HSV-2阴道感染并诱导小鼠阴道粘膜上皮细胞凋亡

最近的报道指出,某些病毒有诱导体外细胞凋亡的作用,借以限制病毒的扩散.为探讨HSV-2在体内诱导细胞凋亡的效果及其形态学特点,用HSV-2 333株感染小鼠阴道,于感染后不同天数处死动物,取其阴道,固定于10%中性福尔马林,TUNEL末端标记染色显示凋亡的细胞,光镜下进行原位观察.结果显示感染后的第1天粘膜上皮内即出现大量的凋亡细胞,第2天至11天凋亡细胞的数量及在上皮内的分布范围达最高水平.早期的凋亡细胞见于感染后所有标本,其核染色质形态及分布似正常细胞,但它被TUNEL标记染成棕黄色;晚期的凋亡细胞亦见于所有标本,其胞核缩小,染色质浓缩并在核周边集聚,核中心空化.载有凋亡细胞的上皮在阴道粘膜上分布很广,最广的可占全阴道上皮的2/3.同时可见HSV-2引起的上皮细胞坏死及疱疹形成,二者均由凋亡细胞包围.凋亡细胞不断地由上皮表面脱落至阴道,未见凋亡小体及吞噬现象.结果提示,HSV-2 333株阴道感染可同时诱导细胞坏死及凋亡,细胞凋亡可能在限制病毒产生子代及限制感染区域扩展起重要作用.     

人单核细胞系U_（937）和T细胞系HSB_2细胞的HSV－1感染

用ＨＳＶ－１接种人单核细胞传代株Ｕ＿（９３７）和Ｔ淋巴细胞传代株ＨＳＢ＿２细胞,通过对病毒滴度、细胞增殖与病变、病毒抗原表达及病毒ＤＮＡ的动态观察,研究了ＨＳＶ－１感染两种细胞的特点。结果发现接种病毒后,Ｕ（９３７）细胞仅允许病毒短时间低滴度复制,培养上清中病毒滴度在１２天内降至测不出水平,细胞经过１～２周后增殖受抑、死亡增多,然后又逐渐恢复;用ＬＰＳ持续刺激则能提高病毒感染滴度,延长感染时间,形成短期持续感染。ＨＳＢ＿２细胞允许病毒复制至较高滴度,呈现急性杀细胞性感染;用ＰＨＡ持续刺激也不改变细胞感染的特点。     

几种细胞因子对HSV—1感染单核巨噬细胞的影响

以ＨＳＶ－１接种人单核细胞系Ｕ９３７和小鼠腹腔巨噬细胞，并在接种前后分别以不同的细胞因子处理，经病毒滴定，免疫荧光试验检测病毒抗原及多聚酶链反应技术检测病毒基因研究了细胞因子对ＨＳＶ－１感染单核巨噬细胞的影响。结果证实，ＴＮＰ－α５０ｕ／ｍＬ，Ｍ－ＣＳＦ２００ｕ／ｍＬ，ＩＦＮ－γ１０００ｕ／ｍＬ，ＩＦＮ－α１０００ｕ／ｍＬ均能增强单核巨噬细胞对ＨＳＶ－１的抗性，加速细胞对ＨＳＶ－１ＤＮＡ的清除，抑     

UV-C照射诱导体外血管平滑肌细胞凋亡模型的建立

应用常规细胞培养超净台紫外消毒灯（２２０Ｗ,２２０Ｖ,５０Ｈｚ）发射的ＵＶ－Ｃ波段的紫外光源（２５４ｎｍ）,垂直照射距离其１０ｃｍ处的大鼠主动脉平滑肌细胞,发现经照射后细胞出现典型的凋亡形态学改变,如细胞变圆,染色质浓缩,细胞膜出泡,出现凋亡小体等;细胞面积,核面积及核／胞面积比均显著降低;且提取细胞ＤＮＡ的琼脂糖凝胶电泳呈现梯状图谱。从形态学和生化指标方面证明了ＵＶ－Ｃ照射可诱导体外血管ＳＭＣｓ     

汉滩病毒感染诱导热休克蛋白70表达

为了解汉滩病毒感染后细胞的应激反应及ＨＳＰ７０的表达与病毒复制的关系,在汉滩病毒Ａ９株感染Ｖｅｒｏ－Ｅ６细胞后,用免疫组织化学及核酸分子原位杂交法,对细胞ＨＳＰ７０基因的表达进行了检测。结果表明,汉滩病毒感染细胞４ｈｙ后即可诱导Ｖｅｒｐ－Ｅ６细胞表达ＨＳＰ７０,表达可持续至感染后５ｄ且ＨＳＰ７０在细胞内的分布也有改变。提示汉滩病毒可直接诱导ＨＳＰ７０的高表达。     

