细胞凋亡与心脏

以往认为细胞死亡与坏死为同一含义,最近这一错误概念已被纠正,目前已知细胞死亡有两种形式:一是病理性细胞死亡,即坏死(necrosis);另一种是生理性细胞死亡,即程序化细胞死亡(programmed cell death,PCD)或凋亡(apoptosis)。凋亡是形态学概念;而PCD是功能上的概念。两者常交叉被用来表示一种生理性细胞死亡。这种死亡是由基因控制的细胞主动死亡,存在蛋白质的主动合成过程。凋亡不同于坏死,它表现为细胞质和核固缩,然后形成凋亡小体,但胞膜不破裂,最后凋亡小体被邻近实质细胞或     

急性淋巴细胞白血病白细胞的蛋白质组研究

目的:在建立人外周血白细胞蛋白质组分析方法的基础上,对健康志愿者和急性淋巴细胞白血病(ALL)患者白细胞的蛋白质组进行差异性分析,寻找与ALL相关的蛋白质。方法:收集10例健康志愿者和10例ALL患者外周血标本,分离白细胞,提取白细胞蛋白质,双向电泳分离蛋白质,PDQuest2D软件对双向电泳图谱进行差异分析,基质辅助的激光解析飞行时间质谱鉴定差异蛋白质。结果:利用PDQuest2D软件对健康志愿者与ALL患者外周血白细胞蛋白质双向电泳图谱进行差异分析,根据基质辅助的激光解析飞行时间质谱鉴定结果确定了与ALL相关的蛋白质有AnnexinA5、cytoskeleton14等。结论:经质谱鉴定和生物信息学分析,确定了蛋白质AnnexinA5、cytoskeleton14与ALL的发生有着密切的关系,为寻找该类型白血病的药物治疗靶点及早期诊断的分子标志物提供了良好的实验依据。     

药物性肝损伤的肝细胞死亡方式及治疗药物研究进展

药物性肝损伤（DILI）是临床常见的肝损伤类型,是严重的药物不良反应之一。细胞死亡是DILI的重要特征,药物可通过诱导内质网应激和激活死亡受体等方式激活凋亡通路,诱导肝细胞凋亡或坏死,诱发肝损伤。除凋亡和坏死外,DILI过程中还伴随着自噬、焦亡和铁死亡。自噬可以清除受损的蛋白质以及细胞器,是肝细胞存活的重要机制,但也可能诱导肝细胞死亡。焦亡和铁死亡是最近发现的细胞死亡方式,其在DILI中的作用尚未完全阐明。阻断肝细胞死亡通路,是治疗DILI的重要手段。水飞蓟素、柚皮素、人参皂苷等可以抑制肝细胞死亡通路,是DILI的潜在治疗药。针对不同细胞死亡方式的机制和特点,研究改善肝细胞死亡的药物对治疗DILI具有重要意义。总结了DILI中肝细胞死亡的机制,并论述了潜在的药物治疗,旨在为DILI的治疗提供参考。     

基于非凋亡性程序性死亡的抗肿瘤研究现状

细胞的存活和死亡是多细胞生物的生长和发育过程中基本现象,参与了诸多疾病发生、发展的重要病理过程。诱导细胞死亡是目前临床化疗药物抗肿瘤作用的主要机制。其中,凋亡是最为广泛研究的程序性细胞死亡。最近研究发现和定义了一些非凋亡的程序性细胞死亡形式,如程序性坏死、铁死亡、焦亡等。是否可以利用这些非凋亡程序性细胞死亡实现抗肿瘤?本文以天然产物诱导的程序性坏死为例,探讨这一可行性与存在问题。     

