共查询到18条相似文献，搜索用时 218 毫秒
1.
试验旨在对不同毒力水貂阿留申病毒（Aleutian mink disease virus,AMDV）在猫肾细胞（feline kidney cell,CRFK）中的增殖规律及其诱导细胞凋亡情况进行比较研究。将标准毒株AMDV-G及分离到的野毒株AMDV-DL124、AMDV-DL125、AMDV-QD2、AMDV-QD3、AMDV-ZJ3接种CRFK细胞,应用间接免疫荧光、实时荧光定量PCR、TCID50测定技术研究病毒在细胞中的复制及表达情况,同时检测病毒诱导的细胞凋亡情况。间接免疫荧光结果显示,5株野毒株荧光着色趋势差异不大,均在感染后12 h出现荧光,随感染时间延长荧光增多,AMDV-G荧光出现时间比野毒株晚,但病毒感染后72 h几乎所有细胞均出现荧光;实时荧光定量PCR结果显示,基因组复制趋势大致相同,AMDV-DL125感染后3 h复制开始,AMDV-G感染后24 h复制才开始并呈快速增长趋势,但感染后72 h均达到峰值。TCID50检测结果表明,0~12 h为病毒感染潜伏期,AMDV-G感染后60 h达到峰值,野毒株均在感染后72 h达到峰值,但是6株病毒均能在感染后48~72 h维持较高的感染滴度,其后随细胞崩解而降低。SPSS 23.0统计软件分析凋亡检测结果显示,与对照组相比,野毒株感染细胞后2~12 h诱导细胞凋亡差异显著（P<0.05）,AMDV-G诱导细胞凋亡差异明显低于野毒株,但是诱导细胞凋亡时间较野毒株长,在感染后24 h仍对细胞凋亡有较明显的诱导作用,但是各病毒诱导的细胞凋亡主要集中在2~12 h。该结果为AMDV的培养、鉴定及致病机理研究提供一定参考。 相似文献
2.
旨在揭示山羊副流感病毒3型(CPIV3)感染气管上皮细胞后是否可诱导细胞发生凋亡,并对细胞凋亡的信号通路进行初步探究。本研究将CPIV3病毒液接种气管上皮细胞,在12、24、36、48、72和96 h收集培养物上清检测病毒增殖滴度;通过形态学观察CPIV3诱导气管上皮细胞病变(CPE)情况;采用Annexin V-FITC/PI双染细胞凋亡检测试剂盒和Caspase-3、Caspase-8和Caspase-9活性检测试剂盒检测凋亡水平及相关指标;荧光定量PCR检测细胞凋亡分子mRNA表达水平;Western blot分析激活型Caspase-3蛋白表达变化情况。结果显示,CPIV3在气管上皮细胞中的增殖呈上升趋势,96 h能达到10~(4.50)TCID_(50)·mL~(-1);形态学观察发现,病毒接种后48 h出现细胞收缩变圆、脱落等CPE现象;流式细胞术检测及Caspase活性检测表明,感染组细胞出现细胞凋亡,48 h后细胞凋亡率达19.66%,且Caspase-3、Caspase-8和Caspase-9活性随着时间延长逐渐升高;死亡受体凋亡途径和线粒体凋亡途径细胞凋亡因子mRNA表达上调。Western blot分析揭示,激活型Caspase-3蛋白在病毒感染过程中被活化。本研究证实CPIV3感染可诱导气管上皮细胞凋亡,且Caspase途径在病毒诱导细胞凋亡的过程中发挥重要作用。 相似文献
3.
《中国兽医杂志》2017,(11)
为了分析TLR4在绵羊肺炎支原体介导肺泡上皮细胞凋亡中的分子机制,以绵羊肺泡上皮细胞为材料,用不同浓度的抑制剂TAK-242与细胞孵育,用MTT法检测细胞活性,用试剂盒检测Caspase-3和Caspase-8的活性,以及用实时定量PCR检测TLR4和TNF-αmRNA的表达水平,接着用流式细胞仪检测细胞的凋亡率。结果:当抑制剂TAK-242浓度为1 nmol/L、5 nmol/L、10 nmol/L不影响细胞活性,而当浓度为20 nmol/L和30 nmol/L时细胞活性显著降低(P<0.05);在感染比例(MOI)为10的情况下,在12 h和24 h支原体可以提高TLR4 mRNA的表达水平;绵羊肺炎支原体可显著提高Caspase-3和Caspase-8的活性,而TAK-242能够显著降低Caspase-3和Caspase-8的活性(P<0.01);在感染时间为24 h,支原体可以提高促TNF-αmRNA的表达水平,而抑制剂TAK-242在10 nmol/L的浓度下可以降低支原体介导的TNF-αmRNA的表达水平;在24 h支原体引起的细胞的细胞凋亡率为16.7%±1.81%,而抑制剂TAK-242处理组引起的细胞凋亡率为11.7%±1.32%。表明TLR4在绵羊肺炎支原体诱导肺泡上皮细胞凋亡中发挥重要作用。 相似文献
4.
