巨噬细胞凋亡及其调控

巨噬细胞通过介导和调控自身及其他细胞凋亡而实现其免疫调节和效应细胞功能.引起巨噬细胞凋亡的原因有生物、化学、病理、自身等因素.不仅巨噬细胞自身凋亡和凋亡调控有其特点,更为有趣的是,巨噬细胞可根据需要：介导或抑制自身凋亡;介导或抑制其他细胞凋亡;抑制自身凋亡,介导其他细胞凋亡.这可能是巨噬细胞在免疫调节,特别是肿瘤免疫中发挥重要作用的基础.     

细胞感染与细胞凋亡

本文叙述了多种细菌及其毒素感染后对淋巴细胞,巨噬细胞,中性粒细胞和单核细胞凋亡的影响,初步探讨了可能的机制及其意义。     

凋亡素2配体对放射线诱导95-D细胞凋亡的影响的研究

目的:探讨凋亡素2配体(Apo-2L)对放射线诱导肺癌95-D细胞凋亡的影响。方法:MTT法检测不同浓度凋亡素2配体在体外对肺癌95-D细胞的抑制率,将细胞分为4组,对照组、凋亡素2配体组、单纯照射组、凋亡素2配体+放射照射组,流式细胞仪检测各组凋亡率及细胞周期。结果:凋亡素2配体对95-D细胞的体外抑制作用明显,随着药物浓度的增大及时间的延长,抑制率明显增高(P0.05)。流式细胞术显示凋亡素2配体与放射线联用能够使95-D细胞的凋亡率提高,与单用凋亡素2配体组及单纯放疗组相比,凋亡率差异显著(P0.05)。结论:凋亡素2配体在体外具有抑制95-D细胞增殖的作用并能够促进细胞的凋亡,同时凋亡素2配体联合放射线可以明显提高肺癌95-D细胞的凋亡率。     

病原微生物与巨噬细胞凋亡

病原微生物与机体细胞间的相互关系一直是一热点问题,病原微生物之所以成为病原,一定有其成为病原的理由。不论是细菌、真菌、病毒,还是其代谢产物大都能诱导宿主细胞,特别是免疫系统中某些细胞(如巨噬细胞)凋亡,这也许为其抵抗机体免疫系统的免疫防御及免疫监视,以便在宿主体内生存,进而为大量繁殖开辟了一条道路。     

鸡胚巨噬细胞AcP酶变化及AcP酶与凋亡细胞的关系

本研究采用电镜及酶细胞化学的方法观察了鸡胚脾脏不同胚龄组巨噬细胞溶酶体酸性磷酸酶（AcP酶）的变化、凋亡实验组巨噬细胞及其AcP酶与凋亡细胞的关系。取10天、13天和17天鸡胚脾脏,按Gomori法显示AcP酶,各胚龄脾脏巨噬细胞AcP酶细胞化学反应阳性,按AcP酶染色阳性做溶酶体计数,结果显示随着胚龄的增加溶酶体数随之增加,尤以第17天组溶酶体数增加最为明显,所得数据经统计分析表明各胚龄组间溶酶体数的差异有统计学意义。凋亡实验组采用放线菌酮诱导15天鸡胚脾脏细胞凋亡,结果显示凋亡细胞为各类幼稚血细胞,以幼稚淋巴细胞为主。巨噬细胞未见凋亡,而是吞噬了大量的凋亡细胞和凋亡小体,AcP酶反应颗粒不仅出现在巨噬细胞的溶酶体、吞噬体,还见于高尔基复合体、内质网等。细胞AcP酶反应强度数字化结果表明：凋亡组酶活性显著高于对照组,差别有统计学意义,提示胚胎巨噬细胞在凋亡细胞出现时AcP酶活性增强,说明巨噬细胞吞噬和消化凋亡细胞或凋亡小体是通过AcP酶等活性物质来实现的。     

活性氧诱导细胞凋亡

细胞凋亡是细胞主动的衰老和死亡方式。细胞凋亡过程极其复杂,其生化机制还不十分清楚。用活性氧如H_2O_2、ONOO及脂质过氧化物等可直接诱导某些细胞发生凋亡,抗氧化剂对细胞凋亡有抑制作用。细胞内产生活性氧的部位主要是线粒体电子传递链、黄嘌呤—黄嘌呤氧化酶途径、磷脂酶A_2激活的花生四烯酸代谢途径及精氨酸-NO合成酶途径。外源性活性氧或通过内源性活性氧的生成,导致细胞内氧化还原状态的失衡,诱导某些基因的表达,引起凋亡。     

大剂量地塞米松快速高效诱导巨噬细胞凋亡

运用透射电镜、DNA琼脂糖凝胶电泳、流式细胞术、原位末端标记法(TUNEL)染色等技术,检测了大剂量地塞米松处理小鼠腹腔巨噬细胞的各种变化.结果显示,大剂量地塞米松处理的巨噬细胞发生胞体皱缩、染色质凝聚、胞质浓缩;TUNEL染色呈阳性;0.5 h后DNA凝胶电泳即呈梯状条带;流式细胞术作周期分析出现凋亡峰等明显的凋亡特征.并且随处理时间延长凋亡率升高.结果表明,大剂量地塞米松快速、高效诱导小鼠腹腔巨噬细胞凋亡.     

