猕猴早期胚胎脑的发育与凋亡相关蛋白P53的表达

目的探讨猕猴早期胚胎脑的发育及细胞凋亡相关蛋白P53的表达.方法建立猕猴早孕模型,获取早期胚胎,采用单克隆抗体链霉素亲生物蛋白过氧化酶 (SP)免疫组织化学方法,检测P53蛋白的表达.结果在猕猴25d、40d和55d胚胎脑均检测到P53免疫反应阳性神经细胞,随着胎龄的增加,P53免疫反应阳性表达率逐渐增大.结论在早期猕猴胚胎脑发育的不同时期内P53蛋白均有表达,并在胚胎脑发育过程中表达逐渐增加,调控脑的发育.     

小鼠胚胎癌细胞来源的分化细胞合成IGF-Ⅱ

胰岛素样生长因子-Ⅱ(IGF-Ⅱ)是一种促有丝分裂多肽,对哺乳类胚胎生长和发育起着重要作用。然而,在早期胚胎发育阶段,有关这种生长因子的产生、功能及其对胚胎细胞生长、分化的调节作用所知甚少。本文用纯化大鼠8.5 KDa增殖刺激活性因子(multiplicationstimulating activity,MSA)作免疫原,制备了多克隆抗体。以同位素在体外代谢标记PC 13小鼠多能胚胎癌(EC)细胞及其经维生素A酸(RA)诱导分化的内胚层样END细胞,在分化的PC 13 END细胞培液中检测到与IGF-Ⅱ抗体起免疫沉淀的物质,而未分化的PC 13 EC细胞并不分泌这种物质,证明了从EC细胞来源的分化细胞能合成和分泌IGF-Ⅱ蛋白。经SDS-聚丙烯酰胺凝胶电泳分析,这种免疫沉淀物质为分子量35 KDa和14-15 KDa的三种形式蛋白。35 KDa分子可能是一种IGF_Ⅱ前体蛋白或具IGF-Ⅱ专一性的载体蛋白。用免疫酶染色方法,在另一株多能B 7-2 EC克隆细胞中,同样证明经环六亚甲基双乙酰胺(HMBA)诱导分化的内胚层样细胞的胞质中染有与IGF-Ⅱ抗体起反应的物质;而且在HMBA诱导处理24小时即已出现这种染色反应。随着药物处理时间延长,除了细胞形态进一步呈梭形和细胞质突起更显著外,免疫反应强度未见有明显变化,表明IGF-Ⅱ的合成在EC细胞诱导分化的较早阶段就开始。根据这些结果,推测早期胚胎发育中内胚层细胞可能产生和分泌IGF-Ⅱ蛋白。     

小鼠早期胚胎发育过程中细胞凋亡及凋亡基因表达的检测

小鼠早期胚胎发育过程中凋亡现象大量存在,细胞凋亡与凋亡基因表达有关。应用彗星电泳法检测小鼠早期胚胎凋亡情况;应用巢式RT-PCR、免疫组化的方法检测了Bcl-2家族成员(Bax、Bcl-2、Bak、Bcl-xl)的表达变化情况。结果显示:随着胚胎细胞数目的增加,凋亡比率逐渐增大;Bax表达量在整个过程中基本不变,Bcl-2表达量逐渐上调,Bak、Bcl-xl的表达量逐渐降低。对小鼠早期胚胎发育过程中的基因表达研究对于揭示早期胚胎发育的机制有重大的意义。     

小鼠早期胚胎内细胞凋亡的一个量化模型

凋亡是早期胚胎（八细胞阶段后,特别是桑椹胚至囊胚期）进入非线性发育期的最重要诱因和调节因素．本文利用双光子激光扫描显微术,获得小鼠植入前胚胎的三维荧光图像;然后建立量化模型,经过一定图像处理,统计了凋亡小体在胚胎内的三维空间分布,并为探索凋亡的动力学机制做出一些尝试．研究发现,胚胎内细胞凋亡现象绝大部分（70％）发生在滋养外胚层,且胚胎质心与几何中心的偏离在32细胞期前后出现台阶式跃迁,这和囊胚腔的出现导致胚胎细胞分布对称性破缺的理论预测一致．     

