代谢型谷氨酸受体5在小鼠肝原代细胞和肝癌细胞中的表达及作用

目的 代谢型谷氨酸受体5(mGlu5)在肝组织中具有表达,并且在肝脏的病理过程中发挥重要的调节作用。本课题组以往的研究结果表明,mGlu5的激动剂和阻断剂影响人肝癌细胞系HepG2的生长、凋亡、浸润和转移等功能,提示mGlu5在肝癌的形成和发展中发挥一定的作用。为进一步验证mGlu5在肝癌中的作用,本研究在此基础上选用小鼠肝原代细胞和小鼠来源的肝癌细胞系Hepa1-6为实验模型,通过比较mGlu5在两者中的表达及功能的差异,深入探讨mGlu5影响肝癌的内在机制。 方法 采用噻唑蓝法、免疫印迹法分别检测了mGlu5对小鼠肝癌细胞活力的影响,两种细胞中mGlu5的表达及mGlu5的功能。 结果 在小鼠肝原代细胞和肝癌细胞中均有mGlu5的表达,在肝癌细胞中的表达量高于小鼠肝原代细胞;激活Hepa1-6中的mGlu5所需DHPG(mGlu5的激动剂)剂量低于激活小鼠肝原代细胞中mGlu5所需剂量;激活mGlu5促进ERK信号通路激活, 在Hepa1-6细胞中,100 mol/L DHPG即可使ERK信号通路激活,而在小鼠肝原代细胞中,激活ERK信号通路需要300 mol/L DHPG。激活mGlu5不影响两种细胞中的JNK以及p38信号通路。激活mGlu5能增加Hepa1-6细胞活力。 结论 mGlu5在肝细胞和肝癌细胞中的表达及功能存在差异,此差异可能是肝细胞癌发生发展的原因之一。     

重症心力衰竭的急诊救治及长期治疗新对策(4)

6.3 β-受体阻滞剂 6.3.1 β-受体阻滞剂的作用机制慢性心力衰竭中交感神经兴奋性显著增高,通过β1受体介导,去甲肾上腺素引起细胞内cAMP活性增加和钙离子超负荷,造成心肌细胞功能异常和坏死.     

缺氧复氧后肾小管上皮细胞MCP-1表达及N-乙酰半胱氨酸对其的干预研究

目的:研究缺氧复氧后肾小管上皮细胞MCP-1的表达及N-乙酰半胱氨酸(NAC)对其的影响.方法:选用人肾小管上皮细胞(HK2)建立缺氧复氧损伤模型,设正常对照组、单纯缺氧复氧组、不同浓度NAC干预组.流式细胞术检测细胞内氧化应激水平,ELISA法检测细胞上清MCP-1蛋白表达,RT-PCR法测定细胞MCP-1 mRNA表达.结果:缺氧复氧后HK2细胞内氧化应激水平提高,MCP-1蛋白和mRNA表达上调,随缺氧时间的延长,细胞内氧化应激水平逐渐升高,MCP-1蛋白和mRNA表达逐渐增强;NAC呈剂量关系抑制细胞内氧化应激水平,呈剂量关系抑制MCP-1蛋白和mRNA表达的上调.结论:缺氧复氧诱导细胞内氧化应激的产生,诱导MCP-1表达上调;NAC可以通过抑制细胞内氧化应激抑制MCP-1的上调.     

N-甲基-D-天冬氨酸受体在神经系统疾病中的作用研究进展

一、概况 N-甲基-D-天冬氨酸(N-methyl-D-aspartate,NMDA)受体是一种配体门控型离子通道,通过不同的亚单位组成,与胞内多种蛋白相互作用.NMDA受体主要集中在突触后膜,在神经传递中发挥以下几种作用:(1)NMDA受体的活化与突触长度的长效性变化有关;(2)传入神经纤维的组成与靶向神经元的发生发展有关;(3)参与谷氨酸介导的兴奋性神经毒性.NMDA受体已知的亚单位有7种:NR1,NR2A~D,NR3A~B.亚单位NR1(NMDA receptor subunit 1)在所有功能性NMDA受体通道中普遍存在,是NMDA受体发挥功能所必需的.     

