刚地弓形虫培养上清抑制THP-1细胞增殖及诱导凋亡的研究

摘要：目的提取弓形虫体外细胞共培养上清，并研究上清对人急性单核细胞白血病细胞THP-1增殖及凋亡的影响。方法收集对数生长期的THP-1细胞以5X10^7／ml细胞浓度接种于不同培养瓶中，对照组加入含10％胎牛血清的RPMll640，实验组加入相同体积不同数量（2×10^7／ml、4X10^7／ml、8×10^7／m1）弓形虫速殖子培养上清，采用四甲基氮噻唑蓝（MTY）法检测吸光度（A490值）并计算THP-1细胞增殖抑制率；倒置显微镜下观察细胞形态变化；Annexin-V-FITC／PI染色细胞后上流式细胞仪检测各个时间点细胞凋亡率变化，以Western印迹方法分析凋亡相关蛋白Bax、Bcl-2的表达或活性。结果MTY法检测结果弓形虫培养上清呈时间剂量依赖性抑制THP-1细胞株增殖，倒置显微镜下观察处理组细胞有发泡现象和凋亡小体出现。流式细胞仪检测弓形虫感染后的THP-1细胞凋亡率较对照组有升高趋势（P〈0．05），呈量效依赖性，Westernblot检测刚地弓形虫培养上清作用于THP-l细胞48h后实验组的Bax、Bcl-2蛋白表达较对照组的比值分别有明显的升高与降低（P〈0．05）。结论刚地弓形虫速殖子培养上清对体外培养THP-l细胞增殖有明显的抑制作用，并可诱导THP-1细胞凋亡。     

脂多糖致感染性脑水肿大鼠星形胶质细胞的活化和凋亡

目的通过观察脂多糖（Lipopolysaccharide，LPS）致大鼠感染性脑水肿后星形胶质细胞的凋亡、活化及iNOS表达情况，探讨在LPS致脑水肿中星形胶质细胞的作用。方法84只雄性SD大鼠，随机分为3组：空白对照纽（C组，n=12）；生理盐水对照纽（S组，n=12）；感染性水肿组（L组，n=60）。感染性脑水肿组又按颈内动脉注射脂多糖后6h、12h、24h、48h、72h分为5个亚组（n=12）。向颈内动脉注射脂多糖LPS150μg（0．15ml）建立大鼠感染性脑水肿模型。采用HE染色、流式细胞检测和免疫纽化染色分别观察脑组织病理改变、星形胶质细胞凋亡、胶质纤维酸性蛋白（GFAP）和iNOS表达情况。结果与C组和S组比较，L组大鼠星形胶质细胞胞体变大、突起增多；细胞内GFAP和iNOS表达增强，12h达高峰，除72h组，其余亚组均有统计学差异（P〈0．05）；各亚组星形胶质细胞凋亡增多（P〈0．05），24h凋亡最显著。C组和S纽比较无统计学意义（P〉0．05）。结论感染性脑水肿后星形胶质细胞凋亡增加，异常活化，分泌iNOS，参与感染性脑水肿的形成，在脑水肿的发生发展中发挥重要作用。     

胃癌侧群细胞耐药性研究及干细胞相关基因检测

目的研究胃癌侧群（Side Population，sP）细胞对化疗药物5-Fu（氟尿嘧啶）的耐药性及可能机制，并检测干细胞相关基因Nanog、Musashi-1及cD44的表达情况。方法选择人胃癌细胞株sGc-7901，以荧光染料H0echst 33342染色，维拉帕米桔抗对照，应用流式细胞仪分选sP细胞和nonsP细胞。细胞耐药实验比较sP细胞与nonsP细胞对化疗药物5．Fu的耐药性差异；westem_h10t检测ABcG2和bcl-2蛋白表达情况；流式细胞仪分析细胞周期；荧光定量PcR检测两组细胞中干细胞相关基因Nanog、Musashi-1及cD44mRNA的表达差异。结果胃癌细胞株sGc．790l中sP细胞的比例为2．8％，sP细胞对5-Fu的耐药存活率明显高于non-sP细胞（P〈0．05），与nonsP细胞相比，sP细胞高表达耐药蛋白ABcG2和抗凋亡蛋白bcl-2，有更多的细胞处于c0／G1期（P〈0．05），并高表达干细胞相关基因Musashi-1和cD44。结论胃癌sGC_7901细胞株中sP细胞对化疗药物5．Fu的耐药性明显高于nonsP细胞，其耐药机制可能与sP细胞高表达耐药蛋白ABcG2和抗凋亡蛋白bcl-2，有更多细胞处于G0／Gl期有关；Musashi-1和cD44可能是相对特异性的胃癌干细胞标志物。     

