地塞米松诱导小鼠胸腺细胞线粒体去极化与凋亡进程研究

目的研究地塞米松(DEX)诱导小鼠胸腺细胞凋亡进程中线粒体去极化作用特点。方法无菌获取Balb/c小鼠胸腺细胞,设对照组和DEX组;在5 h时点,利用Annexin V-FITC/PI双染流式细胞术检测细胞凋亡;利用DiOC6(3)/PI双染流式细胞术检测细胞线粒体去极化与死亡;利用DiOC6(3)/Annexin V-PE双染流式细胞术检测凋亡过程中的去极化现象。结果在1×10-6mol/L DEX诱导下,小鼠胸腺细胞在5 h凋亡百分率为(36.20±5.11)%,对照组为(4.10±0.98)%,差异显著(P<0.01);DEX组坏死百分率为(4.07±0.24)%,对照组为(1.25±0.25)%,差异显著(P<0.01)。DEX组线粒体去极化增强仍存活的细胞所占百分率为(46.77±6.21)%,显著高于对照组的(12.80±4.55)%(P<0.01)。DEX组线粒体去极化增强且已启动凋亡的细胞为(35.34±4.19)%,显著高于对照组的(7.21±0.61)%(P<0.01)。DEX组线粒体去极化作用增强未凋亡的细胞为(13.68±1.27)%,显著高于对照组的(6.85±0.92)%(P<0.01)。结论DEX诱导小鼠胸腺细胞发生典型细胞凋亡和线粒体去极化增强,在该进程中,线粒体去极化增强发生在细胞膜磷脂酰丝氨酸外翻之前。     

依托泊苷通过线粒体信号通路释放细胞色素C诱导Jurkat白血病细胞凋亡

目的：研究在人类Jurkat白血病细胞株中依托泊苷诱导凋亡的分子机制，揭示由依托泊苷启动的凋亡信号通路。 方法：分别用annexin V-FITC和碘化丙啶（PI）染色，通过流式细胞仪测定annexin V阳性和出现亚二倍体DNA的凋亡细胞。以3,3＇-dihexyloxyacarbocyanine iodide ［DiOC6(3)］为染色剂，采用流式细胞术检测细胞线粒体膜电位的变化。采用离心技术分离细胞的胞浆与线粒体。细胞色素c从线粒体转入胞浆，caspase-3的激活，多聚二磷酸腺苷核糖聚合酶（PARP）的切割等蛋白质的表达由免疫印迹技术（Western blotting）检测。 结果：依托泊苷诱导Jurkat白血病细胞凋亡，细胞凋亡与依托泊苷的作用时间呈线性关系。广谱的caspase抑制剂zVAD.fmk可抑制依托泊苷诱导的DNA片段化和磷脂酰丝氨酸外翻。依托泊苷引起的线粒体膜电位下降早于DNA片段化和磷脂酰丝氨酸外翻，形成明显对照的是zVAD.fmk不能阻断依托泊苷诱导的线粒体膜电位的下降。依托泊苷介导细胞色素c从线粒体释放到胞浆，激活caspase-3，caspase-3的底物PARP被切割。 结论：依托泊苷诱导Jurkat白血病细胞株凋亡的机制是降低线粒体膜电位和释放细胞色素c到细胞浆启动线粒体信号转导通路, 最终激活caspase而导致细胞凋亡。     