FcγRⅢa基因多态性对利妥昔单克隆抗体介导的NK细胞杀伤Raji细胞作用的影响

由于FcγRⅢa的基因多态性可导致NK细胞上Fcγ受体(Fcgamma receptor,FcγR)与抗肿瘤单克隆抗体(monoclonal antibody,McAb)Fc段结合的亲和力不同,NK细胞以抗体依赖细胞介导的细胞毒性(antibody-dependent cell-mediated cytotoxicity,ADCC)对肿瘤细胞的杀伤能力也不同.本研究通过CFSE-PI双荧光染色法检测不同FcγRⅢa基因型的NK细胞对Raji细胞的ADCC效应强度,确定不同FcγRⅢa基因型的NK细胞所介导的AD-CC效应差异.用nest-PCR测定FcγRⅢa表型,用CFSE-PI双荧光染色法染色靶细胞(Raji细胞),用流式细胞术检测NK细胞上CD3-CD56+及FcγRⅢa的表达以及Raji细胞上CD20的表达,计算细胞毒性.结果表明:FcγRⅢa-158V/V基因型的NK细胞ADCC细胞毒性指数为(69.05±2.38)%,FcγRⅢa-158V/F基因型的NK细胞ADCC细胞毒性指数为(39.63±3.86)%,与V/V基因型相比,V/F基因型的NK细胞对Raji细胞的ADCC效应明显较弱,差异具有统计学意义(t=12.950,p=0.000).结论:FcγRⅢa基因多态性可影响利妥昔单克隆抗体介导的NK细胞对Raji细胞的体外ADCC效应.以FcγRⅢa-158V/V基因型的NK细胞ADCC效应较FcγRⅢa-158V/F基因型的NK细胞ADCC效应强.     

N-乙酰半胱氨酸对G/GO诱导hRPE细胞氧化损伤的保护作用

目的探讨N-乙酰半胱氨酸(NAC)对葡萄糖/葡萄糖氧化酶(G/GO)氧化损伤人视网膜色素上皮(hRPE)细胞的影响及其机制。方法用不同浓度的GO在高糖条件下处理hRPE细胞,建立葡萄糖氧化而损伤细胞的模型。应用四甲基偶氮唑蓝(MTT)比色法检测细胞存活率;双氢罗丹明123(DHR)染色后流式细胞仪检测细胞内活性氧(ROS)水平;Western blot检测细胞诱导型一氧化氮合酶(iNOS)的蛋白表达。结果GO(5-20 mU)处理hRPE细胞6h,可呈剂量依赖性地降低hRPE细胞的存活率;加入NAC可明显抑制G/GO引起的细胞存活率降低,减少G/GO引起的细胞内ROS堆积以及iNOS蛋白的表达。结论NAC能显著地减轻G/GO对hRPE细胞的氧化损伤作用;其作用机制可能与减少ROS的堆积,抑制iNOS的活性有关。     

N-乙酰半胱氨酸对人离体中性白细胞释放超氧阴离子的影响

目的用人离体中性白细胞研究N-乙酰半胱氨酸(N-acetylcysteine,NAC)对中性白细胞释放超氧阴离子的影响。方法细胞色素C还原法测定NAC对三种刺激剂介导的中性白细胞释放超氧阴离子量。用抗原抗体法检测丝或苏氨酸磷酸化。结果NAC可呈浓度依赖性抑制N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸(fMLP)和佛波豆蔻醚乙酸盐(PMA)介导的中性白细胞释放超氧阴离子;而对花生四烯酸(AA)介导的中性白细胞释放超氧阴离子无影响。Western blot结果显示NAC可呈浓度依赖性抑制fMLP和PMA介导的丝氨酸或苏氨酸的磷酸化。结论NAC可能通过膜受体通道抑制细胞内的蛋白激酶C及其上游的蛋白质激酶,抑制中性白细胞释放超氧阴离子的产生。     