儿茶素对晚期糖基化终末产物诱导的内皮细胞凋亡的干预作用

目的探讨儿茶素对晚期糖基化终末产物（AGEs）诱导内皮细胞凋亡的干预作用。方法糖孵育法制备晚期糖基化终末产物，分离肾血管内皮细胞，将内皮细胞分成空白对照组、AGE组、浓度分别为10、15、20μg／ml的儿茶素组共五组。实验末，采用AlamarBlue还原法测定同内皮细胞增生活力，TUNEL法测定细胞凋亡率，生化法测定培养上清中·OH、MDA浓度，RT-PCR测定Bcl-2mRNA表达，Western-Blotting测定Bcl-2蛋白活性。结果与对照组相比，AGE组内皮细胞增生活性、Bcl-2基因与蛋白活性显著降低（P均〈0．01）；凋亡率、·OH与MDA浓度显著增加（P均〈0．01）；与AGE组相比，儿茶素各浓度组内皮细胞增生活力与Bcl-2基因与蛋白活性表达增高，凋亡率、·OH与MDA浓度降低；儿茶素各剂量组之间呈浓度梯度效应。结论儿茶素可降低AGEs引起的血管内皮细胞凋亡，其机制可能是通过有效清除活性氧自由基、上调Bcl-2mRNA与活性蛋白表达，阻断内皮细胞内过氧化物的堆积二种途径实现的。     

青蒿水提液对肺癌A549细胞的影响

目的研究青蒿水提液对肺癌A549细胞株增殖的影响和诱导凋亡的情况。方法不同浓度青蒿水提液作用于细胞不同时间，四甲基氮噻唑蓝（MTT）法检测吸光度值（A490nm）并计算增殖抑制率；AnnexinV-FITC／PI荧光染色后流式细胞仪检测细胞凋亡率；并以荧光显微镜观察细胞形态改变情况；蛋白质印迹法分析细胞凋亡相关蛋白Bax、Bcl-2的表这。结果青蒿水提液呈时间和剂量依赖性抑制A549细胞增殖；荧光显微镜下A549细胞出现不同时期凋亡特征性改变；流式细胞仪检测细胞凋亡率随着药物浓度增加而升高；A549细胞株的Bax蛋白表达量增多、Bcl-2蛋白表达量下降。结论青蒿水提液促进体外培养的A549细胞株增殖抑制并诱导凋亡，其机制可能与A549细胞Bax表达上调和Bcl-2表达下调有关。     

齐墩果酸抑制肝癌细胞QGY增殖和诱导凋亡的作用研究

目的研究齐墩果酸对人肝癌细胞QGY增殖的作用及与细胞内钙离子浓度（[Ca2＋]i）关系。方法将浓度分别为40、80、100μg／ml齐墩果酸作用肝癌H细胞QGY24h后，DAPI染色，以荧光显微镜观察细胞形态变化；以11组不同浓度齐墩果酸（5—400μg／ml）作用QGY细胞24h后，用四甲基偶氮唑蓝（Myr）法检测QGY增殖情况；分别以不同浓度齐墩果酸（80、100、120μg／ml）作用QGY细胞24h后，流式细胞仪检测细胞周期改变、细胞凋亡率和[Ca2＋]i。结果细胞增殖被抑制并发生凋亡：不同浓度齐墩果酸能够抑制QGY细胞株增殖，且在5—120μg／mL范围内呈剂量依赖性，药物作用细胞24h、48的Ic50分别为76．27μg／mL和66．56μg／mL；处理组细胞周期在s期产生阻滞、细胞内[Ca2＋]i较对照组显著增加，细胞凋亡率和[Ca2＋]i与药物浓度存依赖关系。结论齐墩果酸能够抑制肝癌细胞QGY增殖和诱导其凋亡；诱导凋亡可能与细胞内[Ca2＋]i增加有关。     

