过表达小鼠UNCV蛋白质对HeLa细胞凋亡的影响

目的研究小鼠UNCV蛋白质对HeLa细胞凋亡的影响。方法将BALB/c小鼠Uncv基因重组质粒转染HeLa细胞,筛选出稳定过表达UNCV蛋白质的HeLa细胞株。通过细胞计数法和流式细胞术,检测过表达UNCV蛋白质对血清饥饿和阿霉素诱导的HeLa细胞凋亡的影响。结果获得稳定正确过表达UNCV蛋白质的HeLa细胞株。细胞计数和流式细胞术结果显示过表达UNCV蛋白质对血清饥饿诱导的HeLa细胞凋亡有抑制作用,对于阿霉素诱导的HeLa细胞凋亡没有显著影响。结论过表达UNCV蛋白质对血清饥饿诱导的HeLa细胞凋亡有抑制作用。     

siRNA沉默SIRT1基因对宫颈癌细胞HeLa增殖和凋亡的影响

化学合成靶向SIRT1基因的小干扰RNA,脂质体法转染人宫颈癌细胞株HeLa,观察小干扰RNA沉默SIRT1基因对HeLa增殖及细胞凋亡的影响。在优化siRNA SIRT1转染条件的基础上,应用RT-PCR和Western blot分别检测各组SIRT1 mRNA、SIRT1蛋白及凋亡相关蛋白的表达;CCK-8法检测细胞增殖抑制率;Hoechst荧光染色法和流式细胞仪检测细胞凋亡。结果表明,siRNA SIRT1转染细胞组SIRT1 mRNA水平和蛋白表达量明显低于对照组;siRNA SIRT1转染组细胞增殖受抑制,细胞凋亡率明显增加;凋亡相关蛋白P53、P21表达上调,Survivin表达下调。上述结果表明:siRNA SIRT1诱导的HeLa细胞凋亡与P53、P21、Survivin通路关系密切,但siRNA SIRT1诱导HeLa细胞凋亡的详尽机制有待进一步研究。     

应用DNA芯片鉴定HPV18 E6-siRNA诱导HeLa 细胞凋亡的分子机制

高危型人乳头瘤病毒（human papillomavirus,HPV）的E6基因在宫颈癌的发生中起关键作用,特异siRNA能有效抑制宫颈癌HeLa 细胞内HPV18 E6基因的表达,诱导肿瘤细胞凋亡.为进一步探讨HPV18 E6-siRNA诱导HeLa 细胞凋亡的分子机制,针对HPV18-E6基因设计siRNA序列,利用人源U6启动子为模板,经PCR表达框架法体外扩增,转染宫颈癌HeLa细胞抑制HPV18 -E6基因表达,从而诱导肿瘤细胞凋亡.对转染前后HeLa细胞总RNA样品进行荧光标记后,与Agilent Human 1A寡核苷酸芯片杂交、扫描、数据分析及标准化处理,确定表达差异的基因并经荧光定量PCR对部分基因进行验证,结合PANTHER数据分析系统,将这些基因按照生物学功能进行归类,查阅GenBank数据库及相关文献,对其结果进行深入分析及讨论.在检测的18 716个基因和EST中,共筛出差异表达基因359个,其中307个基因表达上调,52个基因表达下调,主要包括细胞周期相关基因CCNG1、p21;凋亡相关基因CASP4、CASP6、IGFBP3、DFFA;泛素蛋白酶解途径相关基因E6-AP、UBE2C;角化细胞分化相关基因KRT4、KRT6E、KRT18;抑癌基因RECK、VHL等.研究结果表明,HPV18 -E6基因抑制引起的细胞凋亡效应主要是通过P53信号途径和泛素蛋白酶解信号途径调节细胞周期相关基因和凋亡相关基因的表达,从而抑制HeLa细胞增殖、促进细胞凋亡.同时,抑癌基因的激活,角化细胞分化和免疫相关基因的表达上调,都说明了E6抑制后肿瘤细胞恶性转化程度的下降.     

反义RNA抑制存活素基因的表达对HeLa细胞的影响

为研究存活素（survivin)基因在肿瘤细胞中的应用,通过RT-PCR从HeLa细胞中克隆得到存活素cDNA的编码区,将其反向克隆到可诱导型表达载体pHC中,得到存活素反义RNA表达的载体pHSC,通过脂质体的介导,将pHSC导入HeLa细胞中,经C418（800nmol/L)筛选获得可以诱导表达存活素反义RNA的稳定细胞株HeLa-pHSC。获得的细胞株在2mmol/LZn^2 的诱导下,可以表达存活素反义RNA,从而抑制存活素基因的表达,此时HeLa细胞的增殖受到抑制,对化疗药物的敏感性增加,在G2/M期可以减缓细胞周期的进行性,这表明存活素对维持肿瘤细胞的增殖以及细胞周期的进行具有非常重要的作用。     