电碳酸氢钠协同转运蛋白1(NBCe1)在小鼠皮质星形胶质细胞中的功能性表达依赖于S255-257并受mTOR调节

电碳酸氢钠协同转运蛋白1,NBCe1(SLC4A4)是星形胶质细胞中表达的主要碳酸氢根转运蛋白。它对碳酸氢根高度敏感,并且是细胞内、细胞外和突触p H值的主要调节剂,从而调节神经元兴奋性。尽管已知NBCe1的这些基本功能,然而NBCe1介导的星形胶质细胞对细胞外pH值变化反应的分子机制尚不明确。使用原代小鼠皮质星形胶质细胞培养,本研究探索长期细胞外代谢碱中毒对NBCe1调节的影响;并通过免疫印迹,表面蛋白的生物素化,使用H+敏感染料2, 7-双(羧乙基)-5(-6)-羧基荧光素记录细胞内H+,和磷酸化蛋白质组学分析阐明该调节作用潜在的分子机制。结果显示代谢性碱中毒后NBCe1活性显著下调,而NBCe1的蛋白质丰度或表面表达并不受影响。在碱中毒期间,NBCe1活性下调时细胞内H+变化的速率在野生型星形胶质细胞中降低,但在NBCe1缺陷小鼠的皮质星形胶质细胞中不变。激活mTOR信号传导能够拯救碱中毒诱导的NBCe1活性降低。此外,质谱分析揭示S255-257的组成型磷酸化,而突变分析揭示这些残基对NBCe1转运活性至关重要。本研究证明了一种通过调节NBCe1的功能表达而实现的新的mTOR调节机制。该种机制不仅适用于星形胶质细胞,也适用于上皮细胞中的NBCe1。     

慢性粒细胞白血病细胞胰岛素受体酪氨酸蛋白激酶活性的研究

慢性粒细胞白血病细胞胰岛素受体酪氨酸蛋白激酶活性的研究孙茜，陈维信，王殿鸿现已发现许多生长因子受体是催化蛋白质酪氨酸残基磷酸化的酶。胰岛素对于细胞生长的调控作用是通过其受体酪氨酸蛋白激酶的激活及细胞内底物蛋白质的磷酸化来介导发挥的［ＪＢｉｏｌＣｈｅｍ...     

多巴胺受体功能与癫痫的相关研究前沿

多巴胺是中枢神经系统的单胺类神经递质之一,参与多种神经活动的调节。在过去30年中,多巴胺系统一直是许多研究的焦点,它参与了癫痫活动的调节。多巴胺的不同作用是由特异的受体来介导,多巴胺受体分为D1与D2两个亚群。D1亚群包括D1和D5,D2亚群包括D2,D3和D4。D1类受体的存在使兴奋性氨基酸如谷氨酸的神经毒性加强,使γ-氨基丁酸(GABA)浓度降低,同时,可能通过钙离子的内流增加,使动作电位后出现持续性去极化状态,从而使癫痫活动得以加强。D2类受体拮抗兴奋性氨基酸及毒蕈碱所致的兴奋性神经毒性从而降低癫痫的发生。D1与D2类受体参与癫痫的调控都与多巴胺在颅内的传导通路有关。     

Role of intracellular calcium ions and reactive oxygen species in apoptosis induced by ultrasound

Recently, we have reported that ultrasound (US)-induced apoptosis is due to inertial cavitation and that extracellular reactive oxygen species (ROS) generated by inertial cavitation are not directly correlated with the apoptosis (Honda et al. 2002). The molecular mechanism of apoptosis induced by US is not yet sufficiently clear. Here, we examine the role of intracellular calcium ions and the intracellular ROS on apoptosis induced by US. Human myelomonocytic lymphoma U937 cells were exposed to continuous 1-MHz US at an intensity of 4.9 W/cm(2) (I(SPTA)) in the presence of air, and changes of intracellular calcium ion concentration ([Ca(2+)]i) in individual cells by digital imaging, various flow cytometric analyses of endpoints of apoptosis (early apoptosis, secondary necrosis, loss of mitochondria membrane potential, superoxide formation, caspase-3 activation) and DNA fragmentation were explored. Furthermore, the effects of an intracellular calcium ion chelator (BAPTA-AM), an antioxidant (N-acetyl-L-cysteine, NAC), a calcium channel blocker (verapamil), Ca(2+)-free buffer and Levovist were also investigated. These results indicate that: 1. the mitochondria-caspase pathway and the Ca(2+)-dependent pathway play cardinal roles in apoptosis induced by US because BAPTA-AM partially inhibited DNA fragmentation, loss of mitochondria membrane potential and caspase-3 activation; 2. intracellular ROS generated from mitochondria, rather than extracellular ROS (which were directly produced by inertial cavitation in the medium), are involved in the regulation of apoptosis induced by US because addition of NAC after sonication showed effective suppression of the apoptosis; and 3. increase of [Ca(2+)]i appears to be due to nonspecific influx from outside the cells because verapamil is not effective and no increase of [Ca(2+)]i due to sonication could be observed in the Ca(2+)-free buffer.     