胚胎神经管缺陷大鼠模型差异基因的表达

通过构建妊娠合并糖尿病诱发先天性神经管缺陷的SD大鼠模型, 与胚胎不伴有先天性神经管缺陷组大鼠和正常对照组大鼠胚胎进行研究, 提取卵黄囊细胞的mRNA, cDNA 基因芯片技术对表达差异基因进行检测, 应用特异性抗磷酸化抗体进行免疫共沉淀及Western blotting, 对卵黄囊细胞MAP Kinase信号途径蛋白激酶活性进行分析。在神经管缺陷大鼠胚胎卵黄囊细胞和对照组1 200个基因中, 共筛选出表达差异基因79个, 其中42个基因表达上调、37个基因表达下调。同时发现神经管缺陷胚胎卵黄囊细胞出现细胞凋亡特征性的DNA ladder(梯状电泳), 凋亡相关基因 caspase-3、Bax 高表达, 凋亡抑制基因 AKT活性明显受抑; 与正常对照组相比ERK1/2蛋白激酶活性显著下降、JNK1/2活性明显升高。因此, 认为妊娠合并糖尿病诱发胚胎先天性神经管缺陷的发生存在多种差异基因表达, 以及MAP Kinase、凋亡信号传导机制的共同作用。     

热激蛋白对细胞凋亡的调节作用

细胞凋亡是生物发育过程中或在正常生理状态下清除衰老及受损细胞的一种普遍现象。细胞凋亡的发生受胞外或胞内的多种刺激源所诱导,其中热激蛋白是细胞凋亡的调控因子之一。本文着重讨论了热激蛋白在细胞凋亡调节中所发挥的作用。     

热休克蛋白（HSPs）在胚胎发育中作用的研究进展

在胚胎发育,特别是早期胚胎发育时期,基因转录活跃、蛋白质大量合成,细胞的增殖和分化非常剧烈,细胞的环境在不断变化,细胞对外界刺激十分敏感。这个时期的HSPs变化和作用表现得非常突出。本文简要总结了近十年有关热休克蛋白在胚胎发育中作用的研究成果。根据胚胎发育地HSPs的依赖性、HSPs基因表达的发育阶段特异性和组织特异性、干扰胚胎发育中HSPs表达程序以后导致胚胎异常发育等现象推测：（1）热休克基因与和／功调控体形形成、肌肉及神经分化,而在胚胎发育中具有看家基因的功能;（2）热休克蛋白作为伴侣分子,通过介导新合成的和／或可逆变性的蛋白质正确折叠、装配、转动及促进不需要的和／或不可逆变性的蛋白质降解,参与胚胎的正常发育和保护胚胎不受不良刺激的影响。这方面的深入研究,必将有助于阐明胚胎发育、细胞增殖、细胞分化和去分化、细胞转化、生物适应性等的分子机理。     

Ghrelin在绵羊体内卵母细胞和早期胚胎的表达

为了明确ghrelin是否参与了卵母细胞成熟及胚胎早期发育进程,本研究利用免疫荧光技术和实时定量RT-PCR技术检测了绵羊卵母细胞和体内早期胚胎中ghrelin蛋白的表达定位和ghrelin mRNA水平相对表达变化规律。免疫荧光染色结果表明,ghrelin蛋白主要分布于卵母细胞胞质内;实时定量RT-PCR结果揭示绵羊卵母细胞和早期胚胎ghrelin mRNA的相对表达量依据发育阶段的不同而呈现一定变化规律,即在成熟卵母细胞,2细胞胚胎期和8细胞胚胎期显著高于未成熟卵母细胞和4细胞胚胎期(P<0.05),囊胚期表达量最高。卵母细胞和早期胚胎中ghrelin蛋白的表达及ghrelin mRNA特定的表达模式,揭示这一新型分子在绵羊卵母细胞成熟以及胚胎早期发育过程中具有潜在的调控作用。     

sTRAIL基因引起人恶性畸胎瘤细胞与人胚胎干细胞凋亡比较

目的:评估sTRAIL基因引起人胚胎干细胞[human embryonic stem(hES)cells]和人恶性畸胎瘤(human malignant teratoma)细胞NTERA-2凋亡的作用。方法:本室已构建的pAAV-PEG3-sTRAIL质粒,转染NTERA-2和hES细胞,用流式细胞仪和定量PCR检测NTERA-2和hES细胞的早期凋亡及sTRAIL基因的受体的表达。结果:NTERA-2细胞经瞬时转染pAAV-PEG3-sTRAIL质粒后,早期凋亡比例达24.4%±2.1%,sTRAIL的死亡受体DR4、和DR5的表达分别上升2和3倍,而hES细胞转染pAAV-PEG3-sTRAIL质粒后,早期凋亡比例仅2.3%±1.2%,受体DR4、DR5、DcR1和DcR2的表达未有明显变化。结论:pAAV-PEG3-sTRAIL质粒可有效地引起人恶性畸胎瘤细胞NTERA-2的凋亡,但是对人胚胎干细胞尚未发现可见的细胞凋亡现象。     