地塞米松介导胸腺细胞凋亡过程中线粒体和细胞结构蛋白的变化

目的：研究地塞米松(DEX)介导的小鼠胸腺细胞凋亡过程中线粒体质量和结构蛋白变化特点。 方法： 以地塞米松（DEX）诱导小鼠胸腺细胞凋亡为模型，利用Annexin V-FITC/PI双染流式细胞术研究细胞凋亡和坏死，JC-1染色流式细胞术检测线粒体膜电势（△Ψm）和线粒体质量，利用CFDA-SE染色流式细胞术检测细胞结构蛋白变化。 结果： 在1×10-6mol/L DEX诱导下，小鼠胸腺细胞在6 h凋亡比率为(51.25±5.51)%，对照组为(12.03±2.00)%，差异显著（P<0.01）； DEX组坏死比率为(30.25±3.67)%，对照组为(10.11±1.11)%，差异显著（P<0.01）。DEX组在6h时点的线粒体质量显著低于对照组（P<0.01），FL1平均荧光强度分别为（561.62±54.27）和（900.25±38.80）。DEX同时引起线粒体膜电势的显著下降（P<0.01），对照组FL2平均荧光强度为（267.51±26.48），DEX组为（133.17±12.29）。成熟T细胞培养48 h，CFDA-SE法仅检测到亲代单一细胞峰；而在Con A刺激条件下出现3个子代峰。对照组小鼠胸腺细胞在CFDA-SE染色培养6 h条件下，存在(5.25±1.15)％的低荧光强度细胞群，而在DEX刺激下，该群细胞占（47.39±9.76）％，并且在直方图结果上形成明显的细胞峰。 结论： DEX诱导小鼠胸腺细胞凋亡过程中，线粒体质量和细胞结构蛋白均有所下降；CFDA-SE染色流式细胞术可以作为基于细胞结构蛋白变化的凋亡定量检测方法。     

榄香烯诱导晶状体上皮细胞凋亡及其细胞和分子机制

目的：探讨天然药物单体榄香烯 (Ele) 诱导晶状体上皮细胞 (LEC) 凋亡及其细胞和分子机制。 方法: 通过透射电子显微镜观察Ele对体外培养的牛LEC超微结构的影响；采用流式细胞术观察Ele对LEC的DNA含量及线粒体跨膜电位（ΔΨm）的影响。 结果: 透射电镜下可观察到Ele组LEC呈现核染色质凝集、固缩、边集、核碎裂等典型的细胞凋亡形态学改变。Ele组LEC细胞核的DNA含量显著低于空白对照组，随药物作用时间延长而更低（P<0.01）；Ele组细胞质线粒体ΔΨm低于空白对照组，且在药物作用早期即已明显低于空白对照组（P<0.01）。 结论: ① Ele能显著诱导晶状体上皮细胞凋亡。② Ele通过使LEC细胞核DNA含量下降，诱导细胞凋亡。③ Ele使细胞质内线粒体ΔΨm下降，使细胞进入不可逆性凋亡的过程。线粒体ΔΨm下降是Ele 诱导LEC凋亡的早期事件。④ Ele诱导LEC凋亡是通过细胞核和细胞质两种途径。⑤ Ele诱导LEC凋亡的作用可能是其减轻晶状体后囊混浊发生和发展、防治后发性白内障的细胞和分子生物学机制。     

右美托咪定对乳大鼠心肌细胞线粒体活性氧和细胞凋亡的影响

目的:探讨右美托咪定(Dexmedetomidine,DEX)对线粒体介导的乳大鼠心肌细胞氧化应激通路的作用。方法:取新生乳大鼠(3~4天)的心肌进行原代培养,培养24h后分为对照组(C组)、H_2O_2组(终浓度500μM,H组)、右美托咪定组(5μM DEX,D组)、联合用药组(500μM H_2O_2+5μM DEX,DH组)。各组细胞分别给予相应药物刺激6h,采用流式细胞仪测定活性氧自由基(Reactive oxygen species,ROS)水平及心肌细胞凋亡变化;并通过ELISA法测定各组乳大鼠心肌细胞线粒体介导的凋亡因子Caspase-3,Caspase-9表达的变化。结果:与H_2O_2组相比,DEX明显抑制H_2O_2介导的乳大鼠心肌细胞的ROS水平增加(P0.05);下调线粒体介导的下游凋亡因子Caspase-3,Caspase-9的表达(P0.05),从而减少H_2O_2诱导的凋亡(P0.05)。结论:右美托咪定通过抑制心肌细胞活性氧水平以及下调线粒体介导的Caspase-3和Caspase-9的表达发挥抑制凋亡作用,对心肌细胞有一定的保护作用。     

