高温诱导HeLa细胞凋亡的相关机理的研究

目的研究不同高温作用后HeLa细胞bcl-2、bax和p53表达的变化.方法人子宫颈癌细胞系(HeLa细胞)分为37℃、40℃、43℃三个温度组,每组经1h相应的温度处理后,以免疫组织化学的方法检测bcl-2、bax和p53的表达并经图象分析仪检测反应产物的光密度并进行统计分析.结果 40℃、43℃组的Bcl-2的平均光密度明显低于37℃组,而43℃组Bax的平均光密度明显高于37℃组和40℃组,P53随着温度的升高平均光密度也随之升高.形态学观察:43℃组均见明显的细胞凋亡.结论温和性高温使HeLa细胞周期受阻,P53表达上调诱导bax基因表达、抑制bcl-2基因的表达,从而导致细胞凋亡.     

痘苗病毒载体的特点及应用

痘苗病毒具有宿主范围广,安全,高效表达,插入外源基因的容量大等诸钦优点,是继反转录病毒,腺病毒载体之后又一广泛应用的载体系统。本文对痘苗病毒载体的特点及其应用作了综述。     

重组人凋亡诱导因子基因的构建、表达及其对HeLa细胞的促凋亡作用

凋亡诱导因子（apoptosis-inducing factor, AIF）定位于细胞的线粒体膜间隙.当凋亡信号刺激时,AIF分子从线粒体释放到胞浆,然后转位到细胞核内,引起染色体核周边凝集和DNA呈大片段断裂（～50 kb）.用RT-PCR法分段克隆得到人全长AIF基因,经改造截去其N端线粒体定位信号编码序列,代之以不同长度的绿脓杆菌外毒素(PE)转膜结构域序列.把这些重组基因克隆入pIRES2-EGFP真核表达载体,脂质体法转染HeLa细胞,通过荧光显微镜观察、共聚焦显微镜观察、电镜观察等方法检测了多种重组人AIF基因的表达及其对细胞生长的影响.证明了重组人AIF基因的表达可引起HeLa细胞死亡,为肿瘤的杀伤提供了新的策略.     

口蹄疫病毒诱导宿主细胞凋亡的研究

本文报道了口蹄疫病毒（Foot-and-Mouth Disease Virus,FMDV）在体外诱导PK－15细胞凋亡的研究结果,采用Hoechst33258荧光探针、DNA凝胶电泳、脱氧核糖核酸转移酶介导的制品末端标记（TUNEL）技术均检测到了典型的细胞凋亡,结果显示：使用感染性滴度为4.8lgTCID50/mL的口蹄疫病毒感染PK－15细胞,在培养32h后,荧光探针检测呈现典型的凋亡细胞核固缩和梅花状碎裂核,并伴随有凋亡小体出现,调亡率约为20％;DNA凝胶电泳显示ladder梯带;末端标记检测到强绿色荧光标记物结合于凋亡细胞核上。研究结果提示：口蹄疫病毒可以在体外诱导宿主细胞凋亡,细胞凋亡是其致细胞病变死亡的重要途径之一。     

口蹄疫病毒诱导宿主细胞凋亡的研究

本文报道了口蹄疫病毒（Foot-and-Mouth Disease Virus,FMDV）在体外诱导PK-15细胞凋亡的研究结果。采用Hoechst33258荧光探针、DNA凝胶电泳、脱氧核糖核酸转移酶介导的缺口末端标记（TUNEL）技术均检测到了典型的细胞凋亡。结果显示使用感染性滴度为4.8lgTCID50/mL的口蹄疫病毒感染PK-15细胞,在培养32?h后荧光探针检测呈现典型的凋亡细胞核固缩和梅花状碎裂核,并伴随有凋亡小体出现,凋亡率约为20%;DNA凝胶电泳显示ladder梯带;末端标记检测到强绿色荧光标记物结合于凋亡细胞核上。研究结果提示口蹄疫病毒可以在体外诱导宿主细胞凋亡,细胞凋亡是其致细胞病变死亡的重要途径之一。     