猕猴胚胎干细胞的诱导分化和凋亡

采用单层培养法研究维生素A酸(RA)、神经生长因子(NGF)、上皮生长因子(EGF)和碱性成纤维生长因子(bFGF)对猕猴胚胎干细胞系R366．4的诱导分化和凋亡的作用。结果表明：①不添加任何生长因子的条件下,细胞分化不定向,各种细胞所占的比例表现出明显的随机性;②添加单一生长因子能促进细胞的分化进程,并使某一类或某几类的分化细胞比例上升,RA和NGF均能促进神经样细胞的形成,EGF促进内皮样细胞的形成,bFGF提高成纤维样细胞的比例;③在分化的过程中伴有细胞早期和晚期凋亡的发生,RA和NGF可增加细胞凋亡的数量。这种由生长因子诱导的动物胚胎干细胞的分化可能存在种间差异。     

Apoptosis in lens development and pathology

The ocular lens is a distinct system to study cell death for the following reasons. First, during animal development, the ocular lens is crafted into its unique shape. The crafting processes include cell proliferation, cell migration, and apoptosis. Moreover, the lens epithelial cells differentiate into lens fiber cells through a process, which utilizes the same regulators as those in apoptosis at multiple signaling steps. In addition, introduction of exogenous wild-type or mutant genes or knock-out of the endogenous genes leads to apoptosis of the lens epithelial cells followed by absence of the ocular lens or formation of abnormal lens. Finally, both in vitro and in vivo studies have shown that treatment of adult lens with stress factors induces apoptosis of lens epithelial cells, which is followed by cataractogenesis. The present review summarizes the current knowledge on apoptosis in the ocular lens with emphasis on its role in lens development and pathology.     

Superinfection-induced apoptosis and its correlation with the reduction of viral progeny in cells persistently infected with Hz-1 baculovirus.

Differential induction of necrosis or apoptosis was found upon challenge of cells of the insect Spodoptera frugiperda productively or persistently infected with Hz-1 baculovirus, respectively. Unlike parental SF9 cells, which were essentially all killed by virally induced necrosis, persistently infected cells underwent a process of massive cell death by apoptosis; cells which were not killed by apoptosis then reestablished a cell monolayer. Upon viral challenge, the yield of viral progeny was reduced greatly in persistently virus-infected cells but not in parental cells. Immunolabelling of individual cells revealed that upon viral challenge, production of viral progeny was detectable only in necrotic cells and not in apoptotic cells. These results indicated that induction of apoptosis greatly reduces the yield of viral progeny in cells persistently infected with Hz-1 baculovirus. This is the first report of apoptosis induction in persistently infected cells upon viral superinfection.     

Apoptosis may contribute to false-positive results in the in vitro micronucleus test performed in extreme osmolality, ionic strength and pH conditions

Conditions in which clastogens produce positive responses have been increasingly challenged, and several situations have been described in which clastogenic responses would be considered not to be relevant. For example, extreme culture conditions lead to high variations of pH, osmolality or ionic strength. Apoptosis is induced in extreme culture conditions and contributes to false-positive results in the in vitro micronucleus test performed with CTLL-2 cells. These cells can enter apoptosis when exposed to apoptosis stimuli or after IL-2 deprivation, whereas the CTLL-2 Bcl2 cell line is protected from apoptosis due to the over-expression of the apoptosis inhibitor Bcl2 in bcl2-transfected CTLL-2 cells. The two cell lines were treated in extreme culture conditions of either pH or osmolality or were submitted to high ionic strength. The apoptosis level was measured in parallel with the in vitro micronucleus test using the annexin V-FITC method. Data obtained in the two cell lines suggested that apoptosis caused by extreme culture condition induces the formation of micronucleated cells, which leads to false-positive results in the in vitro micronucleus test.     