转TK基因的人结肠癌细胞对多种原药敏感性的研究

构建了含有单纯疱疹病毒胸苷激酶基因（ＨＳＶ－ＴＫ）的重组逆转录病毒载体ＬＴＫＳＮ,经ＰＡ３１７细胞包装后,感染人结肠癌细胞株ＬｏＶｏ．用Ｇ４１８筛选到稳定表达ＨＳＶ－ＴＫ基因的细胞克隆ＬｏＶｏ／ＬＴＫＳＮ,ＬｏＶｏ／ＬＴＫＳＮ与野生型ＬｏＶｏ细胞相比,生长曲线无明显差异,细胞形态亦无改变,细胞毒试验证明ＬｏＶｏ／ＬＴＫＳＮ对ＧＣＶ的敏感性很高,半杀伤浓度ＩＣ５０为０．５μｍｏｌ／Ｌ,比野生型细胞提     

几种细胞因子对HSV─1感染单核巨噬细胞的影响

以ＨＳＶ─１接种人单核细胞系Ｕ＿（９３７）和小鼠腹腔巨噬细胞,并在接种前后分别以不同的细胞因子处理。经病毒滴定、免疫荧光试验检测病毒抗原及多聚酶链反应技术检测病毒基因,研究了细胞因子对ＨＳＶ─１感染单核巨噬细胞的影响。结果证实,ＴＮＦ─α５０ｕ／ｍＬ　、Ｍ─ＣＳＦ２００ｕ／ｍＬＩＦＮ─γ１０００ｕ／ｍＬ、ＩＦＮ－α１０００ｕ／ｍＬ均能增强单核巨噬细胞对ＨＳＶ─１的抗性,加速细胞对ＨＳＶ─１ＤＮＡ的清除,抑制病毒抗原的表达;并能拮抗ＬＰＳ对ＨＳＶ─１在单核巨噬细胞中表达的促进作用。     

几种性传播微生物与人工流产术后不孕关系的研究

应用聚合酶链反应（ＰＣＲ）、单克隆抗体免疫荧光法和特异性培养等方法对３０例人工流产术后不孕妇女和３９例正常妇女进行了五种性传播微生物包括ＨＳＶ－２、ＨＰＶ、ＣＴ、ＵＵ、ＮＧ进行了检测。研究表明：五种ＳＴＭ在人工流产术后不孕妇女生殖道中的混合感染、不论隐性或显性感染,不论是五种ＳＴＭ中任何两种或两种以上的感染,都可能在人流术后不孕中起着重要作用。尤其是性传播病毒ＨＳＶ－２和ＨＰＶ在其中的作用机理值得进一步探讨。     

基因工程干扰素（IFN—α2b）抗柯萨奇和单纯疱疹病毒效应

本文采用微量细胞病变抑制法在Ｗｉｓｈ 及Ｖｅｒｏ 细胞上研究了ＩＦＮ—α２ｂ 抗ＣｏｘＢ３ 型及ＨＳＶ—１ 型和ＨＳＶ—Ⅱ型病毒的作用。结果表明,安达芬和干扰能ＩＨＮα２ｂ 在Ｗｉｓｈ 或Ｖｅｒｏ 细胞上５００ＩＵ／ｍｌ 分别可以抗３ｌｏｇ４ 或１０００ＴＣＩＤ５０ ,５０ＩＵ／ｍｌ 可以抗２ｌｏｇ４ 或１００ ＴＣＩＤ５０ ,５ＩＵ／ｍｌ 可以抗１ｌｏｇ４ 或１０ ＴＣＩＤ５０ 的ＣｏｘＢ３ 型或ＨＳＶ—１ 型和ＨＳＶ—Ⅱ型病毒感染细胞。提示安达芬和干扰能均显示明显的抗病毒效应,且二者无明显差别     