蛇毒神经生长因子对大鼠肝星状细胞增殖、凋亡及肝纤维化相关蛋白质表达的影响

目的探讨眼镜蛇毒神经生长因子(NGF)对大鼠肝星状细胞(HSC-T6)增殖、凋亡以及蛋白质表达差异的影响,进一步为蛇毒NGF抗肝纤维化提供依据。方法实验分为对照组(单纯HSC-T6培养)和实验组(NGF进行干预)。应用MTT检测眼镜蛇毒NGF对HSC-T6细胞增殖抑制率;流式细胞技术检测眼镜蛇毒NGF对HSC-T6细胞凋亡的影响;双向电泳技术观察HSC-T6细胞蛋白质表达变化;质谱鉴定差异表达的蛋白。结果 MTT结果显示NGF对HSC-T6细胞增殖具有明显抑制作用(5 mg.L-1NGF时抑制率为48.2%±1.9%,P<0.01);流式细胞仪也有同样的发现,NGF(5 mg.L-1)干预组的凋亡率21.15%±3.31%明显高于对照组的2.7%±1.55%(P<0.05);比较分析空白对照组和NGF作用组的2-DE图谱,找到差异蛋白质点47个,其中与对照组相比在NGF作用组表达上调22个,下调25个;质谱鉴定出9个蛋白质,其中4个表达上调,5个表达下调。结论蛇毒NGF能抑制大鼠肝星状细胞(HSC-T6)的增殖,诱导其凋亡,并且影响HSC-T6细胞蛋白质的表达。     

人椎间盘纤维环软骨细胞外基质小分子蛋白的双向电泳和质谱鉴定研究

目的初步研究双向电泳和质谱鉴定在人椎间盘纤维软骨的蛋白质组特性研究中的价值和作用,为进一步研究椎间盘疾病的治疗寻找有效途径。方法取人的正常椎间盘的纤维环组织,针对细胞外基质的小分子蛋白进行蛋白萃取,通过固相pH梯度(IPG)等电聚焦和梯度聚丙烯酰胺双向电泳分别分离纤维环的蛋白质,分析电泳图像,使用基质辅助激光解吸附飞行时间质谱仪(MALDI)对蛋白质点进行分析鉴定。结果双向电泳显示,纤维环中的胶原结合蛋白在pH3~10,相对分子质量在22000~250000范围内能够被很好地分离,对各蛋白质点进行质谱仪鉴定,确定了其中的17种蛋白质。结论双向电泳可有效地分离椎间盘纤维环组织中的细胞外基质小分子的胶原结合蛋白质,通过质谱仪鉴定可以确定其组成成分,为进一步研究椎间盘病变中胶原结合蛋白的改变及作用打下基础。     

顺铂处理肝癌细胞SMMC-7721的蛋白质组学分析

目的:用蛋白质组技术分析顺铂(cisplatin,DDP)作用于肝癌SMMC-7721细胞后细胞内蛋白质组的变化.方法:采用四氮唑蓝还原反应法(MTT)测定细胞生长抑制率.顺铂(10 mg·L-1)作用肝癌SMMC-7721细胞24 h,分别收集顺铂处理组与对照组细胞,提取细胞内总蛋白并采用双向电泳(2-DE)分离,Image Master 2D Platinum软件分析顺铂处理前后差异表达蛋白质,切取明显差异点,基质辅助激光解吸飞行时间质谱(MALDI-TOF-MS)鉴定差异蛋白.结果:顺铂可抑制肝癌SMMC-7721细胞的生长,其抑制作用具有剂量及时间依赖性.质谱鉴定出4个已知序列的差异蛋白点.结论:顺铂处理后肝癌SMMC-7721细胞内差异表达的蛋白可能与顺铂的抗肿瘤作用机制有关.     