三氧化二砷诱导细胞凋亡对线粒体的影响

线粒体作为细胞能量的“动力工厂”,在细胞的生理过程中起着重要的作用,本文采用三氧化二砷（Ａｓ２ Ｏ３） 诱导ＭＧＣ－ ８０３ 细胞凋亡,通过罗丹明１２３ 和Ｈｏｅｃｈｅｓｔ３３２５８ 对细胞进行荧光标记,利用具有光子计数成像能力的超高灵敏度荧光显微镜观察凋亡细胞与未加入Ａｓ２ Ｏ３ 的对照组细胞的区别,发现Ａｓ２ Ｏ３ 在诱导细胞凋亡时可引起线粒体内膜电位差减小     

肿瘤光动力疗法诱导细胞凋亡机制研究进展

肿瘤光动力疗法（Photodynamic Therapy,PDT）是利用光敏剂分子接受光照后产生多种活性氧物质（reactive oxyger,ROS),使细胞结构和功能受到损伤,而导致细胞凋亡的一种独特的肿瘤治疗方法,已受到越来越多的重视。本文对近几年有关PDT诱导肿瘤细胞凋亡方面的研究进展作了综述性介绍。     

激光扫描共聚焦显微镜在激光照射诱导细胞凋亡研究中的应用

激光扫描共聚焦显微镜结合荧光探针以及荧光共振能量转移技术,已成为近年来应用在活细胞中研究大分子行为的一种非常有效的研究工具。本文介绍这一技术在激光照射诱导细胞凋亡的研究中的应用。     

脂多糖诱导植物细胞产生NO的光学分子成像检测

脂多糖（lipopolysaccharides, LPS）是革兰氏阴性细菌细胞壁的主要成分,被植物感知后,启动植物防御反应。利用荧光探针分子成像及激光共聚焦扫描显微镜技术,在位、直观检测了LPS诱导下,拟南芥细胞产生重要信号分子一氧化氮（ NO）的时空特征。LPS诱导细胞产生大量NO,这些NO主要定位在细胞膜周围,且是在LPS处理90 min后出现。NO合成酶抑制剂L-单甲基精氨酸能明显抑制LPS诱导的NO生成,说明LPS诱导NO产生是NO合成酶途径依赖的。该研究结果有助于深入理解LPS作用机制以及NO信号传导通路的全貌,并为生物物理技术在相关植物生理研究中的应用提供一定的借鉴作用。     

Tim4- and MerTK-Mediated Engulfment of Apoptotic Cells by Mouse Resident Peritoneal Macrophages

Apoptotic cells are swiftly engulfed by macrophages to prevent the release of noxious materials from dying cells. Apoptotic cells expose phosphatidylserine (PtdSer) on their surface, and macrophages engulf them by recognizing PtdSer using specific receptors and opsonins. Here, we found that mouse resident peritoneal macrophages expressing Tim4 and MerTK are highly efficient at engulfing apoptotic cells. Neutralizing antibodies against either Tim4 or MerTK inhibited the macrophage engulfment of apoptotic cells. Tim4-null macrophages exhibited reduced binding and engulfment of apoptotic cells, whereas MerTK-null macrophages retained the ability to bind apoptotic cells but failed to engulf them. The incubation of wild-type peritoneal macrophages with apoptotic cells induced the rapid tyrosine phosphorylation of MerTK, which was not observed with Tim4-null macrophages. When mouse Ba/F3 cells were transformed with Tim4, apoptotic cells bound to the transformants but were not engulfed. Transformation of Ba/F3 cells with MerTK had no effect on the binding or engulfment of apoptotic cells; however, Tim4/MerTK transformants exhibited strong engulfment activity. Taken together, these results indicate that the engulfment of apoptotic cells by resident peritoneal macrophages proceeds in two steps: binding to Tim4, a PtdSer receptor, followed by MerTK-mediated cell engulfment.     