阻断ERK途径对地塞米松诱导胸腺细胞凋亡中线粒体膜电势的影响

目的： 研究阻断ERK途径对地塞米松（DEX）诱导的胸腺细胞凋亡中线粒体膜电势的影响。 方法： 利用PD098059（PD）阻断小鼠胸腺细胞ERK途径，分别设对照组（control）、单纯PD组（PD only）、DEX组和PD＋DEX组；在3 h、5 h和7 h时点，利用Annexin V-FITC/PI双染流式细胞术检测细胞凋亡；在3 h、7 h和11 h，利用JC-1染色流式细胞术检测线粒体膜电势（△ψm）变化。 结果： 在1 μmol·L-1 DEX刺激下，小鼠胸腺细胞在3 h、5 h和7 h凋亡率分别为(19.63±0.35)%、(41.84±1.67)%和(67.00±2.43)%，对照组分别为(4.98±0.39)%、(6.08±0.33)%和(9.31±0.34)%，差异显著（P<0.01）；相同时点下，PD only组细胞凋亡率分别为(7.95±0.60)%、(10.69±0.48)%和(22.20±1.24)%，显著高于对照组（P<0.01）；PD＋DEX组在3 h和5 h时点细胞凋亡率显著高于DEX组（P<0.01），而在7 h时点，两组差异不显著（P>0.05）。在3 h、7 h和11 h，DEX组△ψm降低的细胞比率分别为(21.23±1.43)%、(55.34±1.78)%和(70.88±2.87)%，对照组分别为(5.25±1.22)%、(8.01±0.97)%和(12.88±1.10)%，差异显著（P<0.01）；相同时点下，PD only组△ψm降低的细胞比率分别为(11.09±2.00)%、(16.21±2.25)%和(21.15±3.70)%，显著高于对照组（P<0.01）；PD＋DEX组在3 h和5 h时点细胞凋亡率显著高于DEX组，分别为(30.55±2.99)%和(65.22±4.32)%（P<0.01），11 h时点两组差异不显著（P>0.05）。 结论： DEX诱导小鼠胸腺细胞凋亡至少部分通过ERK途径，阻断ERK途径在该凋亡过程中具有重要生物学意义。     

槲皮素抑制异烟肼诱导的L-02细胞线粒体氧化应激性DNA损伤

目的:探讨活性氧(ROS)介导的线粒体氧化损伤在异烟肼(INH)诱导L-02细胞DNA损伤中的作用及槲皮素对细胞的保护作用。方法:建立体外培养INH致肝细胞L-02损伤的模型,将细胞分为对照(control)组、INH组、槲皮素低剂量(Que low)及高剂量(Que high)组。利用彗星试验评价细胞DNA损伤;制备L-02细胞线粒体,应用荧光探针DCFH-DA和rhodamine 123检测细胞线粒体ROS水平及线粒体膜电位(ΔΨm);采用TBA法测定丙二醛(MDA)含量;应用黄嘌呤氧化酶法测定超氧化物歧化酶(SOD)的活性;采用Western blotting法检测细胞中Bcl-2和Bax蛋白表达,计算Bax/Bcl-2值。结果:INH可诱导L-02细胞DNA损伤,使细胞线粒体ROS水平、细胞MDA含量及Bax/Bcl-2值明显增高,并使细胞ΔΨm值和SOD活性明显下降。而槲皮素能减轻细胞DNA损伤,减少细胞ROS水平,增加细胞ΔΨm值,降低细胞MDA含量,增加SOD活性,减少Bax/Bcl-2值。结论:INH可通过诱导细胞线粒体氧化应激导致L-02细胞DNA损伤。槲皮素能减轻INH诱导L-02细胞的DNA损伤,对L-02细胞具有保护作用,可能与其抑制ROS介导的线粒体氧化损伤有关。     