肿瘤细胞的凋亡途径、非凋亡途径死亡的特征及新治疗策略

细胞死亡途径存在缺陷是肿瘤的特征之一。肿瘤发展与细胞凋亡的抗性密切相关，但肿瘤细胞也通过非凋亡机制发生死亡，例如坏死、衰老、自吞噬(autophagy)及丝裂灾变(mitotic catastrophe)等。借助药物对凋亡途径和非凋亡途径的调控有可能发展新的肿瘤治疗手段，非凋亡分子机制的研究可能揭示抗癌药物新的作用机制，发现新的靶标以至新的抗癌药物，因而日渐受到关注。     

肿瘤坏死因子诱导凋亡配体(TRAIL)抗肿瘤治疗研究进展

癌症是全球死亡率最高的疾病.严重影响了癌症患者的生活质量.细胞凋亡又称程序化细胞死亡,是多细胞有机体为调控机体发育、维护内环境稳定、由基因控制的细胞主动死亡过程.许多研究表明,肿瘤坏死因子诱导凋亡配体(TRAIL)能通过与死亡受体结合选择性引起肿瘤细胞凋亡而对正常细胞无明显影响,有望成为一种新的抗肿瘤制剂.本文就近几年TRAIL抗肿瘤研究的进展作一综述.     

奇果菌素促进宫颈癌HeLa细胞凋亡的蛋白质组研究

目的寻找与奇果菌素促进宫颈癌HeLa细胞凋亡相关的蛋白质。方法随机将宫颈癌HeLa细胞分成两组,一组为对照组,另一组以奇果菌素(剂量为半数抑制率)处理24 h。提取蛋白质做双向电泳(2-dimensional electrophoresis,2-DE),基质辅助激光解吸/离子化飞行时间质谱仪(matrix-assisted laser desorption/ionization time-of-flight mass spectros-copy,MALDI-TOF-MS)鉴定2组细胞间有明显差异表达的蛋白质。结果奇果菌素作用于HeLa细胞的蛋白质组,质谱鉴定出6种蛋白质表达差异有显著性,分别是:塌陷反应介导蛋白1(CRMP-1)、转胶蛋白2(Transgelin-2)、磷酸化应激诱导蛋白1(StIP1)、腺苷酸环化酶(ATPase)、磷酸甘油酸变位酶1(PGM-1)和原肌球蛋白4(TPM-4);其中StIP1表达下调,其他蛋白均上调。结论奇果菌素能够通过诱导宫颈癌HeLa细胞发生凋亡而发挥抑制HeLa细胞生长作用,奇果菌素可能通过上调或下调上述蛋白质的表达参与促进宫颈癌HeLa细胞的凋亡。     

Ⅰ期非小细胞肺癌相关血清差异表达蛋白的研究

目的:寻找Ⅰ期非小细胞肺癌(NSCLC)与健康人血清的差异表达蛋白.方法:收集天津市胸科医院经手术、病理证实的6例Ⅰ期NSCLC患者血清,等量混合后设为肺癌组;同期15例健康体检血清,等量混合后设为健康对照组.利用双向凝胶电泳(2-DE)和基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF-MS)分离、筛选和鉴定2组间的差异表达蛋白.结果:成功获得了2组的双向凝胶电泳图谱,PDquest软件分析显示,2组间差异大于2倍的蛋白质点共有128个,质谱鉴定后确定了10种蛋白质,其中7种表达量上调,分别为α1-酸性糖蛋白、C1酯酶抑制物、血管紧张素原、转铁蛋白、转甲状腺素蛋白、间-α-胰蛋白酶抑制剂重链H2及无花果酶-3;3种表达量下调,分别为纤连蛋白、补体C4-A及补体C3.结论:应用2-DE及MALDI-TOF-MS在Ⅰ期NSCLC血清中鉴定出4种与肿瘤关系密切的差异表达蛋白,可作为开发NSCLC早期诊断标志物的候选蛋白.     