犬细小病毒YBYJ株体外诱导MDCK宿主细胞凋亡的研究 总被引：1，自引：0，他引：1
为研究犬细小病毒(CPV)在体外能否诱导MDCK宿主细胞凋亡,本研究应用前期分离到的CPV YBYJ强毒株接种MDCK细胞,通过倒置显微镜观察CPV导致MDCK宿主细胞病变(CPE)情况,采用细胞凋亡DNA Ladder抽提试剂盒和Annexin V-FITC/PI双染细胞凋亡检测试剂盒和Caspase-3活性检测试剂盒检测,观察CPV诱导MDCK宿主细胞凋亡情况。结果显示,CPV YBYJ强毒株可导致MDCK宿主细胞产生明显CPE,并出现明显的DNA Ladder,Annexin V-FITC/PI双染为阳性,Caspase-3活性显著升高,提示CPV在体外能够诱导MDCK宿主细胞凋亡。 相似文献
5.
为研究金黄色葡萄球菌小菌落突变株(S.aureus small colony variants,SASCVs)与正常株对原代培养的乳腺上皮细胞的侵袭情况,并观察对比其对细胞结构的损坏程度和对乳腺上皮细胞的影响,本试验对由云南农业大学动物医学实验教学中心实验室分离的一株SASCV进行体外感染小鼠乳腺上皮细胞试验,将分离出的一株SASCV及质控菌株ATCC 25923分别注入实验室体外培养的小鼠乳腺上皮细胞内,采用普通光镜和扫描电子显微镜观察质控菌株ATCC 25923及SASCVs诱导乳腺上皮细胞凋亡的细胞形态学变化;用流式细胞仪(Annexin V-FITC/PI双染法)定量检测质控菌株ATCC 25923及SASCVs感染乳腺上皮细胞凋亡的诱导作用,观察细胞的变化。结果显示,质控菌株ATCC 25923及SASCVs感染小鼠乳腺上皮细胞3 h后,可诱导乳腺上皮细胞发生凋亡,显示为细胞核皱缩,染色质边缘化和细胞浆内空泡增多等典型的凋亡特征。Annexin V-FITC/PI双染法检测后发现,与对照组相比,感染组细胞凋亡率明显升高,并且阳性对照组的细胞凋亡率明显高于试验组。结果表明,SASCVs在侵染细胞的过程中达到一定数量不再上升;SASCVs的凋亡率明显的较正常金黄色葡萄球菌低。因此,SASCVs也可诱导乳腺上皮细胞凋亡,具有细胞凋亡的典型形态特征。本试验结果为以后预防和控制SASCVs感染奶牛慢性乳房炎提供试验依据。 相似文献
6.
动物冷应激过程中肝脏因发生氧化应激而受损。本试验采用过氧化氢(H2O2)诱导buffalo大鼠肝细胞(BRL)凋亡,建立体外氧化应激的凋亡损伤模型,为深入研究冷应激对肝脏损伤的机理奠定基础。试验采用不同浓度H2O2刺激BRL细胞2h,运用WST-1法检测细胞生长活力、Annexin-V/FITC-PI双染技术检测细胞凋亡率及Western blot方法检测Caspase-3的表达量来筛选BRL细胞凋亡的H2O2作用浓度。结果显示,与空白对照组相比,150μmol/L H2O2作用后,细胞增殖率明显下降,Caspase-3的蛋白表达量增加,流式细胞仪检测的凋亡率升高,同时细胞坏死较其他H2O2处理组低。结果表明,150μmol/L H2O2作用BRL细胞2h可模拟氧化应激造成的凋亡损伤。 相似文献
7.
探讨牛磺胆酸对小鼠胸腺细胞凋亡影响的机理。采用流式细胞术检测了牛磺胆酸对凋亡过程中磷脂酰丝氨酸外翻的影响;采用分光光度法检测了牛磺胆酸对凋亡特异性蛋白酶Caspase-9、Caspase-3活性变化的影响。结果显示,高剂量牛磺胆酸可显著抑制磷脂酰丝氨酸外翻,抑制细胞凋亡,而低剂量牛磺胆酸则可显著提高caspase-9、caspase-3活性,诱导细胞凋亡。牛磺胆酸对小鼠胸腺细胞早期凋亡呈现抑制作用,而在凋亡中后期则呈凋亡诱导作用。 相似文献
8.