高氧所致氧化应激状态下肺泡上皮细胞Toll样受体的表达及其功能研究

目的 观察高氧暴露后肺泡上皮细胞内活性氧(ROS)水平与Toll样受体(TLRs)基因、蛋白表达变化及其与信号通路功能之间的关系,探讨高氧所致肺损伤炎症反应的可能机制.方法 将体外培养的人肺腺癌A549细胞株分为空气对照组、高氧组、N-乙酰半胱氨酸(NAC)预处理组;高氧组细胞在＞90％O2的高氧环境中暴露2、6、12、24及48 h,NAC预处理组细胞予以ROS清除剂NAC预处理后高氧暴露6h.于相应时间点用流式细胞术检测细胞内ROS含量以及TLR2/4蛋白在细胞中的分布和表达;用逆转录-聚合酶链反应(RT-PCR)法检测TLR2/4 mRNA表达;用酶联免疫吸附法(ELISA)检测细胞上清液中白细胞介素(IL-6、IL-8)的含量.结果 A549细胞有TLR2/4表达,并且以细胞质内表达为主.与空气对照组比较,高氧组暴露2h细胞内ROS含量(荧光强度)即明显增高(11.820±3.123比7.223±1.170,P＜0.01),随高氧暴露时间延长呈进行性增高,于48 h达高峰(113.837±5.247,P＜0.01);高氧暴露2 h TLR2/4 mRNA表达至高峰(TLR2 mRNA:1.820±0.056比1.263±0.023;TLR4 mRNA:2.080±0.220比1.317±0.107,均P＜0.01),随高氧暴露时间延长,TLR2/4 mRNA表达虽仍有增多,但无显著变化;高氧暴露后TLR2/4蛋白表达显著增高,6h表达至高峰(TLR2蛋白:8.370±1.548比3.930±0.277;TLR4蛋白:25.803±5.783比8.867±2.230,均P＜0.01),以细胞质内表达为主;随高氧暴露时间延长,细胞上清液中IL-6(ng/L)、IL-8(ng/L)水平呈进行性增高,于48 h达高峰(IL-6:2 213.41±69.99比9.76±1.47;IL-8:11 520.38±429.93比159.56±20.80,均P＜0.01).在NAC预处理情况下,高氧刺激后,细胞内ROS水平明显降低(14.050±1.257比31.180±2.336,P＜0.01),细胞TLR2/4 mRNA和蛋白表达显著降低(TLR2 mRNA:1.270±0.061比1.683±0.025; TLR4 mRNA:1.507±0.058比1.650±0.139;TLR2蛋白:3.458±0.258比8.370±1.548;TLR4蛋白:11.611±3.403比25.803±5.783,均P＜0.05),细胞上清液中IL-6、IL-8水平显著下降(IL-6:8.42±0.70比73.51±16.70;IL-8:134.94±5.19比772.82±96.05,均P＜0.05),与空气对照组比较差异均无统计学意义.结论 A549细胞在高氧暴露后,细胞内ROS能够激活人肺泡上皮细胞TLR2/4的表达,导致前炎症细胞因子IL-6和IL-8的大量释放.     

细胞内活性氧在糖基化终末产物促人腹膜间皮细胞分泌单核细胞趋化因子1中的作用

目的观察糖基化终末产物(advanced glycation end products,AGEs)对人腹膜间皮细胞(humanperitoneal mesothelial cell,HPMC)分泌单核细胞趋化蛋白1(monocyte chemoattractant protein-1,MCP-1)的作用及细胞内活性氧族(reactive oxygen species,ROS)在其中的作用。方法分别用不同浓度的糖基化人血清白蛋白(advanced glycation end product s-human serum albumin,AGE HSA)及抗氧化剂N乙酰-L半胱氨酸(N acetyl-L cysteine,NAC)作用于细胞,用逆转录多聚酶链反应(RT PCR)法和酶联免疫吸附法(E L I S A)测定HPMC中MC P 1的表达;再以氧化敏感的荧光染料2,7-二氢二氯荧光素(2,7-dichlorofluorescin,DCFH)染色,流式细胞仪测定ROS强度。结果 AGE HSA能使细胞内ROS水平明显升高,呈现浓度依赖效应;AGE HSA同时以时效和量效方式促进HPMC中MCP-1的表达;而NAC能够明显抑制AGE-HSA所导致的细胞内ROS升高,同时抑制HPMC中MCP-1的分泌。结论 AGE HSA可能部分通过诱导细胞内ROS,促进HPMC表达MCP-1。     