甲基苯丙胺致PC12细胞的差异蛋白表达

目的探讨甲基苯丙胺(METH)作用于PC12细胞后的蛋白质表达变化。方法 METH2.5 mmol·L-1作用PC12细胞24 h后,提取细胞总蛋白质,丙酮沉淀法纯化蛋白,Braford法对蛋白质进行定量,并对蛋白质进行双向凝胶电泳,Image scannerⅢ透射扫描仪获取凝胶电泳图谱。应用Image Master 7.0软件对获得的双向凝胶电泳图谱进行差异性蛋白质点分析,并对相应差异蛋白点用高端基质辅助激光解析-飞行时间(MALDI-TOF)串联质谱仪进行差异蛋白质鉴定。结果应用双向凝胶电泳结合质谱分析技术,METH作用PC12细胞24 h后,共鉴定出18个差异表达蛋白质点,其中8个差异蛋白点在METH作用后表达增强,10个蛋白点表达减弱。这些蛋白质主要包括细胞骨架相关蛋白、分子伴侣、氧化应激和凋亡相关的蛋白以及与能量代谢相关的酶类。结论 METH诱导PC12细胞18个蛋白差异表达。     

重组hFGF-10腺病毒对HaCat细胞影响的蛋白质组学研究

研究重组腺病毒导入人成纤维细胞生长因子10 (hFGF-10) 对HaCat细胞蛋白质组的影响, 由鉴定的差异蛋白质推测hFGF-10对HaCat细胞作用的可能机制。采用双向凝胶电泳结合串联飞行时间质谱技术, 对空载体腺病毒Ad感染细胞和重组腺病毒rAd-hFGF-10感染细胞的总蛋白图谱上的差异蛋白点进行鉴定, 并通过半定量RT-PCR和Western blotting在转录和翻译水平对差异蛋白进行确证。结果显示, 获得了蛋白质分离效果较好的双向凝胶电泳图谱, 鉴定了4种与细胞凋亡、细胞骨架调控、蛋白质降解等相关的差异蛋白质, 并在转录和翻译水平确证了差异蛋白质VDAC2在导入目的基因hFGF-10的HaCat细胞中表达上调, 提示VDAC2可能在hFGF-10的生物学功能中发挥作用。     

Non-apoptotic functions of caspases in cellular proliferation and differentiation

The cysteinyl aspartate-specific proteases (caspases) have been identified as key players in the cellular process termed programmed cell death or apoptosis. During apoptosis, activated apoptotic caspases cleave selected target proteins to execute cell death. Additionally to their established function in cell death, a variety of recent publications have provided increasing evidence that apoptotic caspases also participate in several non-apoptotic cellular processes. Activated caspases exhibit functions during T-cell proliferation and cell cycle regulation, but are also involved in the differentiation of a diverse array of cell types. In some cell types, their differentiation can be morphologically viewed as a kind of incomplete apoptosis. Analysis of well-known apoptotic targets of caspases implicates that the cleavage of a limited number of selected substrates plays a major role during non-apoptotic functions of caspases. Selective substrate cleavage might be regulated by activation of anti-apoptotic factors, via a compartmentalized activation of caspases, or through limited activity of caspases during apoptosis-independent functions. The increasing evidence for caspase function in non-apoptotic cellular events suggests that caspases play a much more diverse role than previously assumed.     

Detection of SIV DNA in rhesus macaque polymorphonuclear neutrophils.

The extent of infection of monkey polymorphonuclear neutrophils (PMN) by simian immunodeficiency virus (SIV) has not yet been determined. Using the polymerase chain reaction (PCR) technique, we detected the presence of SIVmac239 DNA in rhesus macaque-derived PMN after 24 hrs of in vitro incubation of the cells with SIVmac239. Infection by SIVmac239 also down-regulated the expression of the bcl-2 apoptosis-blocking gene in the infected PMN. These SIVmac239-induced PMN intracellular alterations were correlated with an accelerated decrease in PMN viability over a period of 120 hrs compared to non-infected PMN. Evidence of chromatin condensation characteristic of programmed cell death (apoptosis) was also observed in SIVmac239-infected PMN. The results of this study provide a mechanism for the reduced chemotaxis/phagocytosis activities of PMN of SIVmac239-infected macaques and suggest that PMN is one of the target cells for SIVmac239 infection.     

