钾通道阻滞剂四乙基铵敏感性钾电流在肺高压大鼠肺动脉平滑肌细胞中的变化

目的研究大鼠肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells,PASMCs)中电压依赖性钾通道(voltagedepending potassium channel,K_V)电流在肺高压(pulmonary hypertension,PH)中的变化,及钾通道阻滞剂四乙基铵(tetraethylammonium,TEA)对K_V电流的影响。方法采用全细胞膜片钳技术记录PASMCs的K_V电流,观察PH大鼠中K_V电流的变化,以及TEA对K_V的影响。结果野百合碱(monocrotaline,MCT)、慢性低氧(chronic hypoxia,CH)明显减小了大鼠PASMCs上记录到的K_V电流密度;TEA干预能够明显降低正常和PH大鼠PASMCs的K_V电流,并且TEA对K_V电流的抑制作用在PH大鼠中明显减小。结论在CH、MCT诱导的两种PH大鼠模型中,PASMCs的K_V开放程度被明显抑制,而且其对应的TEA敏感性K_V电流明显减小。     

三氧化二砷诱导宫颈癌细胞凋亡的研究

目的:研究三氧化二砷能否诱导宫颈癌细胞凋亡。方法:AnnexinV/PI双染,流式法检测早期凋亡;倒置显微镜观察、HE染色检测凋亡过程的形态学变化;琼脂糖凝胶电泳检测凋亡晚期出现的DNALadder。结论:适合浓度的三氧化二砷可以诱导宫颈癌细胞发生明显凋亡。     

白藜芦醇诱导HeLa细胞凋亡的实验研究

目的:观察白藜芦醇对宫颈癌HeLa细胞凋亡作用.方法:应用四甲基偶氮唑蓝(MTT)比色法检测白藜芦醇对HeLa细胞的抑制效应,并采用末端标记法(TUNEL)对凋亡细胞进行定量检测.结果:白藜芦醇对HeLa细胞48h的抑制效应为20.15%,72h的抑制效应为38.60%.TUNEL检测结果为白藜芦醇促进HeLa细胞凋亡.结论:白藜芦醇抑制HeLa细胞增殖,对指导临床药物治疗宫颈癌具有重要意义.     

三氧化二砷诱导实体瘤细胞凋亡的研究进展

近年来国内学者在研究三氧化二砷（As2O3)治疗急性早幼粒细胞白血病的作用机理时，发现其具有诱导白血病细胞凋亡的效应，而且更多的研究证实，这种效应不仅存在于血液肿瘤细胞，还存在于某些实体瘤，如胃癌细胞、胰腺癌细胞、食管癌细胞等，提示三氧化二砷具有一个相对较广的诱导凋亡效应谱。本文综述了近年来国内学者对三氧化二砷诱导某些实体瘤细胞，如肺癌细胞、肝癌细胞、胰腺癌细胞、结肠癌细胞、食管癌细胞、胃癌细胞、舌癌细胞、宫颈癌细胞、恶性淋巴瘤细胞、骨髓瘤细胞、神经母细胞瘤细胞凋亡的研究状况。     

大黄素对大鼠近端结肠平滑肌细胞电压依赖性钾通道的影响

目的研究大黄素对大鼠近端结肠电压依赖性钾离子通道的影响，以探讨其增强结肠运动的机制。方法 采用全细胞膜片钳技术测定电压依赖性钾离子通道快速激活型钾电流及延迟整流型钾电流。结果大黄素(1～30 μmol·L^-1)浓度依赖性地阻断延迟整流性钾通道，加快电流失活，其阻断作用不需要钾通道的开放。30 μmol·L^-1大黄素可抑制快速激活型钾电流。5 μmol·L^-1大黄素对钾通道的激活动力学及失活动力学没有影响，但30 μmol·L^-1大黄素使其激活动力学曲线明显右移，斜率常数由(13.0±0.6)上升至(19.6±2.5) mV，同时也使失活动力学曲线明显右移。结论大黄素可阻断延迟整流型钾通道及快速激活型钾通道，其阻断作用不是开放阻断。     

三氧化二砷诱导人宫颈癌细胞凋亡及bcl-2高表达对其影响

采用脂染素（ｌｉｐｏｆｅｃｔｉｎ）介导的ＤＮＡ 转染,Ｎｏｒｔｈｅｒｎ 印迹, ＭＴＴ和克隆形成实验,流式细胞术和细胞凋亡原位检测法探讨Ａｓ２Ｏ３ 对人宫颈癌ＳｉＨａ 和ＨｅＬａ 细胞的生物学效应和ｂｃｌ－２ 高表达对这一效应的影响． 结果表明,Ａｓ２Ｏ３ 处理的ＳｉＨａ 和ＨｅＬａ 细胞,存活率和克隆形成能力明显降低,细胞周期的Ｇ１ 期前有低于２ 倍体的凋亡峰,细胞被阻滞在Ｇ２／Ｍ 期,有细胞凋亡时出现的ＤＮＡ 断裂． 而Ａｓ２Ｏ３ 处理的ｂｃｌ－２ 高表达的ＳｉＨａ 和ＨｅＬａ 细胞存活率和克隆形成能力增加,凋亡率减少,Ｇ２／Ｍ 期阻滞程度显著降低． 结果表明,Ａｓ２Ｏ３ 可诱导ＳｉＨａ 和ＨｅＬａ 细胞凋亡,Ｇ２／Ｍ 期阻滞可能是其诱导凋亡的原因之一;ｂｃｌ－２ 高表达可能通过减轻Ｇ２／Ｍ 期阻滞的程度而部分阻止Ａｓ２Ｏ３ 诱导的细胞凋亡．     

