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.     

受体介导内吞对巨噬细胞膜电位、胞浆和溶酶体pH的影响

本文利用荧光标记方法测定了刀豆素Ａ、麦芽凝集素、酵母多糖刺激引起的巨噬细胞膜电位、胞浆ｐＨ溶酶体ｐＨ的变化。结果显示三种配体均导致细胞膜电位超极化,胞浆ｐＨ降低、溶酶体ｐＨ或高,三个生理参量趋于稳定时间稍有不同。胞浆ｐＨ的降低可能有抑制内吞的作用,溶酶体ｐＨ上升是触发溶酶体内容物外排的基本因素。内吞引起的这些变化是细胞代谢过程中自我调节和保护的表现。     

UVB诱导NIH 3T3细胞凋亡时的信号转导机制:Ⅱ.神经酰胺所致细胞内Ca2+和pH的变化

利用粘附式细胞仪 (ACAS -570)结合相应的荧光探针分别测定了外源性神经酰胺诱导NIH3T3细胞凋亡时胞浆游离Ca2 水平和UVB照射NIH3T3细胞所致细胞内 pH的变化以及神经酰胺的生成对这一变化的影响。结果表明 :1.神经酰胺能够导致NIH3T3细胞胞浆游离Ca2 升高 ;胞浆Ca2 升高既来源于胞外钙 ,又来源于胞内钙池 ,但外钙内流是引起和维持胞内Ca2 处于高水平的必要条件 ;NIH3T3细胞钙池上存在着两种受体 :IP3 受体和Ryanodine受体 ,其中IP3受体较占优势。2.UVB照射导致NIH3T3细胞凋亡时胞浆碱化并持续约2小时左右恢复正常 ,pH的变化参与了细胞凋亡的过程并受到神经酰胺生成的调控。这可能是UVB照射启动了磷脂肌醇通路激活磷脂酶C ,导致神经酰胺的生成、Ca2 动员和蛋白激酶C的活化 ,从而激活Na /H 对流引起胞浆碱化。所以 ,胞浆游离Ca2 的增加和 pH的升高不是两个孤立的事件。     

A549/DDP细胞的抗凋亡特性与其pHi和[Ca2+]i变化相关

以l临床用药剂量(30μmol/L)的顺铂处理敏感的人肺腺癌细胞A549和抗顺铂的A549/DDP细胞,在相同条件下培养.对培养不同时间的两株细胞DNA琼脂糖凝胶电泳分析的结果表明,前者在培养12h后即呈现DNA梯子,而后者在培养48h后却未见凋亡特征.用流式细胞计检测其凋亡峰也获得相似的结果.当测定与凋亡相关的生物化学和生物物理变化时发现对顺铂敏感的A549细胞的线粒体膜电势显著降低,胞内更加酸化且胞内钙离子浓度升高;而抗顺铂药物的A549/DDP细胞的线粒体膜电势和pH值却维持在相对较高的水平,而其胞内钙离子浓度随培养时间的延长下降.这些结果表明,抗顺铂的人肺腺癌A549/DDP细胞的抗凋亡特性与其胞内的相对碱化和较高的线粒体膜电势,以及胞内钙离子浓度的降低相关.这些特性可能参与了A549/DDP的顺铂耐药性的调节.     

A549/DDP细胞的抗凋亡特性与其pHi和［Ca2+］i变化相关

以临床用药剂量(30 μmol/L)的顺铂处理敏感的人肺腺癌细胞A549和抗顺铂的A549/DDP细胞,在相同条件下培养.对培养不同时间的两株细胞DNA琼脂糖凝胶电泳分析的结果表明，前者在培养12 h后即呈现DNA梯子，而后者在培养48 h后却未见凋亡特征.用流式细胞计检测其凋亡峰也获得相似的结果.当测定与凋亡相关的生物化学和生物物理变化时发现对顺铂敏感的A549细胞的线粒体膜电势显著降低，胞内更加酸化且胞内钙离子浓度升高；而抗顺铂药物的A549/DDP细胞的线粒体膜电势和pH值却维持在相对较高的水平，而其胞内钙离子浓度随培养时间的延长下降.这些结果表明，抗顺铂的人肺腺癌A549/DDP细胞的抗凋亡特性与其胞内的相对碱化和较高的线粒体膜电势,以及胞内钙离子浓度的降低相关.这些特性可能参与了A549/DDP的顺铂耐药性的调节.     

