感染流行性感冒病毒的小鼠的疗效观察

治疗流行性感冒(简称流感)对年老体弱者有着重要意义。由于流感病毒的一个新亚型出现后,往往要经历十几年至20多年的流行,因此选出具有亚型内共同抗原决定簇的McAb,用于治疗流感是可行的,文献也曾有过报道。前文已报道关于甲3型流感病毒McAb的建立,本文用抗甲3型流感病毒上海/32/84血凝素McAb,与1977年以来分离的甲3型流感     

Friend小鼠白血病病毒（Fr.MuLV）对新生金地鼠的感染性研究

一般认为,Ｆｒｉｅｎｄ小鼠白血病病毒（Ｆｒｉｅｎｄｍｕｒｉｎｅｌｅｕｋｅｍｉａｖｉｒｕｓ：Ｆｒ．ＭｕＬＶ）属同种嗜性白血病病毒（ｅｃｏｔｒｏｐｉｃＭｕＬＶ）,只感染小鼠或大鼠,分别引起小鼠发生白血病和大鼠发生呆小病;但对小鼠或大鼠以外的动物（包括人等）无感染性。为进一步探讨该病毒的感染范围,本文进行了该病毒对金地鼠的感染性研究。结果表明,该病毒可在新生金地鼠体内增殖,引起新生金地鼠出现体重减轻、脾萎缩、并发早死等呆小病样症状,病理学检查发现接种该病毒后新生金地鼠的脾脏、胸腺呈明显的病理变化。提示Ｆｒ．ＭｕＬＶ对新生金地鼠具有感染作用,并产生病毒增殖和引起呆小病样症状,为阐明Ｆｒ．ＭｕＬＶ的感染范围和致病作用提供了实验依据。     

A COMPREHENSIVE STUDY ON APOPTOSIS INDUCTION BY AZADIRACHTIN IN Spodoptera frugiperda CULTURED CELL LINE Sf9

The induction of apoptosis by azadirachtin, a well‐known botanical tetranortriterpenoid isolated from the neem tree (Azadirachta indica A. Juss) and other members of the Meliaceae, was investigated in Spodoptera frugiperda cultured cell line (Sf9). Morphological changes in Sf9 cells treated by various concentrations of azadirachtin were observed at different times under light microscopy. Morphological and biochemical analysis indicated that Sf9 cells treated by 1.5 μg/mL azadirachtin showed typical morphological changes, which were indicative of apoptosis and a clear DNA ladder. The flow cytometry analysis showed the apoptosis rate reached a maximum value of 32.66% at 24 h with 1.5 μg/mL azadirachtin in Sf9 cells. The inhibition of Sf9 cell proliferation suggested that the effect of azadirachtin was dose dependent and the EC₅₀ at 48 and 72 h was 2.727 × 10⁻⁶ and 6.348 × 10⁻⁹ μg/mL, respectively. The treatment of azadirachtin in Sf9 cells could significantly increase the activity of Sf caspase‐1, but showed no effect on the activity of Topo I, suggesting that the apoptosis induced by azadirachtinin Sf9 cells is through caspase‐dependent pathway. These results provided not only a series of morphological, biochemical, and toxicological comprehensive evidences for induction of apoptosis by azadirachtin, but also a reference model for screening insect cell apoptosis inducers from natural compounds.     

雷公藤内酯醇体外抗肿瘤作用和诱导细胞凋亡的研究

以不同浓度的雷公藤内酯醇（ＴＬ）处理肿瘤细胞,同时与植物抗癌药物紫杉醇进行比较,结果表明,ＴＬ具有很强的抗肿瘤活性,其作用明显强于紫杉醇。对某些肿瘤细胞如卵巢癌和肾癌细胞等,作用尤其明显。以抗凋亡蛋白Ｂｃｌ－２的裂解以及ｃａｓｐａｓｅ－３抑制剂阻止其裂解研究了ＴＬ诱导细胞的凋亡作用。     

冷休克诱导肿瘤细胞凋亡的动力学特征

应用扫描电镜观察、末端DNA转移酶介导的脱氧UTP缺口末端标记技术（TUNEL）和流式细胞术研究低温冷休克诱导人乳癌细胞系（ZR－75－30）细胞凋亡,并对其细胞凋亡的动力学特征测定、分析。结果显示：－5℃、0℃、4℃冷休克均可诱导ZR－75－30细胞凋亡,但细胞凋亡进程受实验细胞预培养时间;冷休克温度、时间;细胞冷休克后恢复培养时间的影响,这些影响因素具有明显的动力学特征。经过对冷休克诱导ZR－75－30细胞凋亡的动力学特征分析、比较,优选出研究冷休克诱导肿瘤细胞凋亡的实验体系：即细胞经37℃细胞凋亡的动力学特征分析、比较, 优选出冷休克诱导肿瘤细胞凋亡的实验体系：即细胞经37℃预培养36h→冷休克（-5℃24h,0℃36h,4℃48h）→细胞冷休克后于37℃恢复培养18－24h→细胞凋亡检测→检测结果综合分析。应用该实验体系要使细胞凋亡率达50－60％,且重复性尚佳。优化体系的深入研究冷休克诱导细胞凋亡的信号传递及其分子机制提供实验模型。     

EB病毒介导人鼻咽上皮细胞永生化早期阶段的生物学观察

EB病毒转化人鼻咽上皮细胞,建立体外多阶段细胞模型有利于从细胞和分子水平对肿瘤发病机制作深入研究.我们利用与鼻咽癌密切相关的EB病毒和TPA的协同作用,观察原代人胚鼻咽上皮细胞逃避老化期后其生物学特性的变化.结果表明,EB病毒感染的人胚鼻咽上皮细胞在原代培养后期老化相关半乳糖苷酶(SA-β-Gal)表达降低,形态学上发生改变,出现转化灶样集落,群体倍增时间降低,体外培养寿命延长,表明EB病毒促使部分原代人胚鼻咽上皮细胞逃避老化期、进入永生化早期阶段.这些研究资料为进一步阐明上皮细胞永生化分子机制及建立人鼻咽上皮细胞永生化模型提供实验依据.关键词EB病毒人鼻咽上皮细胞老化期永生化