人结肠癌CW-2干细胞获取方法研究

目的运用3种不同的方法分离纯化人结肠癌CW-2干细胞，并对其分离纯化效率进行比较，探讨获得癌干细胞的有效方法。方法采用单纯无血清悬浮培养、无血清悬浮培养联合化疗药物、流式细胞分选技术分别富集人结肠癌细胞株CW-2干细胞；然后运用流式细胞术、NOD—SCID小鼠致瘤实验和Transwell侵袭实验分析比较3种方法的富集效率。结果无血清悬浮培养细胞．无血清悬浮培养联合化疗药物处理细胞和流式细胞仪分选技术分选后细胞中具有结肠癌干细胞特性的CD44＋EPCAM_细胞分别为（59．39±4．55）％、（74.36±6．78）％、（86．43±8．43）％；3群细胞的成瘤能力和侵袭能力都存在显著统计学差异（P值〈0．05）：流式细胞分选技术分选后细胞〉无血清悬浮培养联合化疗药物处理细胞〉单纯无血清悬浮培养细胞。结论流式细胞分选技术富集癌干细胞的能力强于单纯无血清悬浮培养和无血清悬浮培养联合化疗药物，无血清悬浮培养联合化疗药物又强于单纯无血清悬浮培养。     

早期母子隔离应激对大鼠学习记忆能力的影响

目的探讨早期母子隔离应激对幼鼠学习记忆能力的影响。方法30只SD新生大鼠用于实验，实验随机分成3组，正常对照纽、母子隔离15分钟纽、母子隔离3小时纽。在生后40天，三组大鼠进行Morris水迷宫及记录海马脑片长时程增强电位（LTP）实验。结果Morris水迷宫测试，与正常组比较母子隔离3h组大鼠寻找平台需要的潜伏期延长（P〈0．05），而母子隔离15分钟组大鼠寻找平台需要的潜伏期与正常组比较则无明显变化（P〉0．05）；海马脑片长时程增强电位提示：正常对照组条件刺激（CS）前后fEPSP斜率变化率为：64．7±22．9％；母子隔离3h组为：35．3±14．2％，低于正常组（P〈0．01）；母子隔离15min组为：66．3±29．7％，与正常组比较无显著性意义（P〉0．05）。结论幼鼠生后早期过度的应激导致大鼠的学习记忆能力减弱。     

darbufelone对胃癌SGC-7901细胞裸鼠移植瘤血管生成的影响

目的观察环氧合酶-2／5-脂氧合酶双重抑制剂darbufelone对人胃癌皮下移植瘤血管生成的影响，并初步探讨其机制。方法建立裸鼠实体瘤模型，随机分为darbufelone组和对照组，darbufelone及生理盐水分别连续灌服4周。测量肿瘤质量、体积，计算抑瘤率；免疫组化检测CD34并计算微血管密度；RT—PCR法及Western blot法分析移植瘤组织中MMP-9、VEGF的表达。结果darbufelone可明显抑制裸鼠移植瘤的生长，质量抑瘤率为58．42％，体积抑瘤率为67．13％。darbufelone组的微血管密度（MVD）（15．36±0．30）明显低于对照组（29．47±0．63）（P〈0．05）；darbufelone组肿瘤组织中VEGF及MMP-9在基因水平及蛋白水平的表达（P〈0．05）。结论darbufelone能有效抑制裸鼠移植瘤的生长，减少移植瘤组织中VEGF及MMP-9的表达，抑制肿瘤的微血管生成，具有抗血管生成的作用。     

金丝桃素抑制慢性病毒性心肌炎小鼠心肌细胞凋亡及Fas／FasL蛋白表达

目的观察金丝桃素对慢性病毒性心肌炎（VMC）小鼠心肌细胞凋亡及Fas／FasL蛋白表达的影响。方法Balb／C小鼠多次接种心肌柯萨奇段病毒复制慢性VMC模型，首次感染病毒45天后将存活小鼠分为模型组、金丝桃素组及氯沙坦组，同时设正常对照组．分别给予相应药物干预30天，采用原位末端标记法检测心肌细胞凋亡及免疫组化方法检测Fas／FasL蛋白表达。结果模型组心肌细胞凋亡率较正常组显著增加（P〈0．05），金丝桃素组和氯沙坦组凋亡率较模型组显著降低（P〈0．05）。模型组Fas／FasL蛋白表达较正常组显著增多（P〈0．01），金丝桃素组和氯沙坦组较模型组显著降低（P〈0．01）。结论金丝桃素保护心肌细胞与抑制VMC心肌细胞的凋亡．下调Fas／FasL蛋白表达有关。     