《中国预防兽医学报》2016,(12)
为研究高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)Hu N4株诱导断奶仔猪胸腺细胞凋亡的分子机制,本实验通过免疫荧光技术、流式细胞术、荧光定量PCR和western blot等方法检测HP-PRRSV感染仔猪胸腺内细胞凋亡相关因子Caspase-8、Caspase-9和Caspase-3表达量的变化。结果显示:HP-PRRSV感染仔猪后,胸腺内表达凋亡相关蛋白Caspase-8、Caspase-9和Caspase-3的细胞比例显著升高,并且凋亡相关蛋白Caspase-8、Caspase-9和Caspase-3表达量也明显升高。以上结果表明:HP-PRRSV感染仔猪引起胸腺细胞凋亡是通过Caspase依赖性通路介导。本研究为进一步了解HP-PRRSV感染机制提供了实验依据。 相似文献
9.
猪伪狂犬病毒感染PK15细胞对细胞凋亡及相关凋亡因子的影响 总被引：1，自引：0，他引：1
《中国兽医学报》2016,(4):553-557
为研究PRV感染PK-15细胞对其细胞凋亡及相关凋亡因子的影响,本试验采用体外细胞培养技术在不同时间点收集细胞、应用噻唑蓝(MTT)法检测细胞的增殖活性、流式细胞仪测定细胞凋亡率、实时荧光定量PCR法检测凋亡相关基因caspase-3,Bcl-2,Bax和Bcl-xl的表达情况。结果显示,PRV感染24 h后可明显抑制细胞的增殖,这种抑制与时间密切相关,与感染剂量无显著差异。流式细胞仪检测也显示PRV能明显提高其凋亡率,PT-PCR显示PRV感染后caspase-3,Bax表达量明显上调,Bcl-xl,Bcl-2表达量明显下调。结果表明,PRV在感染过程中对细胞的生长的影响与时间紧密联系,能够诱发细胞凋亡,Bax,Bcl-2,Bcl-xl,caspase-3,起着重要的调控作用。 相似文献
10.
为了研究人乳铁蛋白对宫颈癌Hela细胞的生长抑制作用及其对细胞凋亡、p53蛋白表达和Caspase-3活性的影响,试验采用MTT方法检测细胞的生长抑制率、DNA片段化试验检测细胞凋亡、流式细胞术测定细胞周期、Western-blot检测p53蛋白表达、Caspase-3活性检测试剂盒检测Caspase-3的活性.结果表明:人乳铁蛋白对宫颈癌Hela细胞的抑制呈现时间和剂量依赖性,有73%的Hela细胞停滞在G0～G1期;Western-blot检测结果显示人乳铁蛋白可以上调p53蛋白的表达; 人乳铁蛋白作用48 h后Caspase-3的活性是0小时时的5.5倍,二者差异极显著(P<0.01).结果说明人乳铁蛋白能显著抑制宫颈癌Hela细胞生长,其抗肿瘤机制与诱导细胞凋亡、调控细胞周期、上调p53表达、活化Caspase-3有关. 相似文献
11.
This study was aimed to compare the proliferation and apoptosis characteristics of different virulence of Aleutian mink disease virus(AMDV) in feline kidney cell (CPFK). CRFK cells were infected separately with the standard strain of AMDV-G and the wild strains of AMDV-DL124, AMDV-DL125, AMDV-QD2, AMDV-QD3 and AMDV-ZJ3. Indirect immunofluorescence,Real-time quantitative PCR and TCID50 were used to detect the replication and expression of the viruses in the cells, at the same time the apoptosis induced by the viruses was detected. The results of IFA showed that CRFK cells which infected separately by wild strains appeared the fluorescence after 12 h of infection. With the extension of the infection time, the fluorescence of cells increased, but the fluorescence of AMDV-G was later than the wild strains, almost all of the cells were appeared the fluorescence after 72 h of infection. The Real-time quantitative PCR results showed that the tendency of genome replication of different virus were approximately the same. The genome replication began at 3 h after the AMDV-DL125 infection,the rapid increase of genome replication of AMDV-G occurred at 24 h after the infection, the genome replication of whole viruses reached a peak after 72 h of infection. The results of TCID50 showed that the latent period of viruses infection were 0 to 12 h after infection, AMDV-G reached the peak after 60 h of infection. However, the wild strains at 48 to 72 h could maintain a higher infection titer and reached peak at 72 h after infection, then decreased with the cell disintegration. Apoptosis detection results which analysis by SPSS 23.0 statistical analysis software showed that, compared with control group,cell apoptosis was significantly higher after 2 to 12 h of cells infected by wild strains induced (P<0.05), cell apoptosis of AMDV-G was significantly lower than wild strains, however, the time of cell apoptosis induced by AMDV-G was longer,the apoptosis was still significant after 24 h of infection. But the apoptosis caused by virus infection was mostly concentrated in 2 to 12 h after infection. The results would provide some references for the study on the culture and identification and pathogenic mechanism of AMDV. 相似文献
12.