PDCD4基因在过氧化氢诱导喉癌细胞凋亡中的作用

目的探讨过氧化氢诱导喉癌细胞Hep-2凋亡过程中PDCD4基因表达的变化。方法以体外培养的喉癌细胞Hep-2为实验材料,不同浓度的过氧化氢作用于Hep-2细胞,噻唑蓝(MTT)比色法测定细胞生存率,采用吖啶橙染色、Ho33342/PI荧光双染进行形态学观察,RT-PCR及Western blot检测PDCD4 mRNA水平及蛋白表达的变化,评价在过氧化氢诱导喉癌细胞Hep-2凋亡过程中PDCD4基因的作用。结果过氧化氢(200μmol/L)作用Hep-2细胞24h,能够显著抑制细胞增殖,并诱导细胞凋亡,同时引起pdcd4 mRNA水平显著上调,PDCD4蛋白表达显著增加。结论本研究首次报道PDCD4基因可能在氧化胁迫诱导喉癌细胞凋亡中起关键作用。     

土贝母苷甲经miR-21/ PDCD4 途径诱导胶质瘤U251细胞凋亡

目的:研究土贝母苷甲(TBMS I)对胶质瘤U251细胞的抗肿瘤作用以及探究土贝母苷甲对miR-21及其靶基因PDCD4基因表达的影响。方法:U251细胞在体外进行培养,四甲基偶氮唑盐(MTT)法检测不同浓度的土贝母苷甲对细胞增殖的影响;Hoechst33258染色观察细胞核形态的变化;实时荧光定量PCR检测miR-21和PDCD4基因的表达情况;Western blot检测PDCD4蛋白的表达情况。结果:土贝母苷甲能够显著抑制U251细胞的增殖,其抑制作用呈剂量和时间依赖性。Hoechst33258染色观察到土贝母苷甲处理组细胞的细胞核形态表现出典型的凋亡特征。PCR结果显示:随着土贝母苷甲浓度增加,miR-21的表达逐渐降低(P0.05),PDCD4基因表达显著增加(P0.05)。Western blot结果提示:与阴性对照组相比,15、30μg/mL土贝母苷甲显著上调了PDCD4蛋白的表达(P0.05)。结论:土贝母苷甲能够显著抑制人胶质瘤U251细胞的增殖并诱导细胞发生凋亡,其机制可能与下调miR-21的表达和上调PDCD4的表达有关。     

DNA损伤诱导的长链非编码RNA-UCA1促进HeLa细胞增殖

尿路上皮癌抗原1 (UCA1)是一种长链非编码RNA,在多种肿瘤内高表达.然而,其在宫颈癌细胞和组织中的表达报告颇不一致,且功能尚未确定.本文探索UCA1在宫颈癌HeLa细胞中的生物学功能.实时定量PCR（qRT-PCR）结果显示,UCA1、p21和p53 mRNA在阿霉素（doxorubicin,DOX）或γ射线照射的HeLa细胞中表达上调|相反,敲减p53表达则可抑制DOX诱导的UCA1上调.表明DNA损伤诱导的UCA1可能与p53有关.转染结合CCK8检测HeLa细胞增殖活力结果显示,与对照比较,过表达UCA1促进HeLa细胞增殖,干扰UCA1表达则减缓细胞增殖.此外,流式细胞术结果显示,过表达UCA1导致阿霉素诱导的凋亡率下降;siRNA抑制UCA1表达后引起细胞G2/M期比例上升,S期下降,且阿霉素诱导的细胞凋亡率上升.上述结果说明,DNA损伤诱导的UCA1可促进HeLa细胞增殖,减少细胞凋亡.然而,是否DNA损伤诱导的UCA1上调依赖p53尚需进一步实验证明.     

SIRT6基因沉默对人肝癌细胞凋亡的影响及其机制研究

该文旨在探讨慢病毒介导的沉默信息调节因子6(silent information regulator 6,SIRT6)基因沉默对人肝癌细胞凋亡的影响及其机制。逆转录PCR(RT-PCR)和Western blot分别检测人肝癌细胞系(SK-Hep-1、Huh-7、PLC/PRF/5、Hep G2)和永生化肝细胞系(MIHA)中SIRT6基因的表达水平;利用慢病毒介导的sh RNA干扰技术靶向沉默SIRT6的表达,并通过RT-PCR和Western blot验证其沉默效率;流式细胞术检测SIRT6基因沉默对人肝癌细胞凋亡的影响,进一步应用RT-PCR和Western blot检测SIRT6基因沉默对凋亡抑制蛋白基因(inhibitor of apoptosis proteins,IAPs)家族m RNA和蛋白质水平的影响;最后,应用流式细胞术分析X连锁凋亡抑制蛋白基因(X-linked inhibitor of apoptosis protein gene,XIAP)在SIRT6基因沉默诱导的肝癌细胞凋亡中的作用。结果显示,SIRT6基因在人肝癌细胞系中表达上调;慢病毒介导的sh RNA能抑制人肝癌细胞中SIRT6基因的表达;沉默SIRT6基因的表达能诱导人肝癌细胞凋亡,并降低XIAP的m RNA和蛋白质水平;过表达XIAP能逆转SIRT6基因沉默所诱导的人肝癌细胞凋亡。该研究结果提示,SIRT6基因沉默可能通过调节XIAP的表达从而诱导人肝癌细胞凋亡。     