HCV非结构蛋白(NS2/NS3)基因表达载体的构建及其一级结构分析

应用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）技术,从ＨＣＶ感染者血清中扩增编码ＨＣＶ病毒蛋白酶的ＮＳ２－ＮＳ３ｃＤＮＡ片段,在其５′和３′端分别引入ＥｃｏＲⅠ和ＸｂａⅠ限制性内切酶位点,定向克隆至真核表达载体ｐｃＤＮＡ３,构建重组载体ｐｃＤＮＡ－ＮＳ２３,重组表达载体经限制性内切酶消化鉴定．用ＳＰ６和Ｔ７通用引物对目的基因片断进行序列分析．序列同源性分析结果表明,与ＨＣＶ－Ｊ、ＨＣ－Ｃ２有高度的同源性,与ＨＣＶ－１、ＨＣＶ－Ｊ６、ＨＣＶ－Ｊ８同源性差,提示所克隆的基因属ＨＣＶⅡ型．该区内重要的功能位点如Ｚｎ２＋依赖性金属蛋白酶催化中心、丝氨酸蛋白酶催化中心等均高度保守     

SARS—CoV感染Vero E6细胞诱导细胞凋亡

为了确定SARS冠状病毒(SARS—CoV)感染Vero E6是否引起细胞凋亡,我们利用细胞DNA琼脂糖电泳,感染细胞的间接荧光染色和Hoechst 33258细胞核染色,以及流式细胞仪分析等方法证明了SARS-CoV感染的Veto E6具有典型的凋亡细胞学和生物化学特征。实验证明具有细胞凋亡特征的所有细胞均为处于感染晚期的细胞。表现明显细胞病变(CPE)的细胞大多已经出现核质凝缩或形成凋亡小体进入细胞凋亡的过程。可以断定SARS-CoV感染Vero E6细胞诱发了细胞凋亡。     

Early passage neonatal and adult keratinocytes are sensitive to apoptosis induced by infection with an ICP27-null mutant of herpes simplex virus 1

Herpes simplex virus 1 (HSV-1) is a enveloped, double stranded DNA virus that is the causative agent of various diseases including cold sores, encephalitis, and ocular keratitis. Previous research has determined that HSV-1 modulates cellular apoptotic pathways. Apoptosis is triggered in infected cells early in infection; however, later in the infection the apoptotic response is suppressed due to the expression of several viral apoptotic antagonists. This sets us a delicate balance between pro- and anti-apoptotic processes during the lytic phase of infection. Several studies have demonstrated that the apoptotic balance can be shifted during infection of certain cell types, leading to apoptosis of the infected cells (HSV-1-dependent apoptosis). For example, HEp-2 cells infected with an ICP27-null recombinant HSV-1 virus undergo HSV-1-dependent apoptosis. Differences in the sensitivity to HSV-1-dependent apoptosis have been revealed. Although many tumor cells have been found to be highly sensitive to this apoptotic response, with the exception hematological cells, all primary human cells tested prior to this study have been shown to be resistant to HSV-1-dependent apoptosis. Here, we demonstrate that early passage neonatal and adult human keratinocytes, which are usually the first cells to encounter HSV-1 in human infection and support the lytic stage of the life cycle, display membrane blebbing and ballooning, chromatin condensation, caspase activation, and cleavage of cellular caspase substrates when infected with an ICP27-null recombinant of HSV-1. Furthermore, caspase activation is needed for the efficient apoptotic response. These results suggest that apoptotic machinery may be a target for modulating HSV-disease in patients.     

疱疹病毒Ⅱ型感染SH-SY5Y细胞的生物学效应

目的：探讨疱疹病毒Ⅱ型（HSV-2）感染人神经母细胞瘤细胞株SH-SY5Y的生物学效应。方法：病毒液接种SH-SY5Y细胞后,用相差和电子显微镜观察感染细胞的形态变化,RT-PCR检测病毒在细胞中的增殖,MTT法检测病毒感染对细胞增殖的影响,流式细胞仪测定感染后的细胞凋亡状况。结果：相差显微镜显示细胞病变,从24～72h,细胞变性、坏死的程度和数量随感染时间延长而增加;电镜结果显示感染24h后,细胞核染色质固缩,出现多核巨细胞,线粒体内嵴紊乱、断裂,出现不同程度的自噬化、溶酶体化、空泡化,并可见大量鹰眼样已包装成熟的病毒颗粒及正在包装的病毒粒子;HSV-2LAT基因RT-PCR扩增表明,病毒能在SH-SY5Y细胞中增殖;凋亡检测显示HSV-2在体外细胞感染中并未使细胞出现凋亡现象;感染后24、48及72h,SH-SY5Y细胞的抑制率分别为11.3%、31.2%和63.1%,与对照组相比均存在显著性差异（P〈0.05）;分别用0.1、1、10MOI的病毒感染SH-SY5Y细胞,上述不同组在24、48、72h时细胞形态变化基本一致,感染结果相似,各组之间病毒毒力无明显差异（P〉0.05）。结论：初步在人神经母细胞瘤细胞株SH—SY5Y中建立了HSV-2感染的细胞模型,并研究了感染对细胞生物性状的影响,为探讨HSV-2的潜伏与激发机制、了解HSV-2的致病机制打下基础。     