小鼠巨噬细胞膜快速超极化和凋亡

 利用激光扫描共聚焦显微技术、流式细胞术、TUNEL染色技术、FRAP技术等对大剂量地塞米松诱导小鼠腹腔巨噬细胞凋亡过程中膜通透性、膜脂流动性、膜电位等膜生物物理性状改变进行了研究 .结果显示 ,大剂量地塞米松诱导小鼠腹腔巨噬细胞快速凋亡 .凋亡巨噬细胞膜脂流动性升高 ,尤为显著的是 ,膜电位快速超极化 ,胞浆游离 Ca2 + 加速超极化 .结果表明 ,细胞膜电位变化与巨噬细胞这一非兴奋细胞凋亡密切相关     

巨噬细胞吞噬过程中膜磷脂流动性和受体流动性的关系

本文用ＦＲＡＰ（ｆｌｕｏｒｅｓｃｅｎｃｅｒｅｃｏｖｅｒｙａｆｔｅｒｐｈｏｔｏｂｌｅａｃｈｉｎｇ）技术,测量了静息状态和刀豆素Ａ刺激不同时间后巨噬细胞膜磷脂、ＣｏｎＡ受体扩散系数和荧光恢复率的变化。结果显示ＣｏｎＡ刺激后膜磷脂和ＣｏｎＡ受体的扩散系数和荧光恢复率均较静息状态的巨噬细胞明显降低,磷脂流动性的变化与ＣｏｎＡ受体流动性的变化呈正相关。提示受体介导内吞导致的膜磷脂流动性的降低,可能是由于配体与细胞膜上受体结合形成配体－受体复合体,增加了受体的负荷,使受体的流动性降低,进而使膜磷脂的流动性降低。巨噬细胞内吞过程中膜磷脂和ＣｏｎＡ受体流动性的降低,可能还与ＣｏｎＡ刺激后巨噬细胞胞浆ｐＨ值有关。     

穿透支原体LAMPs诱导NF-kB激活介导小鼠巨噬细胞凋亡

研究穿透支原体(Mpe)脂质相关膜蛋白(LAMPs)能否诱导小鼠巨噬细胞凋亡,并阐明其可能的分子机制,以了解Mpe潜在的致病性.用Annexin-V-FITC凋亡检测试剂盒和DNA Ladder方法检测Mpe LAMPs诱导体外培养的小鼠巨噬细胞系Raw264.7细胞的凋亡.以间接免疫荧光和Western blotting方法检测经Mpe LAMPs处理的小鼠巨噬细胞NF-κB的激活和NF-kB抑制剂吡咯啉烷二甲基硫脲(PDTC)对细胞凋亡的影响.结果表明:Mpe LAMPs能诱导小鼠巨噬细胞发生早期或晚期凋亡;Mpe LAMPs能诱导激活小鼠巨噬细胞的NF-κB,使其从细胞浆中转位到细胞核内;PDTC能显著地抑制经处理的小鼠巨噬细胞的NF-κB的激活,且能抑制Mpe LAMPs诱导的巨噬细胞发生凋亡.因此,Mpe LAMPs诱导小鼠巨噬细胞凋亡可能与NF-kB的激活有关,因而Mpe可能是一个重要的致病因素.     

ATP对巨噬细胞内吞和溶酶体pH的影响

ＦＩＴＣ－ｄｅｘｔｒａｎ标记培养的小鼠腹腔巨噬细胞溶酶体,ＣｏｎＡ－ＦＩＴＣ标记细胞内吞。用激光扫描共聚焦显微镜测量伴刀豆球蛋白（ＣｏｎＡ）、ＡＴＰ引起的巨噬细胞溶酶体ｐＨ动态变化和ＡＴＰ对细胞内吞ＣｏｎＡ－ＦＩＴＣ的影响。结果显示ＣｏｎＡ引起巨噬细胞溶酶体ｐＨ迅速增加,６ｍｉｎ左右达到峰值（ｐＨ５．７）;ＡＴＰ刺激３０ｍｉｎ后再加入ＣｏｎＡ,溶酶体ｐＨ无明显变化（ｐＨ４．０）;同时加入ＡＴＰ和ＣｏｎＡ,５ｍｉｎ左右溶酶体ｐＨ降到最低点（ｐＨ４．１）;ＡＴＰ对巨噬细胞内吞ＣｏｎＡ－ＦＩＴＣ有明显的抑制作用。探讨了受体介导内吞与溶酶体ｐＨ的关系。     

