Smac对骨肉瘤细胞系Saos-2生长及耐药性的影响

目的：观察Smac表达对骨肉瘤细胞Saos-2生长和耐药性的影响。方法：构建smac和反义smac表达载体pcDNA-smac和pcDNA—anti-smac，G418筛选出分别稳定转染pcDNA—$mac和pcDNA—anti—$mac的骨肉瘤细胞Saos—2。利用流式细胞仪检测各转染细胞的细胞周期分布；绘制各细胞在6d内的生长曲线以观察各细胞生长情况的变化。同时采用MTT法检测各转染Saos—2细胞对依托泊苷的敏感性。结果：与转染空载体pcDNA3．0的Saos—2细胞相比，转染反义smac的Saos—2细胞G1／G0期比例升高，细胞生长自第4天起明显加快。转染smac的Saos—2细胞的细胞周期及生长曲线无明显差别；依托泊苷作用细胞后，与转染空载体pcDNA3．0的Saos—2细胞相比，转染反义smac的Saos—2细胞的存活率增高；而转染smac的Saos—2细胞的存活率降低。结论：抑制Smac在细胞中表达，可使Saos—2细胞生长加快，增强对依托泊苷的耐受性，Smac在细胞中的过表达可提高Saos—2对依托泊苷的敏感性。     

锌和维生素E对工型糖尿病大鼠心肌凋亡促进因子Smac表达的作用

目的：探讨锌和维生素E对I型糖尿病大鼠心肌凋亡促进因子Smac表达的作用及对糖尿病大鼠心肌细胞保护作用的机制。方法：按体重将大鼠随机分为正常对照组（10只）和实验组（40只）,实验组动物采用腹腔注射链脲左菌素（streptozotocin,STZ）,60mg／k一次注射,制备糖尿病大鼠模型。将40只实验组大鼠按血糖值参考体重分为4组：糖尿病对照组,糖尿病补锌组,糖尿病补VE组,糖尿病补Zn＋VE组。6周后将各组大鼠处死,切取心室肌组织,进行免疫组织化学染色,观察各组心肌组织Smac的表达水平,利用HPIAS-2000图像分析系统测定Smac在以上各组中表达的平均光密度和平均阳性面积率。结果：正常对照组心肌细胞内Smac呈阴性表达,糖尿病对照组心肌细胞内Smac呈阳性表达;糖尿病补锌组和糖尿病补VE组心肌细胞内Smac呈弱阳性表达;糖尿病补Zn＋VE组心肌细胞内Smac呈阴性表达。图像分析结果显示：糖尿病补Zn＋VE组与糖尿病对照组、糖尿病补锌组及糖尿病补VE组之间Smac的平均光密度及阳性面积率的差异有显著性意义（P〈0．05）;糖尿病补Zn＋VE组与正常组之间Smac的平均光密度及阳性面积率的差异无显著性意义（P〉O05）。结论：Zn和VE联合使用可减低糖尿病导致大鼠心肌细胞凋亡的作用。     

NF-κB沉默联合苦参碱诱导人肝癌细胞HepG2凋亡和相关分子的表达

目的 探讨NF-kB沉默联合苦参碱(matrine,MT)诱导人肝癌细胞HepG2凋亡及相关分子的表达.方法 构建NF-kB/P65 siRNA真核表达载体转染人肝癌细胞HepG2,RT-PCR检测NF-kB/P65基因沉默效果,并筛选出NF-kB/P65基因沉默稳定转染细胞株.将实验分为对照组、苦参碱组(1.5 g/L)、稳转细胞组和联合组(稳转细胞十苦参碱),流式细胞仪检测细胞凋亡率;分别用RT-PCR法和蛋白质印迹法检测细胞NF-kB/P65、CD95(Fas)、Smac、Survivin的mRNA和蛋白水平表达.结果 苦参碱组NF-kB/P65、CD95、Smac和Survivin的表达上调,与对照组比较差异具有统计学意义(P＜0.05);稳转细胞组CD95和Smac的表达上调,与对照组比较差异有统计学意义(P＜0.05),NF-kB/P65和Survivin的表达下调,与对照组、苦参碱组比较差异有统计学意义(P＜0.05);联合组CD95和Smac的表达上调,与对照组、苦参碱组、稳转细胞组比较差异有统计学意义(P＜0.05),NF-kB/P65和Survivin的表达下调,与苦参碱组比较差异有统计学意义(P＜0.05).各组细胞凋亡率分别为3.21％、6.25％、11.82％、21.06％,组间差异均有统计学意义(P＜0.05).结论 NF-kB沉默联合苦参碱诱导人肝癌细胞HepG2凋亡可能与CD95和Smac的表达上调及NF-kB/P65和Survivin的表达下调有关.     