13.
Background
Aleutian disease in mink is caused by infection with Aleutian mink disease virus (AMDV). In Sweden, the infection most commonly causes classical Aleutian disease in which the immune system fails to neutralize the virus and the infection becomes persistent. Diagnosis of AMDV infection is based on serological methods that detect virus-specific antibodies. Traditionally counterimmunoelectrophoresis (CIEP) has been the preferred method, but in order to enable automation interest has been paid to other antibody detecting systems. Recently, at least two different ELISA systems that detect antibodies to AMDV have been manufactured; one is based on an in vitro grown AMDV as antigen, and the other system is based on the AMDV capsid protein VP2 as antigen. The aim of this study was to evaluate the two ELISA systems for detection of antibodies to AMDV using CIEP as the gold standard.Results
When employing the mean optical density of the samples from CIEP negative mink plus three standard deviations as cut-off value, the ELISA with the VP2 antigen had a sensitivity of 99.7% and a specificity of 98.3% compared to CIEP (n = 364). Analysis of samples with the AMDV-G antigen based ELISA employing an assay cut-off value based on the negative control samples, as suggested by the manufacturer, resulted in a sensitivity of 54.3% and a specificity of 93.2% with reference to CIEP as the gold standard (n = 359). When employing the mean optical density of the samples from CIEP negative mink plus three standard deviations as cut-off value, the AMDV-G ELISA had a sensitivity of 37.6% and a specificity of 98.3%.Conclusions
The ELISA system based on VP2 antigen had high sensitivity and specificity, and was concluded to be an alternative to the CIEP as a diagnostic tool for AMDV antibodies. In contrast, the AMDV-G ELISA suffered from low sensitivity when compared to CIEP. 相似文献14.
In order to study the situation of S. aureus small colony variants (SASCVs) and the normal strains hitting the mammary epithelial cells of primary culture, we observed and compared the degree of damage to the cellular structure and the effects on mammary epithelial cells. This test was carried out with a strain of SASCV which was separated by Yunnan Agricultural University Animal Medical Experimental Teaching Center Laboratory via infecting mice mammary epithelial cells in vitro. We put the SASCVs and quality control strains ATCC 25923 respectively into the mammary epithelial cells cultivated in vitro in the laboratory, and observed the morphological change of mammary epithelial cell apoptosis induced by quality control strains ATCC 25923 and SASCVs using ordinary light microscope and scanning electron microscope; At the same time, we also observed the mammary gland epithelial cells apoptosis induced by quality control strains ATCC 25923 and SASCVs using flow cytometry (Annexin V-FITC/PI double staining). The results showed that 3 h after quality control strains and small colony mutant strains infecting the mammary epithelial cells, the mammary epithelial cells could be induced to apoptosis. It showed some typical characteristics of apoptosis such as the nucleus shrivelled, chromatin marginalized, mammary gland epithelial cells could be induced apoptosis, expression was shrinking nucleus, chromatin marginalized, the increase of cell intracytoplasmic vacuoles and so on. After testing using the Annexin V-FITC/PI double staining, we found that compared with the control group, the apoptosis rate of infection group was obviously higher, and the apoptosis rate of the positive control group was obviously higher than that of the experimental group. The results showed that in the process of infecting cells, the SASCVs reached a certain number and then stopped; The apoptosis rate of SASCVs was obviously lower than the normal of S.aureus. Therefore, SASCVs could also induce mammary epithelial cells apoptosis. It had the typical morphological characteristics of apoptosis. It provided experimental basis for the prevention and control of SASCVs chronic mastitis cows. 相似文献
15.