干旱胁迫对玉米幼苗根系中NO信号转导的影响

以玉米(Zea mays L.)幼苗根系为材料,研究其生长发育对内外源NO的感应及在干旱条件下内外源NO对其生长发育的影响.结果表明:干旱抑制根系NO释放,NO的产生可能不依赖于NO合酶系统,而依赖于NADPH硝酸还原酶的酶促反应;用活性氧清除剂NAC、NAC与干旱复合处理均能促使内源NO的产生和释放:其根系生物量的分析表明内源NO的释放明显抑制根系的生长发育.     

Macrophages and Apoptotic cells in Human Colorectal Tumours

The numerical relationship between tumour associated macrophages (TAM) and apoptotic cells in 12 human colorectal tumours was evaluated. TAM were labelled immunohistochemically and apoptotic cells were visualized by counterstaining with haematoxylin and eosin (H&E). The stereological techniques. Cavalieri's estimator of volume and the Disector were used to estimate both tumour volume and numerical density of both cell types. The occurrence of TAM per unit volume of tissue increased with increasing tumour volume to a maximum in a tumour of 110·5 cm³, after which numbers declined. Levels of apoptosis also increased with tumour volume though more erratically than levels of TAM and declined for tumour volumes greater than 80 cm³. This is the first report of an attempt to assess the relationship between apoptotic cells and TAM in human tumours.     

Arginase II Downregulates Nitric Oxide (NO) Production and Prevents NO-mediated Apoptosis in Murine Macrophage-derived RAW 264.7 Cells

Excess nitric oxide (NO) induces apoptosis of some cell types, including macrophages. As NO is synthesized by NO synthase (NOS) from arginine, a common substrate of arginase, these two enzymes compete for arginine. There are two known isoforms of arginase, types I and II. Using murine macrophage-like RAW 264.7 cells, we asked if the induction of arginase II would downregulate NO production and hence prevent apoptosis. When cells were exposed to lipopolysaccharide (LPS) and interferon-γ (IFN-γ), the inducible form of NOS (iNOS) was induced, production of NO was elevated, and apoptosis followed. When dexamethasone and cAMP were further added, both iNOS and arginase II were induced, NO production was much decreased, and apoptosis was prevented. When the cells were transfected with an arginase II expression plasmid and treated with LPS/IFN-γ, some cells were rescued from apoptosis. An arginase I expression plasmid was also effective. On the other hand, transfection with the arginase II plasmid did not prevent apoptosis when a NO donor SNAP or a high concentration (12 mM) of arginine was added. These results indicate that arginase II prevents NO-dependent apoptosis of RAW 264.7 cells by depleting intracellular arginine and by decreasing NO production.     

气体NO处理对蒜苗生长和抗氧化酶活性的影响

以改良的蒜为材料,用不同浓度NO气体（0．1、0．5、1．0μmol·L^-1）在无氧环境中熏蒸大蒜3h后,检测蒜苗生长、光合色素和可溶性蛋白质含量以及抗氧化酶活性的结果表明：0．1和0．5μmol·L^-1NO气体熏蒸的蒜种长成的幼苗,叶中光合色素和可溶性蛋白含量、假茎长、株高和假茎粗均大于未经NO熏蒸的植株,叶中超氧化物歧化酶（SOD）、过氧化物酶（POD）和过氧化氢酶（CAT）的活性均提高;而1．0μmol·L^-1的NO则抑制蒜苗生长,且抗氧化酶活性亦下降。气态NO处理的鳞茎幼苗生长也得到促进,以0．5μmol·L^-1NO处理的效果最佳。     

Apoptotic and Autophagic Effects of Sesbania grandiflora Flowers in Human Leukemic Cells

Background

Identification of cytotoxic compounds that induce apoptosis has been the mainstay of anti-cancer therapeutics for several decades. In recent years, focus has shifted to inducing multiple modes of cell death coupled with reduced systemic toxicity. The plant Sesbania grandiflora is widely used in Indian traditional medicine for the treatment of a broad spectrum of diseases. This encouraged us to investigate into the anti-proliferative effect of a fraction (F2) isolated from S. grandiflora flowers in cancer cells and delineate the underlying involvement of apoptotic and autophagic pathways.