Apoptosis in the pathogenesis of infectious diseases of the eastern oyster Crassostrea virginica

Apoptosis, or programmed cell death, has been reported as being pivotal in infectious diseases of different organisms. The effects of apoptosis on the progression and transmission of the protistan parasites Perkinsus marinus and Haplosporidium nelsoni in the eastern oyster Crassostrea virginica were studied. Oysters were diagnosed for their respective infections by standard methods, and apoptosis was detected using in situ hybridization to detect DNA fragments by end labeling on paraffin sections. A digoxigenin nucleotide probe was used to label the 200 bp fragment produced by apoptosis and detected immunohistochemically using an antidigoxigenin peroxidase conjugate. The probe/DNA fragment complex was stained with a peroxidase substrate and tissues were counterstained with methyl green. Uninfected oysters had large numbers of apoptotic hemocytes present in the connective tissue underlying the stomach, gill, and mantle epithelia, whereas oysters infected with P. marinus had a reduced number of apoptotic hemocytes. The parasite may prevent hemocyte apoptosis in order to yield a greater number of hemocytes in which to house itself. Large numbers of P. marinus cells in some infected oysters were eliminated via apoptosis in the stomach epithelia, disabling the spread of infectious particles through seawater. The oysters infected with H. nelsoni also had reduced numbers of apoptotic hemocytes, while part of the vesicular connective tissue cells were apoptotic. H. nelsoni plasmodia were eliminated via apoptosis in some oysters. Apoptosis may enhance progression and prevent transmission of infectious oyster diseases.     

奶牛乳腺上皮细胞系的建立及高温对细胞超微结构的影响

应用差酶消化法和反复贴壁法在体外建立奶牛乳腺上皮细胞培养方法,以细胞流式术、免疫组化、免疫印迹、超微结构观察等方法对乳腺上皮细胞特性进行检测,并研究高温热刺激对乳腺细胞超微结构的影响。实验结果表明,运用本方法建立的奶牛乳腺细胞系上皮特性及遗传特征完备;41℃1h的高温热刺激可使乳腺细胞染色质浓缩、线粒体肿胀、空泡化,形成凋亡小体,说明高温可以诱发乳腺细胞凋亡。     

奶牛乳腺上皮细胞系的建立及高温对细胞超微结构的影响

应用差酶消化法和反复贴壁法在体外建立奶牛乳腺上皮细胞培养方法,以细胞流式术、免疫组化、免疫印迹、超微结构观察等方法对乳腺上皮细胞特性进行检测,并研究高温热刺激对乳腺细胞超微结构的影响。实验结果表明,运用本方法建立的奶牛乳腺细胞系上皮特性及遗传特征完备;41℃ 1h 的高温热刺激可使乳腺细胞染色质浓缩、线粒体肿胀、空泡化,形成凋亡小体,说明高温可以诱发乳腺细胞凋亡。     

Herpes Simplex Virus Type 1 Renders Infected Cells Resistant to Cytotoxic T-Lymphocyte-Induced Apoptosis

Many viruses interfere with apoptosis of infected cells, presumably preventing cellular apoptosis as a direct response to viral infection. Since cytotoxic T lymphocytes (CTL) induce apoptosis of infected cells as part of the “lethal hit,” inhibition of apoptosis could represent an effective immune evasion strategy. We report here herpes simplex virus type 1 (HSV-1) interference with CTL-induced apoptosis of infected cells and show that HSV-1 inhibits the nuclear manifestations of apoptosis but not the membrane changes. The HL-60 cell line (human promyelocytic leukemia) undergoes apoptosis in response to many stimuli, including incubation with ethanol. After HSV-1 infection (strains E115 and 17+), ethanol-treated cells did not produce oligonucleosomal DNA fragments characteristic of apoptosis, as assayed by gel electrophoresis and enzyme-linked immunosorbent assay. Inhibition was detected 2 h after infection and increased over time. Importantly, HSV-1-infected cells were resistant to apoptosis induced by antigen-specific CD4⁺ CTL, despite the fact that CTL recognition and degranulation in response to infected targets remained intact. Unlike HSV-1, HSV-2 (strains 333 and HG52) did not inhibit DNA fragmentation. In contrast to the inhibition of DNA fragmentation by HSV-1, none of the HSV-1 or -2 strains interfered with the ethanol-induced exposure of surface phosphatidylserine characteristic of apoptosis, as determined by annexin V binding. These results demonstrate that genes of HSV-1 inhibit the nuclear manifestations of apoptosis but not the membrane manifestations, suggesting that these may be mediated via separate pathways. They also suggest that HSV-1 inhibition of CTL-induced apoptosis may be an important mechanism of immune evasion.     