Mechanism of the inhibitory effect of atorvastatin on leptin expression induced by angiotensin II in cultured human coronary artery smooth muscle cells

Leptin contributes to the pathogenesis of atherosclerosis. Ang II (angiotensin II), a proatherogenic cytokine, increases leptin synthesis in cultured adipocytes. Statin suppresses leptin expression in adipocytes and human coronary artery endothelial cells. However, the effect of Ang II and statin on leptin expression in VSMCs (vascular smooth muscle cells), the major cell types?in atheroma, is poorly understood. Thus the aim of the present study was to investigate the molecular mechanism of atorvastatin for reducing leptin expression after Ang II stimulation in VSMCs. VSMCs from human coronary artery were cultured. Ang II stimulation increased leptin protein and mRNA and phospho-JNK (c-Jun N-terminal kinase) expression. Exogenous addition of Dp44mT (2,2'-dipyridyl-N,N-dimethylsemicarbazone) and mevalonate increased leptin protein expression similarly to Ang II. Atorvastatin, SP600125, JNK siRNA (small interfering RNA) and NAC (N-acetylcysteine) completely attenuated the leptin and phospho-JNK protein expression induced by Ang II. Ang II significantly increased ROS (reactive oxygen species) formation in human VSMCs. Addition of atorvastatin and NAC significantly attenuated the formation of ROS induced by Ang II. Addition of atorvastatin and SP600125 inhibited the phosphorylation of Rac1 induced by Ang II. The gel shift and promoter activity assay showed that Ang II increased AP-1 (activator protein-1)-binding activity and leptin promoter activity, while SP600125, NAC and atorvastatin inhibited the AP-1-binding activity and leptin promoter activity induced by Ang II. Ang II significantly increased the migration and proliferation of cultured VSMCs, while addition of atorvastatin, SP600125, NAC and leptin siRNA before Ang II stimulation significantly inhibited the migration and proliferation of VSMCs induced by Ang II. Ang II significantly increased secretion of leptin from human VSMCs, and addition of SP600125, atorvastatin and NAC before Ang II stimulation almost completely inhibited the leptin secretion induced by Ang II. In conclusion, Ang II induces leptin expression in human VSMCs, and atorvastatin could inhibit the leptin expression induced by Ang II. The inhibitory effect of atorvastatin on Ang II-induced leptin expression was mediated by Rac, ROS and JNK pathways.     

ROS在FTY720诱导U266细胞死亡中的作用机制研究

本研究探讨活性氧（ROS）在FTY720诱导多发性骨髓瘤U266细胞株凋亡及自噬中的作用。不同浓度FTY720处理U266细胞24 h后,应用流式细胞仪检测细胞凋亡,应用Western blot检测LC3B的表达。结果显示：应用FTY720可引起U266细胞发生凋亡及自噬,自噬的发生可促进细胞死亡。自噬抑制剂Bafilomycin A1可降低FTY720导致的细胞生存率下降。应用ROS抑制剂NAC或Tiron后,FTY720引起的细胞凋亡减轻,联合应用NAC或Tiron和FTY720后LC3B-Ⅱ表达较单用FTY720明显下降。结论：FTY720可诱导U266细胞发生凋亡及自噬,ROS是FTY720引起的多发性骨髓瘤细胞凋亡及自噬的共同调节剂。     

Upregulated ROS production induced by the proteasome inhibitor MG-132 on XBP1 gene expression and cell apoptosis in Tca-8113 cells