吗啡成瘾和正常大鼠的前额叶皮质表达蛋白质组变化的初步研究

目的建立和比较吗啡成瘾与正常大鼠前额叶皮质(PFC)蛋白质双向电泳图谱,寻找和鉴定吗啡成瘾大鼠PFC中的差异蛋白表达谱。方法以固相pH梯度等电聚焦为第一向和垂直SDS-PAGE为第二向,分别对正常对照大鼠和吗啡成瘾大鼠的PFC蛋白质样品进行二维分离,2-DE图谱经ImageMaster 2D Platinumv5.0软件分析,选取4个差异蛋白点用基质辅助激光解吸附离子化飞行时间质谱(MALDI-TOF-MS)进行鉴定。结果通过对2-DE图谱蛋白斑点的匹配及对比分析,与吗啡成瘾相关的差异表达蛋白斑点为87个;经质谱鉴定出2个有意义的差异表达的蛋白斑点:Snap25亚型β-Snap25突触相关蛋白25、β-肌动蛋白。结论吗啡成瘾组与正常对照组大鼠PFC蛋白表达存在差异;初步鉴定了大鼠前额叶皮质中与吗啡成瘾相关的差异蛋白,其表达的变化可能通过多种途经影响PFC神经元功能,为我们研究阿片类物质依赖作用机制提出了新的思路和方向。     

Cholecystokinin-8-induced hypoplasia of the rat pancreas: influence of nitric oxide on cell proliferation and programmed cell death

The background of cholecystokinin-8 (CCK-8)-induced hypoplasia in the pancreas is not known. In order to increase our understanding we studied the roles of nitric oxide and NF-kappaB in rats. CCK-8 was injected for 4 days, in a mode known to cause hypoplasia, and the nitric oxide formation was either decreased by means of N(omega)-nitro-L-arginine (L-NNA) or increased by S-nitroso-N-acetylpencillamine (SNAP). The activation of NF-kappaB was quantified by ELISA detection, apoptosis with caspase-3 and histone-associated DNA-fragmentation and mitotic activity in the acinar, centroacinar and ductal cells were visualized by the incorporation of [(3)H]-thymidine. Pancreatic histology and weight as well as protein- and DNA contents were also studied. Intermittent CCK injections reduced pancreatic weight, protein and DNA contents and increased apoptosis, acinar cell proliferation and nuclear factor kappaB (NF-kappaB) activation. It also caused vacuolisation of acinar cells. The inhibition of endogenous nitric oxide formation by L-NNA further increased apoptosis and NF-kappaB activation but blocked the increased proliferation and vacuolisation of acinar cells. The DNA content was not further reduced. SNAP given together with CCK-8 increased apoptosis and other pathways of cell death, raised proliferation of acinar cells and strongly reduced the DNA content in the pancreas. Histological examination showed no inflammation in any group. We conclude that during CCK-8-induced pancreatic hypoplasia, endogenously formed nitric oxide suppresses apoptosis but increases cell death along non-apoptotic pathways and stimulates regeneration of acinar cells. Exogenous nitric oxide enhances the acinar cell turnover by increasing both apoptotic and non-apoptotic cell death and cell renewal. In this situation NF-kappaB activation seems not to inhibit apoptosis nor promote cell proliferation.     

Prostaglandin E2-prostanoid EP3 signal induces vascular contraction via nPKC and ROCK activation in rat mesenteric artery