大黄素提高HeLa细胞对三氧化二砷促凋亡敏感性的研究

活性氧(reactive oxygen species,ROS)在三氧化二砷(arsenic trioxide,As2O3)诱导肿瘤细胞凋亡中扮演重要角色。本研究用一种天然蒽醌类物质——大黄素(emodin)作为提高HeLa细胞ROS水平的手段,考察其对As2O3促凋亡敏感性的影响,并探究可能涉及的信号传导机制。结果显示大黄素10μmol／L提高ROS并增加了HeLa细胞在As2O32μmol／L作用下的凋亡率,对正常成纤维细胞却无影响。该联合作用可以促进HeLa细胞线粒体跨膜电位降低;抑制转录因子NF-κB激活。本研究提示：大黄素通过提高ROS介导凋亡信号传导的增强和生存信号传导的抑制,增加HeLa细胞对As2O3促凋亡的敏感性。     

蛋白酶体抑制剂MG132诱导K562和HeLa细胞凋亡程度存在明显差异

蛋白酶体抑制剂MG132诱导人白血病细胞K562和宫颈癌细胞HeLa凋亡,用3个不同浓度的蛋白酶体抑制剂MG132处理人白血病细胞K562和宫颈癌细胞HeLa,通过MTT检测、annexin Ⅴ/ PI 双染法、流式细胞术、酶标仪和Western 印迹分别检测MG132对K562细胞和HeLa细胞的生长效应、细胞凋亡率、细胞内活性氧(ROS)水平和caspase-3活性变化的影响.蛋白酶体抑制剂MG132诱导K562细胞凋亡明显,对HeLa细胞诱导凋亡不明显.结果表明,蛋白酶体抑制剂MG132特异性诱导不同肿瘤细胞凋亡的程度存在明显差异.     

鲍曼不动杆菌Omp34经线粒体途径诱导HeLa细胞凋亡

[目的]构建鲍曼不动杆菌(Acinetobacter baumannii)外膜蛋白34(outer membrane protein 34,Omp34)的表达载体pcDNA3.1/myc-His-Omp34,研究Omp34引起HeLa细胞凋亡的机制。[方法]PCR扩增目的基因Omp34,将其克隆至载体pcDNA3.1/myc-His;菌液PCR和测序筛选阳性克隆;将pcDNA3.1/myc-His-Omp34转染HeLa细胞;反转录PCR和Western Blot检测Omp34在HeLa细胞中的表达;CCK8实验检测细胞增殖抑制率;JC-1探针检测线粒体跨膜电位;透射电镜观察HeLa细胞线粒体结构,Western Blot鉴定HeLa细胞线粒体凋亡相关蛋白。[结果]成功构建pcDNA3.1/myc-His-Omp34真核表达载体;并且Omp34可抑制HeLa细胞增殖,引起线粒体损伤及跨膜电位崩溃,导致促凋亡蛋白Bax和Bad表达升高,抗凋亡蛋白Bcl-2和Bcl-XL表达降低。[结论]成功构建pcDNA3.1/mycHis-Omp34表达载体,并证明Omp34可经线粒体途径导致HeLa细胞凋亡。     

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     

痘苗病毒毒力测定及其免疫血清的制备

目的测试痘苗病毒毒力,制备痘苗病毒免疫血清,为药物评价和病毒检测奠定基础。方法鸡胚绒毛尿囊膜培养痘苗病毒,测定病毒的TCID50和小鼠毒力。将痘苗病毒悬液稀释成100TCID550,0．2％福尔马林灭活,分别在0、7、14d以腹腔注射的方式免疫BALB／c小鼠,用IFA、IEA、ELISA方法评价血清的敏感性和特异性。结果痘苗病毒TCID50／0．05mL=1．8×10^4,小鼠LD50／0．2mL=10^6.8;制备的免疫血清经IFA法测定效价为1：320,IEA法测血清效价为1：160,ELISA测血清效价1：6400。结论确定的细胞和小鼠毒力将为药物测定提供基础比对数据;制备的痘苗病毒免疫血清具有高度的敏感性和特异性,可作为测试对照血清使用。     