地塞米松影响小鼠胸腺细胞c-Myc表达以及凋亡研究

目的: 研究在地塞米松(DEX)诱导的小鼠胸腺细胞凋亡过程中c-Myc信号变化特点及其与下游caspase-3变化的相关性,以进一步探讨c-Myc在小鼠胸腺细胞凋亡过程中的作用机制。方法:以终浓度1 μmol/L地塞米松诱导小鼠胸腺细胞凋亡,通过Annexin V-FITC/PI双染流式细胞术测定30 min、3 h、6 h和9 h凋亡和坏死细胞比率,在6 h时点电镜观察细胞形态,利用SDS-PAGE及Western blot检测0 min、30 min、1 h和3 h细胞内c-Myc和caspase-3信号变化特点。结果:在1μmol/L DEX诱导下,小鼠胸腺细胞在30 min、3 h、6 h和9 h凋亡比率分别为(5.70±0.46)%、(35.79±1.13)%、(50.61±2.15)%和(35.52±1.66)%,对照组分别为(5.97±0.25)%、(10.20±0.71)%、(12.10±0.66)%和(15.45±0.51)%,组间差异显著(P＜0.01)。相应DEX组坏死比率分别为(4.58±0.51)%、(4.66±0.67)%、(25.36±1.64)%和(46.99±2.67)%,对照组分别为(4.38±0.39)%、(4.19±0.73)%、(9.63±1.25)%和(13.38±0.72)%,组间差异显著(P＜0.01);电镜观察结果显示DEX处理6 h见较多典型凋亡细胞。对照组在0 min即可检测到c-Myc的明显表达,30 min略见增多,1 h和3 h呈微弱的下降趋势;DEX组c-Myc在0 min表达强于对照组,30 min最强,而在1 h和3 h显著减弱;对照组caspase-3随培养时间延长而逐渐增多,而DEX组caspase-3在0 min表达增多,30 min最多,而在1 h和3 h显著减少。结论:本研究结果提示DEX诱导的小鼠胸腺细胞凋亡呈现c-Myc介导的细胞凋亡模式,而且在该过程中c-Myc和caspase-3信号存在着反馈抑制调节的可能性。     

榄香烯诱导晶状体上皮细胞凋亡及其机制

目的 :探讨天然药物单体榄香烯 (Elemene,Ele)诱导晶状体上皮细胞 (lensepithelialcell,LEC)凋亡及其机制。方法 :通过透射电子显微镜观察Ele对LEC超微结构的影响 ;采用流式细胞术 (flowcytometry ,FCM)研究Ele对LEC的DNA含量及线粒体跨膜电位 (mitochondrialtransmembranepotential,ΔΨm)的影响。结果 :透射电镜下可观察到Ele组LEC呈现核染色质凝集、固缩、边集、核碎裂等典型的细胞凋亡形态学改变。Ele组LEC细胞核的DNA含量随药物作用时间的延长而下降 ,与空白对照组有显著差异 (P <0 0 1) ;细胞质线粒体ΔΨm在药物作用早…     

地塞米松影响小鼠胸腺细胞线粒体膜电势变化研究

目的：研究地塞米松刺激小鼠胸腺细胞过程中线粒体膜电势（△Ψm）的变化。 方法： 以终浓度1×10－6mol/L地塞米松（DEX）刺激小鼠胸腺细胞，通过JC-1染色流式细胞术，在0、1、3、5、7、9、11、24和27 h时点分别检测小鼠胸腺细胞平均J-aggregate（FL2）和平均J-monomer （FL1）含量，并计算线粒体膜电势（FL2/FL1）。 结果： 本研究中，小鼠胸腺细胞离体后FL1所反映的线粒体质量迅速下降，至5 h对照组达到平台期（913.38±70.11），然后缓慢下降到27 h的（622.80±22.81）。DEX组FL1在1 h和3 h与对照组没有显著差异（P>0.05），而从5 h（660.91±72.95）开始显著低于对照组（P<0.01），并且随培养时间延长呈下降趋势，至27 h的（309.70±53.35）。对照组和DEX组FL2随培养时间延长而降低。1-9 h，对照组和DEX的△Ψm没有显著差异（P>0.05）。而在11、24和27 h，对照组△Ψm分别为（256.41±21.59）、（214.14±23.21）和（146.14±17.97），而DEX组△Ψm显著低于对照组（P<0.01），分别为（138.28±20.59）、（111.61±29.67）和（72.92±17.86）。 结论： DEX诱导小鼠胸腺细胞凋亡过程中，线粒体质量降低和△Ψm下降是早期事件，而细胞内现存线粒体△Ψm下降则是较晚期的事件。在本研究中，线粒体质量下降与现存线粒体膜电势的变化之间关系不紧密。     