Prostaglandin E2 (PGE2) is one major prostanoid produced under inflammatory situation. Although PGE2 is known to induce vascular contraction, its detailed mechanism remains unknown. In the present study, we investigated the signaling pathway underlying PGE2-induced smooth muscle contraction in rat mesenteric artery. PGE2 (0.3-30 μM) concentration-dependently caused contraction in endothelium-denuded artery. RT-PCR showed that this artery expresses mRNAs for all four prostanoid EP receptors (prostanoid EP1-4). Among selective agonists for PGE2 receptors, only a prostanoid EP3 receptor agonist, ONO-AE-248 (0.3-30 μM) induced contraction. Consistently, pretreatment with a prostanoid EP3 antagonist (L-798106, 1 μM) significantly but not completely inhibited the PGE2-induced contraction. Interestingly, pretreatment with a prostanoid FP antagonist (AL8810, 1 μM) or a TP antagonist (SQ29548, 10 nM) also partially inhibited the PGE2-induced contraction. Since ONO-AE-248 (10 μM) did not influence intracellular Ca2+ concentration in mesenteric artery, we next examined the involvement of Ca2+-independent contractile pathway including PKCs and ROCK in prostanoid EP3-mediated contraction. Pretreatment with bisindolyl-maleimide I (a general PKC inhibitor, 1 μM), Ro-31-8425 (a conventional PKC and PKCε inhibitor, 1 μM), rottlerin (a selective PKCδ inhibitor, 1 μM) and Y-27632 (a ROCK inhibitor, 1 μM) but not Go 6976 (a conventional PKC inhibitor, 1 μM) attenuated 10 μM ONO-AE-248-induced vascular contraction. In western blot analysis, we confirmed that the treatment with ONO-AE-248 (10 μM, 30 min) phosphorylated PKCδ (Thr505) and PKCε (Ser729). These results suggest that PGE2 induces vascular smooth muscle contraction via prostanoid EP3, FP and TP receptors in rat mesenteric artery. Prostanoid EP3-mediated contraction is ascribed to Ca2+-independent contractile pathway including PKCδ, ε and ROCK.     

Genomic and proteomic analyses of 1,3-dinitrobenzene-induced testicular toxicity in Sprague–Dawley rats

1,3-Dinitrobenzene (DNB) is an industrial intermediate and testicular toxicant that has been shown to target Sertoli cells. The mechanism of action of DNB in the testis, however, is unclear. To investigate global alterations in gene or protein expression during testicular toxicity, testes from rats treated orally with DNB were subjected to microarray and two-dimensional gel electrophoresis (2-DE) analyses. Histopathological abnormalities were detected in the testes of the DNB-treated rats. Microarray analysis revealed that, during early testicular toxicity, several genes involved in apoptosis, germ cell/Sertoli cell junction, and tight junction signaling pathways were differentially expressed. Based on 2-DE analysis, 36 protein spots showing significantly different expression during early testicular toxicity were selected and identified. Network analysis of the identified proteins revealed that these proteins are associated with cellular development or reproductive system diseases. Collectively, these data will help clarify the molecular mechanism underlying testicular toxicity in DNB-exposed rats.     

Proteomic alteration in lung tissue of rats exposed to cigarette smoke

Cigarette smoke has been widely investigated in terms of epidemiological and pathological studies in relation to human lung diseases. In this study, we conducted a proteomic analysis to characterize the differential protein expression in lung tissue of rats exposed to cigarette smoke. Wistar rats were exposed to cigarette smoke twice a day, 30 min each for 1, 2 and 4 months, respectively. The total protein of lung tissue was extracted for two-dimensional electrophoresis (2-DE) and analyzed with ImageMaster 2D Platinum software. A total of 28 differentially expressed proteins between the control and the smoke-exposed groups were screened and of which 18 were identified by matrix assistant laser desorption ion-top of flight-mass spectrometry (MALDI-TOF-MS) or MALDI- TOF-TOF analysis, revealing 10 up-regulated and 8 down-regulated proteins. The up-regulated expression of two proteins, receptor for advanced glycation endpoints (RAGE) and thioredoxin (Trx), were validated by immunoblotting and found to be consistent with the proteomic analysis. The results presented in this study demonstrate the identification of proteomic pattern as an early indicator of lung damages induced by cigarette smoke. The differentially expressed proteins may be applied as exposure biomarkers in future experimental as well as epidemiologic investigations upon confirmation by a greater sample size and more validate study design for the proteomic research.