UVB诱导NIH3T3细胞凋亡时的信号转导机制:Ⅱ.神经酰胺所致细胞内Ca~(2+)和pH的变化

利用粘附式细胞仪（ＡＣＡＳ－５７０）结合相应的荧光探针分别测定了外源性神经酰胺诱导ＮＩＨ３Ｔ３细胞凋亡时胞浆游离Ｃａ＾２＋水平和ＵＶＢ照射ＮＩＨ ３Ｔ３细胞所致细胞内ＰＨ的变化以及神经酰胺的生民对这一变化的影响。结果表明,１．神经酰胺能够导致ＮＩＨ ３Ｔ３细胞胞浆游离Ｃａ＾２＋升高既来源于胞外叠为源于胞内钙池,但外钙内流是引起和维持胞内Ｃａ＾２＋处于高水平所必要条件,ＮＩＨ ３Ｔ３细胞也上存在着两     

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。     

Intracellular alkalinization in dexamethasone-induced thymocyte apoptosis

Glucocorticoid can induce apoptosis of thymocytes, but its mechanism is not clear yet. In this study, we reported that dexamethasone-induced apoptosis was associated with intracellular alkalinization. Dexamethasone induced a higher percentage of apoptosis in 138 mM than in 50 mM NaCl, total abrogation of apoptosis was noted in NaCl-depleted culture medium. Highest apoptotic rate was observed in medium with pH 7.2, whereas it was partially and completely inhibited at pH 6.5 and pH 6.0, respectively. Intracellular pH was higher in pre-apoptotic thymocytes than non-apoptotic ones. The Na+ /H+ antiporter inhibitor of 5-(N,N'-dimethyl)-amiloride inhibited the dexamethasone-induced increase in pHi and apoptosis of thymocytes. Glucocorticoid antagonist RU486 also blocked the dexamethasone-induced effect. Furthermore, the apoptosis and increase in intracellular pH induced by dexamethasone were inhibited by cycloheximide, actinomycin D. It seems that intracellular pH is increased during the development of thymocyte apoptosis and inhibiting its increment would retard the rate of progression to cell death.     

线粒体PT孔参与甘草诱导MGC-803细胞凋亡的调控

不久前我们从中药中首次筛选发现了甘草能显著诱导胃癌MGC-803细胞凋亡,本文进一步研究甘草诱导MGC-803细胞凋亡过程中凋亡百分率、线粒体膜电位、胞内游离钙、DNA电泳和细胞膜通透性以及染色质DNA凝聚的时相变化,并研究了线粒体PT孔专一抑制剂环孢菌素A(CsA)对凋亡过程的影响.我们观察到,细胞膜通透性增强、胞内游离钙升高和线粒体膜电位下降为细胞凋亡的早期事件,先于凋亡峰出现、染色质凝聚和DNA电泳梯状条带出现,CsA明显抑制线粒体膜电位下降,细胞膜通透性增强和胞内游离钙变化,并极大程度地延迟细胞凋亡过程.结果提示,钙和CsA敏感性的线粒体PT孔开放参与甘草提取物诱导MGC-803细胞凋亡的调控.     

大鼠小脑缺血再灌注损伤的细胞凋亡及尼莫通的保护作用

采用四血管闭塞法制作全脑缺血再灌动物模型, 再灌后４８ 小时取小脑, 石蜡包埋切片。应用末端转移酶介导的缺口末端标记法原位检测到小脑皮质及小脑核有阳性反应的凋亡细胞, 表明细胞凋亡是迟发性神经元损伤的主要形式。缺血前３０ 分钟给以尼莫通能有效地减少细胞凋亡, 尼莫通对小脑缺血再灌注损伤有显著性保护作用     