阿司匹林诱导人宫颈癌HeLa细胞凋亡及机制

目的:探讨阿司匹林对人宫颈癌HeLa细胞凋亡的作用及机制。方法:体外培养HeLa细胞,分别以0.5,1.0,5.0mmol.L-1阿司匹林干预。采用Annexin V-FITC/PI凋亡试剂盒检测凋亡;碘化丙锭(PI)染色法检测细胞周期;Western-blot法检测NF-κB p65蛋白表达。结果:0.5,1.0,5.0mmol.L-1的阿司匹林均能使Hela细胞凋亡率明显增加,S期、G2/M期细胞比例减少,G0/G1期细胞比例显著增加,细胞中NF-κB p65蛋白表达水平明显下降,且上述作用均呈剂量依赖性。结论:阿司匹林在体外可以通过下调NF-κB p65蛋白表达,影响细胞周期,诱导Hela细胞凋亡。     

大鼠肺内动脉平滑肌细胞钾通道的研究

目的　研究大鼠肺内动脉平滑肌细胞钾电流。方法　用急性酶解分离法得到单个大鼠肺内动脉平滑肌细胞,采用膜片钳全细胞记录方式研究钾通道特性。结果　将平滑肌细胞钳制在-70 mV,给予-70～50 mV的斜坡刺激,时间为600 ms。可引出一随电压逐渐增大的电流,在+50 mV时其值为359±31 pA。细胞内用CsCl取代KCl后,该电流几乎完全消失;细胞外用无钙台氏液灌流,电极内液用高浓度的EGTA时,电流可被抑制50%±1%;细胞外给予特异性钙激活钾通道阻断剂TEA和延迟整流钾通道阻断剂4-AP均可使该电流明显下降。结论　大鼠肺内动脉平滑肌细胞的钾电流主要由钙激活钾电流和延迟整流钾电流组成。     

吴茱萸碱诱导人子宫颈癌HeLa细胞凋亡的机制研究

目的研究吴茱萸碱(evodiamine)诱导人子宫颈癌HeLa细胞凋亡的分子生物学机制。方法用结晶紫法和琼脂糖凝胶电泳法以及用两种caspase的蛋白酶抑制剂测定细胞凋亡过程中caspase的信号传导路径。结果吴茱萸碱可诱导HeLa细胞的细胞膜皱缩、细胞质浓集及凋亡小体的形式,并清晰可见以180 bp倍数裂解的DNA梯形电泳带的出现。抑制剂VAD-fmk(caspase家族总抑制剂)和DEVD-fmk(caspase-3抑制剂)能部分抑制HeLa细胞的凋亡。结论Evodiamine诱导人宫颈癌HeLa细胞凋亡;caspase cascade信号传导路径与凋亡密切相关。     

维拉帕米对HeLa细胞12C6+离子辐射敏感性的影响

利用重离子加速器国家重点实验室提供的12C6+束流照射人宫颈瘤(HeLa)细胞,测量辐射结束后细胞克隆率绘制细胞存活曲线。使用10μg/mL的维拉帕米处理细胞作为一组对照,检测维拉帕米对于HeLa细胞辐射敏感性的影响。利用Fluo-3 AM钙离子荧光探针配合流式细胞仪检测重离子照射后的胞内游离钙(IECa2+)水平。实验结果表明0～5 Gy范围内重离子辐射剂量与HeLa细胞存活率负相关,照射结束后胞质游离钙浓度有一定程度的上升。而维拉帕米能显著降低HeLa细胞经重离子辐射后的存活及增殖能力,且胞质游离钙浓度处于较低水平。造成这种现象的原因可能是维拉帕米能降低细胞对非致死性DNA损伤的修复能力。     

糖尿病大鼠冠状动脉平滑肌细胞中大电导钙离子激活钾通道电流和钙离子浓度变化

目的探讨糖尿病大鼠冠状动脉平滑肌细胞中大电导钙离子激活钾通道(BK通道)电流及钙离子浓度的变化。方法 40只SD大鼠随机均分为正常对照组(A组)和糖尿病组(B组)。采用链脲霉素腹腔内注射建立糖尿病大鼠模型,酶消化法分离冠状动脉平滑肌细胞,全细胞膜片钳实验和荧光测定方法分别检测冠状动脉平滑肌细胞BK通道电流和钙离子浓度。结果与A组相比,当刺激电压大于60mV时,B组冠状动脉平滑肌细胞BK通道电流密度明显下降(P<0.05);A组冠状动脉平滑肌细胞内钙离子浓度明显低于B组[(103±23)nmol/L vs.(193±22)nmol/L](P<0.05)。结论冠状动脉平滑肌细胞中BK通道电流下降及细胞内钙离子浓度升高可能是糖尿病冠状动脉功能损伤的重要原因。     