Exposure of Tca-8113 cells to proteasome inhibitor carbobenzoxy-Leu-Leu-leucinal (MG-132) causing apoptosis is associated with endoplasmic reticulum (ER) stress. X-box-binding protein-1 (XBP1) is an important regulator of a subset of genes active during ER stress, which is related to cell survival and is required for tumor growth. The present study is to evaluate the effect of MG-132 on ROS production, XBP1 gene expression, tumor necrosis factor receptor-associated factor 2 (TRAF2), ASK1 and c-jun protein expression in tongue squamous cell carcinoma cell line Tca-8113 cells. ROS production was measured by reactive oxygen species assay. X-box binding protein-1 (XBP1) mRNA was analyzed by real-time–PCR, TRAF2, ASK1 and c-jun protein were investigated by western blot and immunocytochemistry respectively. The result indicated that ROS production, TRAF2, ASK1 and c-jun were elevated in MG-132 treated cells. Giving ROS scavenger N-acetyl-L-cysteine (NAC) largely prevented the effects of MG-132. Furthermore, treating with MG-132 lead to decreased XBP1 mRNA expression but could not completely block the expression of XBP1. Taken together, these findings provide the evidence that MG-132 induced ER stress lead to Tca-8113 cells apoptosis through ROS generation and TRAF2-ASK1-JNK signal pathway activation.     

Study on the effect of cell proliferation and anti-oxidative damage of aldehyde dehydrogenase-2 gene transfected into K562 cells

OBJECTIVE: To construct a eukaryotic expression vector containing aldehyde dehydrogenase-2 (ALDH2) gene and investigate the effects and its possible mechanisms of ALDH2 gene on cell proliferation and anti-oxidative damage in the K562 cells. METHODS: An eukaryotic expression vector containing the ALDH2 gene cloned from human hepatocytes was constructed and transfected into K562 cells by liposome. RT-PCR and Western blot were used to evaluate the expression of ALDH2. MTT assay was used to check the cell proliferation and trypan blue exclusion to check K562 cells damage induced by hydrogen peroxide (H2O2). RT-PCR and fluorescence spectrophotometry were used to determine the expression of heme oxygenase-1 (HO-1) and the generation of intracellular reactive oxygen species (ROS) respectively. RESULTS: RT-PCR and Western blot analysis showed distinct higher ALDH2 protein expression in gene transfected group. The latter group had a higher cell proliferation (P < 0.05) and survival rate against H2O2 induced-oxidative damage, being increased by 7.8 times (IC(50) was 12.3 μmol/L and 1.4 μmol/L for K562-pcDNA3.1-ALDH2 and control cells, respectively, P < 0.01). The HO-1 mRNA expression and the generation of intracellular ROS were downregulated at a specific concentration of H2O2 in the ALDH2 gene transfected group. CONCLUSION: ALDH2 gene transfection can protect K562 cells against oxidative damage, and the downregulation of HO-1 expression and intracellular ROS may be involved in this process.     

ALDH2基因导入对K562细胞增殖和抗氧化损伤的影响

目的 克隆人乙醛脱氢酶2(ALDH2)基因,研究ALDH2基因导入慢性粒细胞白血病细胞系K562细胞后对其增殖和抗氧化损伤的影响.方法 从肝细胞中克隆人ALDH2基因,构建真核表达载体,用脂质体法将其导入K562细胞中,用RT-PCR和Western blot法检测ALD-H2基因的表达,锥虫蓝拒染法和MTT法检测转基因组细胞的增殖水平及对氧自由基引起的氧化损伤的反应;在此基础上,利用RT-PCR以及荧光分光光度法进一步检测氧自由基诱导后转基因组细胞中血红素加氧酶-1(HO-1)的表达和细胞内活性氧类(ROS)的产生.结果 成功克隆人ALDH2基因并将构建的真核表达载体转染K562细胞,RT-PCR和Western blot法检测到ALDH2的高表达.锥虫蓝拒染法和MTT结果显示转基因组细胞增殖水平明显高于对照组(P＜0.05),经基因修饰的细胞对H2O2耐受性增高,H2O2的IC50值提高了7.8倍(IC50值分别为12.3μnol/L和1.4 μmol/L,P＜0.01).HO-1的表达和ROS的产生随H2O2浓度的增大而增加,而一定浓度H2O2诱导后HO-1的表达和ROS的产生在转基因组中显著低于对照组(P＜0.05).结论 ALDH2基因导人K562细胞后可增加对氧自由基引起的细胞损伤的耐受性,起到保护作用,该过程伴随着ROS水平以及HO-1表达的降低.