Small molecule inhibitor of apoptosis proteins antagonists: a patent review

Introduction: The family of inhibitor of apoptosis proteins (IAPs) plays a key role in the suppression of proapoptotic signaling; hence, a small molecule that disrupts the binding of IAPs with their functional partner should restore apoptotic response to proapoptotic stimuli in cells. The continued publication of new patent applications of IAP antagonists over the past 4 years is a testament to the continued interest surrounding the IAP family of proteins.

Areas covered: This review summarizes the IAP antagonist patent literature from 2010 to 2014. Monovalent and bivalent Smac mimetics will be covered as well as two new developments in the field: IAP antagonists coupled to or merged with other targeted agents and new BIR2 selective IAP antagonists.

Expert opinion: In addition to the well-explored scaffolds for monovalent and bivalent Smac-mimetics, some companies have taken more drastic approaches to explore new chemical space – for example, fragment-based approaches and macrocyclic inhibitors. Furthermore, other companies have designed compounds with alternative biological profiles – tethering to known kinase binding structures, trying to target to the mitochondria or introducing selective binding to the BIR2 domain. An overview of the status for the four small molecule IAP antagonists being evaluated in active human clinical trials is also provided.     

Smac/Diablo和actived caspase-3在卵巢浆液性肿瘤中的表达

目的探讨Smac/Diablo和Actived caspase-3在卵巢浆液性肿瘤中的表达。方法采用免疫组化S-P法检测45例卵巢浆液性囊腺癌和24例正常卵巢组织中Smac/Diablo和actived caspase-3表达水平。结果 Smac/Diablo和Actived caspase-3在45例浆液性囊腺癌中的表达率分别为86.7%和53.3%;而在正常卵巢中24例中的表达率分别为58.3%和91.7%。结论 Smac/Diablo表达紊乱导致的actived caspase-3表达的减少可能与卵巢浆液性囊腺癌的发生有关。     

细胞色素C、Smac与胎膜早破相关性的研究

目的探讨细胞色素c（cytochrome c,Cyt-c）及半胱氨酸天冬氨酸蛋白水解酶激活剂（second mitochondria-derived activator of caspase,Smac）在胎膜早破中的表达以及与胎膜早破的关系。方法胎膜早破孕妇40例,20例正常孕妇（无妊娠合并症、并发症）作为对照组,均取胎膜破口处组织（2cm×1.5cm×0.5cm）,常规石蜡包埋切片,采用免疫组织化学方法测定细胞色素C、Smac在胎膜组织中的表达。结果①两组病例的胎膜组织均见Cyt-c、Smac的表达;③Cyt-c实验组平均光密度值为0.18±0.04,对照组为0.10±0.02,差异有显著性（〈0.05）;Smac实验组平均光密度值为0.18±0.03,对照组为0.10±0.03,差异有显著性（〈0.05）;③胎膜早破组Cyt-c、Smac的表达呈正相关（r=0.34,〈0.05）。结论胎膜早破中Cyt-c、Smac高表达,推测胎膜早破的发生与细胞凋亡有关。     

丹参酮ⅡA对人卵巢癌细胞SKOV3增殖与凋亡的影响

目的:探讨丹参酮ⅡA(TanⅡA)对人卵巢癌细胞SKOV3的生长抑制和诱导凋亡作用及其可能机制。方法:培养人卵巢癌细胞SKOV3,经TanⅡA作用后,通过四甲基偶氮唑蓝(MTT)比色法测定丹参酮ⅡA对人卵巢癌细胞SKOV3的增殖抑制作用;流式细胞术检测细胞周期;RT-PCR检测Survivin与Smac/DIABLO mRNA表达水平;免疫荧光细胞化学检测Survivin蛋白及Smac/DIABLO蛋白的表达。结果:1.0～10.0μg/ml TanⅡA对人卵巢癌细胞SKOV3具有生长抑制作用,并具有时间-剂量依赖性。实验组较对照组G1/G2期细胞数量增多,而S期细胞数量减少(P<0.05)。随着TanⅡA干预浓度的增加和作用时间的延长,Survivin mRNA的表达受到明显抑制(P<0.05),而Smac/DIABLO mRNA的表达明显上调(P<0.05)。Survivin蛋白及Smac/DIABLO蛋白均表达于SKOV3细胞,其表达多定位于细胞质中,偶见于细胞核。结论:TanⅡA对人卵巢癌细胞SKOV3具有生长抑制和诱导凋亡作用,其作用机制可能是通过抑制人卵巢癌细胞SKOV3 Survivin和促进Smac/DIABLO的表达而实现的。     