Stimulation of Na+,K+-ATPase of isolated smooth muscle membranes by the Ca2+ channel inhibitors,nimodipine and nitrendipine

Nimodipine (0.015 to 1.5 μM) increased Na⁺, K⁺-ATPase activity by 70–120% in isolated smooth muscle membranes. At 0.015 μM, nitrendipine, but not nifedipine, verapamil or diltiazem, also activated this enzyme. Nimodipine stimulated this Na⁺, K⁺ATPase three times more than nitrendipine at 15 nM. Marked stimulation of Na⁺,K⁺-ATPase by nimopidine was seen in membranes from rat and guinea pig aorta and rat vas deferens, but not in membranes from guinea pig heart or brain. Although it is not known whether these results are applicable to intact cells, the results are consistent with the hypothesis that vasodilation produced by nimodipine and nitrendipine may be due not only to inhibition of Ca²⁺ entry but also to the stimulation of the Na^? pump.     

Purinergic regulation of the epithelial Na+ channel

1. The epithelial Na⁺ channel (ENaC) is a major conductive pathway that transports Na⁺ across the apical membrane of the distal nephron, the respiratory tract, the distal colon and the ducts of exocrine glands. The ENaC is regulated by hormonal and humoral factors, including extracellular nucleotides that are available from the epithelial cells themselves.
2. Extracellular nucleotides, via the P2Y2 receptors (P2Y2Rs) at the basolateral and apical membrane of the epithelia, trigger signalling systems that inhibit the activity of the ENaC and activate Ca²⁺ -dependent Cl⁻ secretion.
3. Recent data from our laboratory suggest that stimulation of the P2Y2Rs at the basolateral membrane inhibits ENaC activity by a signalling mechanism that involves Gβγ subunits freed from a pertussis toxin (PTX)-sensitive G-protein and phospholipase C (PLC) β4. A similar signalling mechanism is also partially responsible for inhibition of the ENaC during activation of apical P2Y2Rs.
4. Stimulation of apical P2Y2Rs also activates an additional signalling mechanism that inhibits the ENaC and involves the activated Gα subunit of a PTX-insensitive G-protein and activation of an unidentified PLC. The effect of this PTX-insensitive system requires the activity of the basolateral Na⁺/K⁺/2Cl⁻ cotransporter.     

Kv通道亚型在15-HETE收缩肺动脉过程中的作用

目的从组织功能及细胞分子水平研究电压门控型钾通道(Kv通道)亚型在15-羟化二十碳四烯酸(15-HETE)致大鼠肺动脉收缩过程中的作用。方法采用组织浴槽血管环法，使用Kv通道阻断剂，确定受15-HETE调控大鼠肺动脉平滑肌细胞(PASMCs)膜上Kv亚型；使用RT-PCR和Western blotting技术观察受15-HETE调控PASMCs 膜上Kv亚型。结果阻断Kv1.1，Kv1.2，Kv1.3和Kv1.6通道并不影响15-HETE诱导肺动脉血管收缩；15-HETE不影响PASMCs膜上Kv1.1和Kv1.2通道蛋白质表达；15-HETE下调PASMCs膜上Kv1.5和Kv2.1通道mRNA和蛋白质表达。结论缺氧可能是通过15-HETE这一介导因子抑制Kv1.5和Kv2.1通道，减少PASMCs膜上功能性Kv1.5和Kv2.1通道数量，导致PASMCs收缩。     

Arsenic trioxide in environmentally and clinically relevant concentrations interacts with calcium homeostasis and induces cell type specific cell death in tumor and non-tumor cells

While arsenic compounds are known as environmental toxicants (especially in drinking water) and as carcinogens, some arsenic compounds, like arsenic trioxide (As2O3), are clinically used in humans to treat some forms of cancer (e.g. leukemia). Although arsenic compounds have been studied intensively, their interactions with living cells are still not fully elucidated. We have previously proposed that modulation of intracellular calcium ([Ca2+]i) homeostasis induced by As2O3 could be an important mechanism to induce cytotoxicity. Here we demonstrate, using human cell models (neuroblastoma (SY-5Y) or embryonic kidney cells (HEK)) and confocal microscopy in combination with the calcium sensitive dye fluo 4-AM, that As2O3 interferes with calcium signaling at low (environmentally and clinically relevant concentrations of 100 pM to 1 microM). Within this concentration range, As2O3 had cell type specific cytotoxic effects, with neuroblastoma cells being more sensitive to As2O3 than HEK 293. In addition, by staining with Hoechst 33347 and counting micronucleated cells as well as apoptotic nuclei, As2O3 was found to increase the rate of apoptosis and DNA damage, which was also cell type specific. These results indicate that the As2O3-induced cell death could be triggered or mediated by [Ca2+]i signals and suggest that low concentrations of As2O3 are able to interfere with specific physiological processes in diverse cell models.