The role of mitochondria in nitric oxide-mediated thymocyte apoptosis

The aim of this study was to analyze whether immunosuppressants could affect NO-induced thymocytes apoptosis. S-Nitroso-N-acetyl-D,L-penicillamine (SNAP, an NO donor) has an apoptotic effect on murine thymocytes. This NO-induced thymocyte apoptosis is associated with a reduced uptake of DiOC(6), a fluorochrome which incorporates into cells depending on their mitochondrial membrane potential (DeltaPsi(m)). The immunosuppressant, cyclosporin A, partially inhibited NO-induced thymocyte apoptosis as well as the reduction of DeltaPsi(m). In contrast, another immunosuppressant, FK506, did not affect NO-induced thymocyte apoptosis. Although FK506 and cyclosporin A inhibit T-cell signal transduction by the same mechanism, their effects on mitochondria may contribute to the differential apoptosis induction ability.     

Nitric oxide induces a decrease in the mitochondrial membrane potential of peripheral blood lymphocytes, especially in natural killer cells

Increased levels of nitric oxide (NO) at an inflammatory site may affect the biological activity of lymphoid cells. To investigate the effects of NO on the immune system, we measured the mitochondrial membrane potential (delta psi m) of the peripheral blood lymphocytes (PBL) cultured with a chemical NO donor. PBL from healthy volunteers were cultured with NOC18, a NO-generating compound, at various concentrations. The delta psi m of the PBL was measured by flow-cytometry using 3,3-dihexyloxacarbocyanine iodide (DiOC6(3)). NOC18 induced a decrease in the delta psi m of the PBL in a dose-dependent fashion, induced an increase in the levels of reactive oxygen species (ROS), and caused these cells to undergo apoptosis. Dual-color staining of the delta psi m and lymphocyte surface markers demonstrated that CD3-CD56+ natural killer (NK) cells were responsive to NO. Trolox, a vitamin E analog, partially reversed the NO-induced decrease in the delta psi m of the PBL. We showed that the delta psi m of peripheral NK cells were decreased by NO, which suggests that abundant NO at an inflammatory site may impair NK cell function.     

Mitochondrial membrane potential change induced by Hoechst 33342 in myelogenous leukemia cell line HL-60

Abstract. Hoechst 33342's effects on apoptosis and mitochondrial membrane potential (delta psi) were investigated in a myelogenous leukemia cell line, HL-60. Delta psi was detected with 2 lipophilic cationic fluorochromes: 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] or 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). Mitochondrial mass was measured with nonyl acridine orange (NAO). Protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) depolarized mitochondria in control experiments. Cell viability was determined by propidium iodide uptake. Hoechst 33342 at 10-20 mg/L decreased fluorescence for DiOC6(3) at 0.5 hr. The fluorescence partially normalized at 3 hr and then progressively decreased at 5-24 hr, resulting in cell shrinkage and death. Mitochondrial mass decreased 40-70% by 1 hr and 70-90% at 24 hr. A lower concentration of Hoechst 33342, 5 mg/L, reduced the delta psi at 0.5 hr, but delta psi returned to control values after 3 hr. Mitochondrial mass decreased 30-40% and then partially normalized, and cell viability was > 92% at 24 hr. Protonophore carbonyl cyanide m-chlorophenylhydrazone lowered delta psi with little cell death. Thus, at high concentration, Hoechst 33342 induces depolarization of delta psi and subsequent apoptosis. Lack of apoptosis at low concentration of Hoechst 33342, despite depolarization of delta psi, indicates that mitochondrial membrane depolarization alone is insufficient to induce apoptosis.     

Nitric oxide induces murine thymocyte apoptosis by oxidative injury and a p53-dependent mechanism.