ADAMTS-2 functions as anti-angiogenic and anti-tumoral molecule independently of its catalytic activity

ADAMTS-2 is a metalloproteinase that plays a key role in the processing of fibrillar procollagen precursors into mature collagen molecules by excising the amino-propeptide. We demonstrate that recombinant ADAMTS-2 is also able to reduce proliferation of endothelial cells, and to induce their retraction and detachment from the substrate resulting in apoptosis. Dephosphorylation of Erk1/2 and MLC largely precedes the ADAMTS-2 induced morphological alterations. In 3-D culture models, ADAMTS-2 strongly reduced branching of capillary-like structures formed by endothelial cells and their long-term maintenance and inhibited vessels formation in embryoid bodies (EB). Growth and vascularization of tumors formed in nude mice by HEK 293-EBNA cells expressing ADAMTS-2 were drastically reduced. A similar anti-tumoral activity was observed when using cells expressing recombinant deleted forms of ADAMTS-2, including catalytically inactive enzyme. Nucleolin, a nuclear protein also found to be associated with the cell membrane, was identified as a potential receptor mediating the antiangiogenic properties of ADAMTS-2.     

Impaired apoptosis in lymphoblasts from Alzheimer’s disease patients: Cross-talk of Ca<Superscript>2+</Superscript>/calmodulin and ERK1/2 signaling pathways

We have analyzed the intracellular signals that allow lymphoblasts from Alzheimer’s disease (AD) patients to escape from serum deprivation-induced apoptosis. The following observations suggested that modulation of ERK1/2 activity by Ca²⁺/calmodulin (CaM) is involved in preventing apoptosis: (i) ERK1/2 activity seems to support lethality in control cells, as PD98059, the inhibitor of the activating MEK prevented cell death; (ii) control cells show a persistent and higher stimulation of ERK1/2 than that of AD cells in the absence of serum; (iii) CaM antagonists have no effects on control cells, but sensitize AD cells to death induced by serum withdrawal and increased ERK1/2 phosphorylation, and (iv) no apoptotic effects of CaM antagonists were observed in AD cells treated with PD98059. These results suggest the existence of an activation threshold of the ERK1/2 pathway setting by Ca²⁺/CaM-dependent mechanisms, which appears to be the critical factor controlling cell survival or death decision under trophic factor withdrawal. F. Bartolomé, N. de las Cuevas: These authors contributed equally to this work. Received 14 February 2007; received after revision 16 April 2007; accepted 23 April 2007     

Hyperthermia-induced apoptosis and the inhibition of DNA laddering by zinc supplementation and withdrawal of calcium and magnesium in suspension culture of tobacco cells

In the present paper we report examination of stereotypic hallmarks of apoptosis in heat-treated tobacco cells. Hyperthermia (44 °C, 4 h) caused apoptosis in 53.6% of cells when assayed 24 h after heat treatment. The induction of apoptosis by heat treatment was confirmed by flow cytometric assay. Cytological observations revealed condensation of the cytoplasm and nucleus, as well as nuclear collapse. DNA ladders were observed in DNA extracted from heat-treated cells, whereas DNA from control cells remained undegraded. The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay revealed that 51.8% of the heat-treated cells (44 °C, 4 h) show positive reaction after a 24-h recovery. When cells were cultured in a medium supplemented with 0.4–5.0 mM ZnSO₄, internucleosomal DNA fragmentation induced by heat shock was completely negated. Strikingly, when cells were cultured in Ca²⁺ and/or Mg²⁺ free medium for 44 h followed by heat treatment, DNA laddering was not observed. The results suggest hyperthermia-induced apoptosis and a correlation between the regula tion of endonucleases and heat shock signal in apoptotic tobacco cells. Received 17 September 1998; received after revision 4 January 1999; accepted 4 January 1999     

Celiac disease patient IgA antibodies induce endothelial adhesion and cell polarization defects via extracellular transglutaminase 2