旨在研究布鲁氏菌外膜蛋白OMP16对小鼠巨噬细胞RAW264.7细胞凋亡和免疫活性的影响及其机制探讨。以OMP16处理RAW264.7细胞为模型,通过MTT以及Hoechst法检测OMP16对细胞活力的影响,通过Western blot检测Caspase-3、GRP78、CHOP的表达情况,流式细胞术检测OMP16对细胞凋亡的影响以及RT-PCR检测免疫因子IL-1β、IL-6和TNF-α的变化。结果表明,OMP16能够影响RAW264.7细胞的活力,且有浓度依赖性;添加OMP16后能够显著增加凋亡标志因子Caspase-3的表达（P<0.001）,并促进RAW264.7细胞的凋亡;作用24、36 h之后,显著引起内质网应激标志蛋白GRP78、CHOP的表达;并且能显著上调IL-1β、IL-6、TNF-α等炎性因子的mRNA表达水平。布鲁氏菌OMP16能够诱导RAW264.7凋亡,OMP16显著引起内质网应激CHOP通路的激活。 相似文献
16.
Yufei Guo Chanjuan Shen Anchun Cheng Mingshu Wang Na Zhang Shun Chen Yi Zhou 《Veterinary microbiology》2009,138(3-4):258-265
Anatid herpesvirus 1 (AHV-1) CH virulent strain was first isolated from an infected duck and it was found that this virus strain could induce cytopathic effect (CPE) in duck embryo fibroblast (DEF). Following AHV-1 infection, DEF showed morphological changes such as cell rounding, improved refractivity and detachment from the culture surface. However, its pathological characteristics were not adequately known. Related studies were performed and the results showed that syncytium formation could be observed as the other type of CPE in AHV-1 infection. Hematoxylin-eosin staining and 4’, 6-diamidino-2-phenylindole (DAPI) staining of infected DEF were each used to visualize the shape and distribution of chromatin within nuclei and nuclear fragmentation was observed. Chromatin condensation and margination, as well as formation of apoptotic bodies were observed by transmission electron microscopy (TEM). DNA ladder formation was detected in AHV-1 infected cells and apoptosis of the infected DEF was also detected by flow cytometry analysis of Annexin V-FITC/PI staining method. Therefore, it was suggested that AHV-1 virulent strain can induce syncytium and apoptosis in DEF. Syncytium formation and apoptosis observed in this study may contribute to the elucidation of AHV-1 pathogenesis. 相似文献
17.
羊口疮(Orf)是由羊口疮病毒(ORFV) 引起的一种急性、接触性和高度嗜上皮性人畜共患的动物传染病,主要感染绵羊和山羊,有时威胁人类。本研究将ORFV(OV/HLJ/04株)纯化后作为试验材料,采用巨噬细胞系RAW264.7作为病毒细胞宿主,RAW264.7在LPS体外激活的情况下,接种ORFV,研究ORFV感染对巨噬细胞凋亡的影响。结果表明,用LPS处理的试验组巨噬细胞形态学改变较大,凋亡量也较大;而没用LPS处理过的试验组巨噬细胞变化则相对较少。Western blotting研究结果显示,细胞凋亡信号Caspase-3与甘油醛-3-磷酸脱氢酶(GAPDH)在24 h时对比,凋亡信号明显减弱,表明在24 h的巨噬细胞凋亡水平有所下降。 相似文献
18.
试验旨在研究添加羧乙基锗倍半氧化物（carboxyethylgermanium sesquioxide，Ge-132）对牛孤雌激活后胚胎发育率、胚胎细胞数、早期胚胎内活性氧（reactive oxygen species，ROS）水平及胚胎内相关凋亡基因的影响。在牛早期胚胎体外培养基中添加不同浓度的Ge-132（0、10、100和200 μg/mL），观察其对牛体外孤雌激活胚胎发育的影响；应用Hoechst对孤雌激活后第8天胚胎进行染色后制作装片，在显微镜下对细胞进行计数；用DCFH-DA染色检测早期胚胎内ROS水平，并用Image J测量荧光强度后对数据进行统计分析，用RT-PCR对胚胎内细胞凋亡相关基因（Caspase-3、Bax、Bcl-xl和Survivin）进行分析。结果显示，10 μg/mL Ge-132处理组胚胎率与对照组间无显著差异（P>0.05），但可降低1细胞期的ROS水平；10 μg/mL Ge-132处理组较对照组早期胚胎细胞数显著增加（P<0.05）；通过检测细胞凋亡相关基因mRNA转录水平发现，与对照组相比，10 μg/mL Ge-132处理组胚胎促凋亡基因Caspase-3表达水平显著降低（P<0.05），抑制细胞凋亡基因Survivin表达水平显著升高（P<0.05）。结果表明，在早期胚胎培养基中添加10 μg/mL Ge-132可降低细胞内ROS水平，减少胚胎中氧化应激诱导的细胞凋亡，从而提高孤雌激活后牛胚胎的发育潜能。 相似文献