Previously, we showed that NO induces thymocyte apoptosis via a caspase-1-dependent mechanism [(1) ]. In the present study, we investigated the role of heme oxygenase, catalase, bax, and p53 in this process. The NO donor, S-nitroso-N-acetyl penicillamine (SNAP), induced DNA fragmentation in thymocytes in a time- and concentration-dependent way. SNAP (100 microM) induced 50--60% apoptosis; higher doses did not increase the rate of apoptosis significantly. SNAP decreased catalase and heme iron (Fe) levels without affecting superoxide dismutase, glutathione, or total Fe stores in thymocytes. SNAP significantly increased the expression of heme oxygenase 1 (HSP-32), p53, and bax but not bcl-2. Treatment with the heme oxygenase inhibitor, tin protoporphyrin IX inhibited SNAP-induced thymocyte apoptosis. Furthermore, thymocytes from p53 null mice were resistant to NO-induced apoptosis. Our data suggest that NO may induce its cytotoxic effects on thymocytes by modulating heme oxygenase and catalase activity as well as up-regulating pro-apoptotic proteins p53 and bax.     

Comparison of signaling pathways involved in apoptosis of a thymocyte hybridoma triggered by a rat thymic medullary epithelial cell line, dexamethasone or T-cell receptor cross-linking

Using an in vitro co-culture assay we found that a rat medullary thymic epithelial cell (TEC) line (TE-R2.5) induces apoptosis of the BWRT8 thymocyte hybridoma (TH) (CD4(hi)CD8(low) alphabetaTCR(hi)). TH apoptosis induced by this TEC line was predominantly mediated by direct cell-cell contacts and was potentiated by cross-linking of the T cell receptor (TCR) by R73 monoclonal antibody (mAb). Dexamethasone (Dx) also triggered TH apoptosis but inhibited death of these cells induced by TE-R2.5 cells or immobilized R73 mAb. The TEC-induced apoptosis was independent of the LFA-1/ICAM-1 interaction but partly depended on a novel 29 kDa molecule expressed on TE-R2.5 cells. All three types of TH apoptosis were followed by the cleavage of poly-(ADP-ribose)-polymerase and were blocked by a caspase inhibitor Z-Val-Ala-Asp(OMe)-CH(2)F.PKC stimulation by phorbol myristate acetate interfered with the TH apoptosis induced by TE-R2.5 and Dx, but did not modulate the effect of R73 mAb. On the contrary, inhibition of calcineurin with cyclosporine A did not influence the apoptosis induced by TE-R2.5 and Dx, but completely prevented the R73-triggered TH cell death. The TE-R2.5-mediated BWRT8 apoptosis was suppressed by Na-orthovanadate, an inhibitor of protein tyrosine phosphatases (PTP) as well as by genistein, a protein tyrosine kinase (PTK) inhibitor, while both compounds potentiated the effect of Dx. Blocking PTP, but not PTK decreased the proapoptotic effect of R73 mAb. These results, including those using a BWRT8 subclone (BWRT8-MDP.2) which is resistant to TCR-triggered apoptosis, but sensitive to apoptosis stimulated by TE-R2.5 and Dx, indicate that TE-R2.5-induced TH apoptosis in our model is different from apoptosis in other TEC co-culture models, published so far.     

Reactive oxygen species regulate activation-induced T cell apoptosis.

Reactive oxygen species (ROS) mediate apoptosis in a number of cell types. We studied the role that ROS play in activated T cell apoptosis by activating T cells in vivo and then culturing them for a short time. Activated T cells died independently of Fas and TNF alpha. Their death was characterized by rapid loss of mitochondrial transmembrane potential (delta psi(m)), caspase-dependent DNA fragmentation, and superoxide generation. A superoxide dismutase mimetic, Mn (III) tetrakis (5, 10, 15, 20-benzoic acid) porphyrin (MnTBAP), protected T cells from superoxide generation, caspase-dependent DNA loss, loss of delta psi(m), and cell death. These results indicate that ROS can regulate signals involved in caspase activation and apoptosis and may contribute to peripheral T cell deletion.