Production of Modified Vaccinia Ankara Virus by Intensified Cell Cultures: A Comparison of Platform Technologies for Viral Vector Production

Modified Vaccinia Ankara (MVA) virus is a promising vector for vaccination against various challenging pathogens or the treatment of some types of cancers, requiring a high amount of virions per dose for vaccination and gene therapy. Upstream process intensification combining perfusion technologies, the avian suspension cell line AGE1.CR.pIX and the virus strain MVA-CR19 is an option to obtain very high MVA yields. Here the authors compare different options for cell retention in perfusion mode using conventional stirred-tank bioreactors. Furthermore, the authors study hollow-fiber bioreactors and an orbital-shaken bioreactor in perfusion mode, both available for single-use. Productivity for the virus strain MVA-CR19 is compared to results from batch and continuous production reported in literature. The results demonstrate that cell retention devices are only required to maximize cell concentration but not for continuous harvesting. Using a stirred-tank bioreactor, a perfusion strategy with working volume expansion after virus infection results in the highest yields. Overall, infectious MVA virus titers of 2.1–16.5 × 10⁹ virions/mL are achieved in these intensified processes. Taken together, the study shows a novel perspective on high-yield MVA virus production in conventional bioreactor systems linked to various cell retention devices and addresses options for process intensification including fully single-use perfusion platforms.     

UV-Induced Apoptosis in Resistant HeLa Cells

Recently, apoptosis (genetically programmed cell death) induced by UV hasbeen documented in some cell culture models. However, the significance ofapoptosis in UV-induced cytotoxicity and resistance is uncertain. In thisstudy, we investigated the induction of apoptosis in HeLa cells and itsrole in acquired UV-resistance. The membrane receptor Fas was induced toassembly, and its immediate downstream target, caspase-8, was induced byUV in a dose- and time-dependent manner. Caspase-10, another possiblecandidate for forming the death-inducing signaling complex with Fas, wasalso activated in a dose- and time-dependent manner. There was significantactivation of caspase 9, 3 and 2 by UV. The apoptotic pathways appeared tobe normal in acquired UV-resistant HeLa cells. In addition, there was a UVdose-dependent induction of chromatin condensation in both parental andUV-resistant cells. However, resistant cells displayed significant reductionin chromatin condensation at lower doses. Inhibition of caspase-3 activation byspecific inhibitor significantly reduced the chromatin condensation in bothcell types, and unexpectedly, the difference between the two cell lines wascompletely eradicated, suggesting that the caspase-3 pathway plays asignificant role in reducing apoptosis in resistant cells. The resultsindicate that UV induces apoptosis by direct activation of apoptoticproteins in HeLa and resistant cells. Although resistant cells displayedpartial inhibition of UV-induced apoptosis through the caspase-3 pathway,there was no consistent difference in the activation of this and relatedcaspase-9 caspases compared to parental HeLa cells.     

人癌胚抗原-重组痘苗病毒的构建和制备

痘苗病毒的基因组庞大,结构复杂而特殊,不可能将外源基因直接插入它的基因组,必须利用一种特殊的痘苗病毒质粒,才能构建成功重组痘苗病毒．在分析了痘苗病毒质粒ｐＪ１２０〔含有我国天花疫苗－痘苗病毒天坛株７６１的启动子和胸苷激酶（ｔｈｙｍｉｄｉｎｅｋｉｎａｓｅ,简称ＴＫ基因）,及含有人癌胚抗原（ｃａｒｃｉｎｏｅｍｂｒｙｎｉｃａｎｔｉｇｅｎ,简称ＣＥＡ）ｃＤＮＡ全序列的质粒ｐ９１０２３Ｂ－ｃｅａ－１７结构的基础上,设计出三步法构建了重组疫苗病毒质粒ｐＪ－ＣＥＡ．经酶切及ＰＣＲ鉴定ｐＪ－ＣＥＡ中ＣＥＡｃＤ－ＮＡ的存在,进一步用同源重组方法构建了表达人ＣＥＡ的重组痘苗病毒,并以人体成纤维细胞作为宿主细胞,对ＣＥＡ－重组痘苗病毒进行了大量培养．再次证实痘苗病毒是良好的真核表达载体,可以高效而准确地表达细胞膜糖蛋白ＣＥＡ．     