DNA synthesis precedes gliotoxin-induced apoptosis

The toxin gliotoxin induces apoptosis or programmed cell death in a variety of immune cells including thymocytes. Apoptosis induced by gliotoxin in thymocytes is unaffected by protein synthesis inhibitors nor is it associated with early changes in intracellular calcium levels (Beaver and Waring, 1994). This work shows that the cell lines P815 and WEHI7 and murine thymocytes when treated with gliotoxin show an early incorporation of tritiated thymidine over the concentration range which causes apoptosis. Proliferating cell nuclear antigen (PCNA), a marker for S phase, is elevated in cells following gliotoxin treatment and S phase DNA content is increased. Thymidine incorporation is inhibited by hydroxyurea, an inhibitor of replicative DNA synthesis not repair. Free radical scavangers have no effect on apoptosis induced by gliotoxin in thymocytes. Hydrogen peroxide-treated cells showed no enhanced thymidine incorporation and no apoptosis. Thus oxidative stress does not appear to be a factor in gliotoxin-induced apoptosis. Thymocytes treated with gliotoxin show increased phosphorylation of a 16.3 kDa protein, and apoptosis is inhibited by the tyrosine kinase inhibitor genistein, which also inhibited the increased thymidine incorporation in P815 cells. We conclude that one mechanism by which gliotoxin can cause apoptosis may be the induction of inappropriate entry of cells into the cell cycle followed by death.     

Distinct activation signals determine whether IL-21 induces B cell costimulation, growth arrest, or Bim-dependent apoptosis

IL-21 costimulates B cell proliferation and cooperatively with IL-4 promotes T cell-dependent Ab responses. Somewhat paradoxically, IL-21 also induces apoptosis of B cells. The present study was undertaken to more precisely define the expression of the IL-21R, using a novel mAb, and the circumstances by which IL-21 promotes B cell growth vs death. The IL-21R was first detected during T and B cell development, such that this receptor is expressed by all mature lymphocytes. The IL-21R was further up-regulated after B and T activation, with the highest expression by activated B cells. Functional studies demonstrated that IL-21 substantially inhibited proliferation and induced Bim-dependent apoptosis for LPS or CpG DNA-activated B cells. In contrast, IL-21 induced both costimulation and apoptosis for anti-CD40-stimulated B cells, whereas IL-21 primarily costimulated B cells activated by anti-IgM or anti-IgM plus anti-CD40. Upon blocking apoptosis using C57BL/6 Bim-deficient or Bcl-2 transgenic B cells, IL-21 readily costimulated responses to anti-CD40 while proliferation to LPS was still inhibited. Engagement of CD40 or the BCR plus CD40 prevented the inhibitory effect by IL-21 for LPS-activated B cells. Collectively, these data indicate that there are three separable outcomes for IL-21-stimulated B cells: apoptosis, growth arrest, or costimulation. We favor a model in which IL-21 promotes B cell maturation during a productive T cell-dependent B cell response, while favoring growth arrest and apoptosis for nonspecifically or inappropriately activated B cells.     

Cell cycle specificity of apoptosis during treatment of leukaemias

This review summarizes our observations on the mechanism of induction of apoptosis in vitro in leukaemic cell lines and in vivo in patients with leukaemia undergoing chemotherapy, in relation to the cell cycle. Multiparameter flow cytometric methods allowed us to identify apoptotic cells and position them with respect to their cell cycle phase. Several antitumor agents of different classes have been characterized in terms of the cell cycle phase specificity of induction of apoptosis. Three types of apoptosis could be distinguished in relation to the initial damage to the cell vis-a-vis cell cycle position: (1) homo-phase apoptosis where the cells underwent apoptosis during the same phase in which they were initially affected; (2) homo-cycle apoptosis, where the cells underwent apoptosis during the same cell cycle in which they were initially affected, i.e., prior to or during the first mitosis, and (3) post-mitotic apoptosis, where cells underwent apoptosis during the cell cycle(s) subsequent to that in which the cell was initially affected, most likely at the G1 or G2 checkpoints of these cycle(s). Four ranges of drug concentration can be distinguished in vitro for most drugs, where either: (1) no immediate effects; (2) cytostasis or post-mitotic apoptosis; (3) homo-cycle or homo-phase apoptosis; or (4) necrosis are observed. Analysis of cell death of blast cells from peripheral blood or bone marrow of over 250 leukaemia patients (AML, ALL, CML in blast crisis) treated with various drugs during routine chemotherapy reveals that in the case of DNA topoisomerase inhibitors (e.g., mitoxantrone, VP-16) apoptosis is often rapid (peaks at 1-2 days after drug administration) and has features of homo-phase apoptosis. In contrast, cell death observed after administration of paclitaxel (taxol) or cytarabine (cytosine arabinoside) occurs later and has features of post-mitotic apoptosis: the cells divide but die in G1 of the subsequent cycle(s).     