地塞米松诱导培养的大鼠大脑皮质星形胶质细胞凋亡

目的　研究地塞米松诱导纯化培养的大鼠大脑皮质星形胶质细胞凋亡的作用。方法　不同浓度的地塞米松(浓度为 10 -3 、 10 -4、 10 -5mol/L)与纯化培养的大鼠大脑皮质星形胶质细胞共同孵育 18小时后 ,吖啶橙染色荧光显微镜观察细胞凋亡形态学改变 ,流式细胞仪检测细胞凋亡和结晶紫比色法酶标仪测定活细胞数。结果　 (1)吖啶橙染色荧光显微镜观察 :10 -4组偶见细胞凋亡 ,10 -3 组可见许多细胞有典型的凋亡形态学改变核固缩 ,深染 ,或肿胀 ,碎裂 ,并可见凋亡小体。 (2 )流式细胞仪检测细胞凋亡 :10 -3 组细胞凋亡率为 15 99% ,与其它三组相比明显增高 ,有显著性差异 (P <0 0 1)。 (3)结晶紫法酶标仪测定活细胞数 :10 -3 组OD值为 0 . 185与其它三组相比明显下降 ,有显著性差异 (P <0 0 1) ,表明活细胞数明显减少。结论　大剂量地塞米松可诱导体外的星形胶质细胞凋亡。     

Intracellular water motion decreases in apoptotic macrophages after caspase activation

Triggering of the macrophage cell line RAW 264.7 with lipopolysaccharide and interferon-gamma promoted apoptosis that was prevented by inhibitors of type 2 nitric oxide synthase or caspase. Using (1)H NMR analysis, we have investigated the changes of the intracellular transverse relaxation time (T(2)) and apparent diffusion coefficient (ADC) as parameters reflecting the rotational and translational motions of water in apoptotic macrophages. T(2) values decreased significantly from 287 to 182 ms in cells treated for 18 h with NO-donors. These changes of T(2) were prevented by caspase inhibitors and were not due to mitochondrial depolarization or microtubule depolymerization. The decrease of the intracellular values of T(2) and ADC in apoptotic macrophages was observed after caspase activation, but preceded phosphatidylserine exposure and nucleosomal DNA cleavage. The changes of water motion were accompanied by an enhancement of the hydrophobic properties of the intracellular milieu, as detected by fluorescent probes. These results indicate the occurrence of an alteration in the physicochemical properties of intracellular water during the course of apoptosis.     

Lithium inhibits growth of intracellular Mycobacterium kansasii through enhancement of macrophage apoptosis

Mycobacterium kansasii (Mk) is an emerging pathogen that causes a pulmonary disease similar to tuberculosis. Macrophage apoptosis contributes to innate host defense against mycobacterial infection. Recent studies have suggested that lithium significantly enhances the cytotoxic activity of death stimuli in many cell types. We examined the effect of lithium on the viability of host cells and intracellular Mk in infected macrophages. Lithium treatment resulted in a substantial reduction in the viability of intracellular Mk in macrophages. Macrophage cell death was significantly enhanced after adding lithium to Mk-infected cells but not after adding to uninfected macrophages. Lithium-enhanced cell death was due to an apoptotic response, as evidenced by augmented DNA fragmentation and caspase activation. Reactive oxygen species were essential for lithium-induced apoptosis. Intracellular scavenging by N-acetylcysteine abrogated the lithium-mediated decrease in intracellular Mk growth as well as apoptosis. These data suggest that lithium is associated with control of intracellular Mk growth through modulation of the apoptotic response in infected macrophages.     

Effect of polyamines on superoxide dismutase activity under dexamethasone-induced apoptosis in rat thymocytes

The intracellular redox state is of importance for cell growth, differentiation, and apoptosis through reactive oxygen species (ROS) functioning as metabolic fine-tuner. Optimal levels of polyamines are necessary for growth, differentiation, and apoptotic cell death while they also protect cell from ROS accumulation. We have carried out studies to find out the interrelation between these two distant metabolic pathways. For that purpose, the glucocorticoid-triggered programmed cell death of rat thymocytes has been used. Our data confirm that SOD activity (which testifies both to the level of ROS generation and antioxidative defense state) changes in response to programmed cell death conditions and to alteration of intracellular polyamines level. Thymocytes death induced by dexamethasone is partially mediated by polyamines content. Our data prove that one of the molecular mechanisms of thymocytes population resistance after dexamethasone treatment is an enhanced level of antioxidant defense. It is evident that in dexamethasone-treated rat thymocytes polyamines modulate signal transduction processes to apoptosis development via changes in cellular redox status.