We have recently found that celiac disease patient serum-derived autoantibodies targeted against transglutaminase 2 interfere with several steps of angiogenesis, including endothelial sprouting and migration, though the mechanism involved remained to be fully characterized. This study now investigated the processes underlying the antiangiogenic effects exerted by celiac disease patient antibodies on endothelial cells, with particular regard to the adhesion, migration, and polarization signaling pathway. We observed that celiac IgA reduced endothelial cell numbers by affecting adhesion without increasing apoptosis. Endothelial cells in the presence of celiac IgA showed weak attachment, a high susceptibility to detach from fibronectin, and a disorganized extracellular matrix due to a reduction of protein cross-links. Furthermore, celiac patient IgA led to secretion of active transglutaminase 2 from endothelial cells into the culture supernatants. Additionally, cell surface transglutaminase 2 mediated integrin clustering in the presence of celiac IgA was coupled to augmented expression of β1-integrin. We also observed that celiac patient IgA-treated endothelial cells had migratory defects and a less polarized phenotype when compared to control groups, and this was associated with the RhoA signaling pathway. These biological effects mediated by celiac IgA on endothelial cells were partially influenced but not completely abolished by R281, an irreversible extracellular transglutaminase 2 enzymatic activity inhibitor. Taken together, our results imply that celiac patient IgA antibodies disturb the extracellular protein cross-linking function of transglutaminase 2, thus altering cell-extracellular matrix interactions and thereby affecting endothelial cell adhesion, polarization, and motility.     

Comparative effects of ouabain, natriuretic factor and ammonium chloride in the toad urinary bladder

Electrical changes and direct effects on Na-K ATPase activity induced by an endogenous digitalis-like natriuretic factor (NF), NH4Cl and ouabain were studied in toad bladders. NF inhibited the SCC and the Na-K ATPase activity in a similar manner to ouabain, but induced a greater increase in calculated direct current resistance (R) (p less than 0.05). NH4Cl was a weak inhibitor of Na-K ATPase activity, although it produced steeper SCC inhibition slopes than those observed with ouabain or NF (p less than 0.01). The data suggested the same mechanism of action of NF and ouabain on the sodium pump, with an additional effect of the former on apical sodium permeability of the cells and/or closure of paracellular routes leading to an increased tissue resistance. In contrast, the effects of NH4Cl were mostly compatible with intracellular inhibition of apical sodium entry into the cell.     

Proliferation status defines functional properties of endothelial cells

Homeostasis of solid tissue is characterized by a low proliferative activity of differentiated cells while special conditions like tissue damage induce regeneration and proliferation. For some cell types it has been shown that various tissue-specific functions are missing in the proliferating state, raising the possibility that their proliferation is not compatible with a fully differentiated state. While endothelial cells are important players in regenerating tissue as well as in the vascularization of tumors, the impact of proliferation on their features remains elusive. To examine cell features in dependence of proliferation, we established human endothelial cell lines in which proliferation is tightly controlled by a doxycycline-dependent, synthetic regulatory unit. We observed that uptake of macromolecules and establishment of cell–cell contacts was more pronounced in the growth-arrested state. Tube-like structures were formed in vitro in both proliferating and non-proliferating conditions. However, functional vessel formation upon transplantation into immune-compromised mice was restricted to the proliferative state. Kaposi’s sarcoma-associated herpes virus (KSHV) infection resulted in reduced expression of endothelial markers. Upon transplantation of infected cells, drastic differences were observed: proliferation arrested cells acquired a high migratory activity while the proliferating counterparts established a tumor-like phenotype, similar to Kaposi Sarcoma lesions. The study gives evidence that proliferation governs endothelial functions. This suggests that several endothelial functions are differentially expressed during angiogenesis. Moreover, since proliferation defines the functional properties of cells upon infection with KSHV, this process crucially affects the fate of virus-infected cells.     

Inhibitors of phosphatidylinositol 3-kinase activity prevent cell cycle progression and induce apoptosis at the M/G1 transition in CHO cells

Phosphatidylinositol 3-kinase (PI3-kinase) activity has been implicated in regulating cell cycle progression at distinct points in the cell cycle by preventing cell cycle arrest or apoptosis. In this study, the role of PI3-kinase activity during the entire G1 phase of the ongoing cell cycle was studied in Chinese hamster ovary (CHO) cells synchronized by mitotic shake-off. We show that inhibition of PI3-kinase activity during and 2 h after mitosis inhibited cell cycle progression into S phase. In the presence of the PI3-kinase inhibitor wortmannin or LY294002, cells were arrested during early G1 phase, leading to the expression of the cleaved caspase-3, a central mediator of apoptosis. These results demonstrate that PI3-kinase activity is required for progression through the M/G1 phase. In the absence of PI3-kinase activity, cells are induced for apoptosis in this particular phase of the cell cycle. Received 7 September 2005; received after revision 26 October 2005; accepted 11 November 2005