Computerized video time-lapse microscopy studies of ionizing radiation-induced rapid-interphase and mitosis-related apoptosis in lymphoid cells

Computerized video time-lapse (CVTL) microscopy of X-irradiated cultures of cells of the murine lymphoma cell lines ST4 and L5178Y-S and the human lymphoid cell line MOLT-4 demonstrated that these cells exhibit a wide disparity in the timing of induction and execution of radiation-induced cell death that included rapid-interphase apoptosis, delayed apoptosis, and postmitotic apoptosis. ST4 cells that received 2.5 or 4 Gy of X radiation underwent rapid-interphase apoptosis within 2 h. Apoptosis commenced with a 10-20-min burst of membrane blebbing followed by swelling for 2-4 h and cell collapse. No apoptotic bodies were formed. After a dose of 1 Gy, approximately 90% of ST4 cells died by rapid-interphase apoptosis, while the remainder completed several rounds of cell division prior to cell death. Postmitotic death of ST4 cells occurred with the same morphological sequence of events as during rapid-interphase apoptosis induced by doses of 1-4 Gy. In contrast, L5178Y-S and MOLT-4 cells that received 4 Gy underwent apoptosis more slowly, with a complex series of events occurring over 30-60 h. Only 3% of L5178Y-S cells and 24% of MOLT-4 cells underwent apoptosis without attempting cell division. The cells became abnormally large during a long G(2)-phase delay, and then most of the cells (76-97%) attempted to divide for the first or second time at approximately 18-30 h postirradiation. However, either mitosis failed or division was aberrant; i.e., the large cells divided into three or four fragments which eventually fused together. This process was followed by several rounds of complex and unpredictable membrane blebbing, gross distortions of shape, fragmentation-refusion events, and formation of apoptotic bodies, after which the cells collapsed at 36-60 h postirradiation.     

Apoptosis may contribute to false-positive results in the in vitro micronucleus test performed in extreme osmolality, ionic strength and pH conditions

Conditions in which clastogens produce positive responses have been increasingly challenged, and several situations have been described in which clastogenic responses would be considered not to be relevant. For example, extreme culture conditions lead to high variations of pH, osmolality or ionic strength. Apoptosis is induced in extreme culture conditions and contributes to false-positive results in the in vitro micronucleus test performed with CTLL-2 cells. These cells can enter apoptosis when exposed to apoptosis stimuli or after IL-2 deprivation, whereas the CTLL-2 Bcl2 cell line is protected from apoptosis due to the over-expression of the apoptosis inhibitor Bcl2 in bcl2-transfected CTLL-2 cells. The two cell lines were treated in extreme culture conditions of either pH or osmolality or were submitted to high ionic strength. The apoptosis level was measured in parallel with the in vitro micronucleus test using the annexin V-FITC method. Data obtained in the two cell lines suggested that apoptosis caused by extreme culture condition induces the formation of micronucleated cells, which leads to false-positive results in the in vitro micronucleus test.     

Association of Nuclear-Localized Nemo-Like Kinase with Heat-Shock Protein 27 Inhibits Apoptosis in Human Breast Cancer Cells

Nemo-like kinase (NLK), a proline-directed serine/threonine kinase regulated by phosphorylation, can be localized in the cytosol or in the nucleus. Whether the localization of NLK can affect cell survival or cell apoptosis is yet to be disclosed. In the present study we found that NLK was mainly localized in the nuclei of breast cancer cells, in contrast to a cytosolic localization in non-cancerous breast epithelial cells. The nuclear localization of NLK was mediated through direct interaction with Heat shock protein 27 (HSP27) which further protected cancer cells from apoptosis. The present study provides evidence of a novel mechanism by which HSP27 recognizes NLK in the breast cancer cells and prevents NLK-mediated cell apoptosis.