Principal Findings

Using MTT based cell viability assay, we evaluated the cytotoxic potential of fraction F2. It was the most effective on U937 cells (IC₅₀∶18.6 µg/ml). Inhibition of growth involved enhancement of Annexin V positivity. This was associated with elevated reactive oxygen species generation, measured by flow cytometry and reduced oxygen consumption – both effects being abrogated by anti-oxidant NAC. This caused stimulation of pro-apoptotic proteins and concomitant inhibition of anti-apoptotic protein expressions inducing mitochondrial depolarization, as measured by flow cytometry and release of cytochrome c. Interestingly, even with these molecular features of apoptosis, F2 was able to alter Atg protein levels and induce LC3 processing. This was accompanied by formation of autophagic vacuoles as revealed by fluorescence and transmission electron microscopy – confirming the occurrence of autophagy. Eventually, F2 triggered caspase cascade – executioners of programmed cell death and AIF translocation to nuclei. This culminated in cleavage of the DNA repair enzyme, poly (ADP-ribose) polymerase that caused DNA damage as proved by staining with Hoechst 33258 leading to cell death.

Conclusions

The findings suggest fraction F2 triggers pro-oxidant activity and mediates its cytotoxicity in leukemic cells via apoptosis and autophagy. Thus, it merits consideration and further investigation as a therapeutic option for the treatment of leukemia.     

红景天苷对LPS诱导的HSC-T6细胞增殖作用及机制研究

目的：观察红景天苷（Salidroside,Sal）对大鼠肝星状细胞（hepaticstellatecell,HSC）一T6增殖的影响,并探讨一氧化氮（NO）在此过程中的作用。方法：选择脂多糖（LPS）活化HSC—T6,采用不同浓度的Sal,作用于经LPS活化的大鼠HSC-T6,24小时后进行如下实验：噻唑兰（MTT）比色法检测HSC—T6增殖;NO荧光探针（DAF-FMDA）检测细胞内NO浓度;Westem-blot检测INOS蛋白水平。结果：在LPS作用于HSC．T6细胞24小时后,与对照组相比,细胞增殖增加（P〈0．01）,在Sal作用下,HSC-T6细胞增殖与LPS组相比受到抑制（P〈0．01）,并呈浓度依赖性;在Sal处理HSC．T6细胞24小时后,细胞内NO水平有所上升,并呈浓度依赖性增加（P〈O．01）;Sal预处理后的HSC—T6细胞,iNOS蛋白表达有所升高（P〈0．01）。结论：Sal对HSC—T6细胞的增殖具有浓度依赖性的抑制作用,NO可能是抑制增殖的因素之一。     

缺氧对世居高原藏族人脐静脉内皮细胞ET和NO水平的影响

目的: 观察世居高原藏族人静脉血ET和NO含量和缺氧对培养脐静脉内皮细胞ET和NO水平的影响,以期从胎儿生长发育的角度探讨人类高原适应的机制.方法: 分别以放射免疫分析法和Greiss法测定世居高原藏族、移居高原汉族和平原汉族人静脉血ET和NO含量.体外培养世居高原藏族和移居高原汉族新生儿UVECs,分为4组:①世居高原藏族人UVECs常氧组(TC);②世居高原藏族人UVECs缺氧组(TH);③移居高原汉族人UVECs常氧组(HC);④移居高原汉族人UVECs缺氧组(HH),收集培养上清液测定ET和NO含量.结果: 世居高原藏族人静脉血NO水平显著高于移居高原汉族人,而ET水平显著低于移居高原汉族人.体外低氧(0.5% O2)条件培养12 h和24 h时,TH组ET浓度显著低于HH组,而HH组与TH组和HC组与TC组相应时间点之间的NO浓度差异无显著性意义.结论: 缺氧时世居高原藏族人UVECs ET分泌增高的程度较低,可能有助于保持相对低的血管张力,有利于胎儿血液供应.     

Effects of exogenous NO supplied with different approaches on cadmium toxicity in lettuce seedlings

The study aimed to test the effects of sodium nitroprusside [SNP, a nitric oxide (NO) donor], supplied with different approaches on cadmium (Cd) toxicity in lettuce seedlings (Lactuca sativa) in a pot experiment. SNP (8.94 mg) was applied into Cd-contaminated soil directly or added into a capsule, a paper bag, starch-coated granules, or foliar application. Cd (50 mg kg^? 1) reduced chlorophyll content, caused oxidative stress, increased Cd accumulation in roots and leaves, and inhibited the uptake of calcium (Ca), magnesium (Mg), and iron (Fe). The addition of exogenous NO in Cd-contaminated soil increased chlorophyll content, improved antioxidant enzyme activities, promoted the uptake of Ca, Mg, and Fe, reduced Cd-induced oxidative damages, and inhibited Cd transferred from roots to shoots. Moreover, SNP supplied with different approaches had varied effects on Cd tolerance of lettuce seedlings. The alleviated effect of SNP applied into soil directly was the worst, and the three SNP slow release materials had better alleviation effects on Cd toxicity. Foliar SNP application had the best effects on increasing Cd tolerance in lettuce seedlings.