Morphological apoptotic characteristics of the post-meiotic micronuclei in Paramecium caudatum

In a previous study, the apoptotic degeneration of meiotic products outside the paroral region of Paramecium caudatum was indirectly demonstrated by means of “apofluor” staining. In this experiment, conjugating pairs and exconjugants of P. caudatum were stained with either “apofluor” or carbol fuchsin or both to find some direct evidence to demonstrate the apoptotic characteristics of this process. As a result, asynchronous meiotic nuclear degeneration was observed. Furthermore, a number of additional meiotic nuclei were found. Disintegrating/dividing meiotic nuclei outside the paroral region were observed, which might be the origin of these additional meiotic nuclei. Condensed chromatin and disintegrated chromatin attached to the nuclear membrane were also observed in degenerating nuclei, which are the typical morphological characteristics of apoptosis. Comparison of the cells stained by the above two methods indicated that “apofluor”-stained meiotic nuclei could not be detected by carbol fuchsin in some cells, which suggests a time lag between meiotic nuclear DNA degradation and their eventual disappearance. In this study, some direct evidence was found to show that the meiotic nuclear degeneration in P. caudatum is of apoptotic nature, which further confirmed our previous study (Yang et al. 2007) and indicated that morphological apoptotic characteristics discovered in multicellular organisms do exist in unicellular eukaryotic ciliate protozoa.     

褐飞虱若虫中肠凋亡细胞的检测

为揭示褐飞虱Niloparvata lugens Stl若虫在发育过程中中肠的凋亡细胞,使用末端脱氧核苷酸转移酶介导的dUTP-生物素断端标记法(TUNEL)进行中肠组织切片检测,结果表明,1～5龄若虫中肠分别存在2%～5%的凋亡细胞。利用4′,6-二脒基-2-苯基吲哚二盐酸(DAPI)染色法检测表明,存在Ⅰ,Ⅱa和Ⅱb期凋亡的细胞核,其特征包括染色体浓缩、边缘化及细胞核碎裂。透射电子显微镜检测结果表明,早期凋亡的细胞呈现染色质浓缩、边缘化特征,晚期凋亡的细胞出现细胞核碎裂、形成凋亡小体及细胞质空泡化等。本研究揭示了在正常发育过程中褐飞虱若虫中肠有少量的细胞发生了凋亡。通过人工干预的方式调控中肠细胞的凋亡进程有可能使之成为防治该水稻害虫的新靶标。     

Herpes simplex virus blocks apoptosis by precluding mitochondrial cytochrome c release independent of caspase activation in infected human epithelial cells

Expression of HSV-1 genes leads to the induction of apoptosis in human epithelial HEp-2 cells but the subsequent synthesis of infected cell protein prevents the process from killing the cells. Thus, viruses unable to produce appropriate prevention factors are apoptotic. We now report that the addition of either a pancaspase inhibitor or caspase-9-specific inhibitor prevented cells infected with an apoptotic HSV-1 virus from undergoing cell death. This result indicated that HSV-1-dependent apoptosis proceeds through the mitochondrial apoptotic pathway. However, the pancaspase inhibitor did not prevent the release of cytochrome c from mitochondria, implying that caspase activation is not required for this induction of cytochrome c release by HSV-1. The release of cytochrome c was first detected at 9 hpi while caspase-9, caspase-3 and PARP processing were detected at 12 hpi. Finally, Bax accumulated at mitochondria during apoptotic, but not wild type HSV-1 infection. Together, these findings indicate that HSV-1 blocks apoptosis by precluding mitochondrial cytochrome c release in a caspase-independent manner and suggest Bax as a target in infected human epithelial cells.