柯萨奇病毒致病毒性心肌炎的细胞凋亡研究

病毒性心肌炎是心血管系统的常见病与多发病。对病毒引起的心肌细胞病理改变及机制的探讨一直是该领域的研究热点。自从科学家报道“凋亡”这种细胞死亡形式以来,为病毒性疾病研究者找到了一条新的研究途径。此后,许多国内外基础医学研究者通过不同方法对细胞受到病毒侵袭后的死亡形式进行研究。目前认为,病毒性心肌炎急性期机体心肌组织的确存在凋亡,     

The Evidence of HeLa Cell Apoptosis Induced with Tetraethylammonium Using Proteomics and Various Analytical Methods

Tetraethylammonium (TEA) is a potassium channel (KCh) blocker applied in the functional and pharmacological studies of the KChs. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, a colorimetric assay to quantitatively measure living cells, demonstrated that TEA reduced the HeLa cell viability dose-dependently. Flow cytometry analysis indicated an increased apoptosis rate of the HeLa cell after exposing to TEA. The patch clamp technique revealed that the K⁺ current of the HeLa cell was inhibited up to 80% when exposed to TEA. In addition, quantitative real-time PCR approach set up cross-talk among the cytotoxicity of TEA, 4-aminopyridine, and anti-cancer drug such as cisplatin. Using comparative proteomics combined with MALDI-TOF MS/MS, 33 significantly changed proteins were found from TEA treatment group; among these proteins, 12 were up-regulated, and 21 were down-regulated. Here we indicated that these proteins were closely connected with many biological functions such as oxidative stress response, signal transduction, metabolism, protein synthesis, and degradation. Both Western blotting and quantitative real-time PCR approaches further verified these differential proteins. Ingenuity Pathways Analysis software, a tool to analyze “omics” data and model biological system, was applied to analyze the interaction pathways of these proteins. The subcellular locations of the differential proteins are also predicted from Uniprot. All results above can help in our understanding of the mechanism of TEA-induced cytotoxicity and provide potential cancer biomarkers. Various experimental results in this study (like those for cisplatin) indicated that TEA is not only a KCh blocker but also a potential anti-cancer drug.     

人白介素6受体基因在痘苗病毒载体系统中的表达

人ＩＬ－６受体是一个在各种细胞上广泛表达的跨膜糖蛋白分子,是ＩＬ－６发挥细胞效应所必需的。本文通过将ＩＬ－６ＲｃＤＮＡ重组到痘苗病毒的ＴＫ基因中构建成重组痘苗病毒ＶＩＬ６Ｒ。细胞原位杂交和ＡＰＡＡＰ染色结果表明,感染ＶＩＬ６Ｒ后的Ｖｅｒｏ细胞中,ＩＬ－６Ｒ在ｍＲＮＡ和蛋白水平上均呈现较强的表达。Ｗｅｓｔｅｒｎｂｌｏｔ分析所表达的分子量为８０ｋＤ,表明所表达的产物是糖基化的。ＩＬ－６结合试验表明,表达的膜ＩＬ－６Ｒ能够结合ｒＩＬ－６,说明它是有功能的。利用ＶＩＬ６Ｒ免疫小鼠后,能够刺激较强的抗体产生。从而为进一步研究ＩＬ－６Ｒ的信号传导和构效关系提供了基础。