大鼠Smac基因的克隆及其在心肌细胞H9C2中的表达

目的：克隆大鼠促凋亡基因Smac,构建真核表达载体pcDNA3.1⁺-rSmac,并在心肌细胞中表达。方法：应用逆转录聚合酶链式反应（RT-PCR）技术从大鼠肾组织中扩增到大鼠Smac基因的CDs片段,构建含Smac基因的真核表达载体pcDNA3.1⁺-rSmac并将此重组质粒、空载体pcDNA3.1⁺、pcDNA3.1⁺-EGFP通过脂质体介导转染大鼠心肌细胞H9C2,荧光显微镜、流式细胞术间接检测转染效率,RT-PCR法检测外源基因的表达。结果：经测序目的基因序列与GenBank（NM_001008292）公布的结果一致,所构建的重组质粒pcDNA3.1⁺-rSmac经PCR和酶切鉴定所释放的片段大小与预期结果相符。荧光显微镜和流式细胞术检测,细胞转染有效,转染效率达38.36%。转染重组载体pcDNA3.1⁺-rSmac 48 h后,H9C2细胞Smac的mRNA水平明显升高。其中,对照组Smac/β-actin为1.19,空载体组Smac/β-actin为1.31,二者之间差异无显著性（P>0.05）;转染pcDNA3.1⁺-rSmac组Smac/β-actin为1.67,与对照组比较差异具有显著性（P<0.01）。结论：克隆大鼠Smac基因成功,成功构建重组真核表达载体pcDNA3.1⁺-rSmac,并在转染后的心肌细胞中表达,为研究Smac基因在心肌细胞凋亡过程中的调控作用奠定基础。     

Denbinobin induces apoptosis in human lung adenocarcinoma cells via Akt inactivation, Bad activation, and mitochondrial dysfunction

Increasing evidence demonstrated that denbinobin, isolated from Ephemerantha lonchophylla, exert cytotoxic effects in cancer cells. The purpose of this study was to investigate whether denbinobin induces apoptosis and the apoptotic mechanism of denbinobin in human lung adenocarcinoma cells (A549). Denbinobin (1-20microM) caused cell death in a concentration-dependent manner. Flow cytometric analysis and annexin V labeling demonstrated that denbinobin increased the percentage of apoptotic cells. A549 cells treated with denbinobin showed typical characteristics of apoptosis including morphological changes and DNA fragmentation. Denbinobin induced caspase 3 activation, and N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a broad-spectrum caspase inhibitor, prevented denbinobin-induced cell death. Denbinobin induced the loss of the mitochondrial membrane potential and the release of mitochondrial apoptotic proteins including cytochrome c, second mitochondria derived activator of caspase (Smac), and apoptosis-inducing factor (AIF). In addition, denbinobin-induced Bad activation was accompanied by the dissociation of Bad with 14-3-3 and the association of Bad with Bcl-xL. Furthermore, denbinobin induced Akt inactivation in a time-dependent manner. Transfection of A549 cells with both wild-type and constitutively active Akt significantly suppressed denbinobin-induced Bad activation and cell apoptosis. These results suggest that Akt inactivation, followed by Bad activation, mitochondrial dysfunction, caspase 3 activation, and AIF release, contributes to denbinobin-induced cell apoptosis.     

MiR-24反义核酸通过BIM-Smac/Diablo途径促进多柔比星对结肠癌细胞的凋亡诱导效应

目的 探讨miR-24在结肠癌中发挥的作用并研究其是否和多柔比星的体外治疗有关。方法 用RT-qPCR法检测miR-24在结肠癌患者血浆中及结肠癌细胞系中的表达水平。MTT法检测miR-24反义核酸对多柔比星杀伤结肠癌细胞能力的影响。利用生物信息学、定量PCR及western blot法验证miR-24是否调节结肠癌细胞BIM的表达。运用JC-1染色、Annexin V染色及western blot法研究miR-24反义核酸影响多柔比星疗效的信号通路。结果 结肠癌患者血浆及结肠癌细胞系中miR-24表达水平显著升高。miR-24反义核酸可显著增强多柔比星对SW480细胞的杀伤活性。定量PCR及western blot实验表明miR-24的靶基因可能为BIM。miR-24反义核酸联合多柔比星可引起SW480细胞线粒体膜电位的丧失并诱导线粒体内Smac/DIABLO的释放,进而引起caspase-3的活化和凋亡的发生。转染BIM siRNA后miR-24反义核酸联合多柔比星对SW480细胞的凋亡诱导效应显著降低。结论 MiR-24反义核酸通过BIM-Smac/Diablo途径促进多柔比星对结肠癌细胞的凋亡诱导效应。