NAIF1对肝癌细胞 HepG2 增殖与迁移能力的影响

目的:在肝癌细胞Hep G2中过表达外源NAIF1(核凋亡诱导因子1),探讨NAIF1的亚细胞定位以及对Hep G2增殖和迁移能力的影响。方法:以真核表达质粒p EGFP-N1为对照组,p EGFP-N1-NAIF1为实验组,瞬时转染肝癌细胞Hep G2,利用免疫印迹方法检测NAIF1蛋白表达效率;以DAPI染核,荧光显微镜下观察绿色荧光蛋白定位,确定NAIF1的亚细胞定位;通过MTT方法绘制细胞增殖曲线;通过transwell小室法检测NAIF1对Hep G2迁移能力的影响。结果:在肝癌细胞Hep G2中,外源表达NAIF1主要定位于细胞核;与对照组Hep G2/p EGFP-N1相比,Hep G2/p EGFP-N1-NAIF1的细胞增殖、迁移能力下降(P<0.05)。结论:外源表达NAIF1蛋白定位于Hep G2细胞核,过表达NAIF1抑制Hep G2的细胞增殖与迁移能力,NAIF1可能作为肝癌治疗的潜在靶点。     

反义及正义表达癌基因c-Ha-ras重组质粒的构建

c-Ha-ras癌基因通过其产物p21蛋白的点突变或过量表达使细胞恶变。国外一些实验室的初步研究表明,导入一段与c-Ha-ras基因转录出的mRNA(sense RNA,正义RNA)顺序互补的RNA(anti-sense RNA,反义RNA),有可能通过阻断DNA复制,RNA转录或蛋白质翻译等过程,减少p21蛋白的合成,从而使转化细胞逆转。为了进一步系统研究反义c-Ha-ras RNA在转化细胞逆转中的     

Gene expression profiling in the silkworm, Bombyx mori, during early embryonic development

We prepared a cDNA library for a microarray from eggs of the silkworm, Bombyx mori, at the germ-band formation (24 hours after fertilization) stage. Using a microarray constructed with 2,445 ESTs, we screened gene expression profiles during germ-band formation at six specific time points in the early embryonic stages (from the unfertilized egg to the formation of abdominal leg appendages), and determined 241 of these cDNAs to represent genes that were expressed differentially during the germ-band formation stage. These differentially expressed genes grouped into two clusters. In the early and late clusters, 203 and 38 genes were upregulated, respectively. In the upregulated clusters, we isolated several genes that were associated with development and cell communication, including egalitarian, RAD23b, innexin 2, and senescence-associated protein. Northern blot hybridization revealed that the expression patterns of 14 genes had changed in each of the stages. In this study, we assessed changes in the levels of gene expression in relation to the germ-band formation stages in whole Bombyx embryos.     

Use of a 15 k gene microarray to determine gene expression changes in response to acute and chronic methylmercury exposure in the fathead minnow Pimephales promelas Rafinesque

This study describes the use of a 15 000 gene microarray developed for the toxicological model species, Pimephales promelas , in investigating the impact of acute and chronic methylmercury exposures in male gonad and liver tissues. The results show significant differences in the individual genes that were differentially expressed in response to each treatment. In liver, a total of 650 genes exhibited significantly ( P < 0·05) altered expression with greater than two-fold differences from the controls in response to acute exposure and a total of 267 genes were differentially expressed in response to chronic exposure. A majority of these genes were downregulated rather than upregulated. Fewer genes were altered in gonad than in liver at both timepoints. A total of 212 genes were differentially expressed in response to acute exposure and 155 genes were altered in response to chronic exposure. Despite the differences in individual genes expressed across treatments, the functional categories that altered genes were associated with showed some similarities. Of interest in light of other studies involving the effects of methylmercury on fish, several genes associated with apoptosis were upregulated in response to both acute and chronic exposures. Induction of apoptosis has been associated with effects on reproduction seen in the previous studies. This study demonstrates the utility of microarray analysis for investigations of the physiological effects of toxicants as well as the time-course of effects that may take place. In addition, it is the first publication to demonstrate the use of this new 15 000 gene microarray for fish biology and toxicology.     

基因芯片在骨骼肌老龄化相关基因表达谱中的应用

对老龄组大鼠 (30月龄 )和年轻对照组大鼠 (3月龄 )的腓肠肌超微结构进行观察 ,可以看到前者肌肉肌纤维萎缩伴有线粒体空泡变性。并进行总RNA抽提、mRNA纯化、探针制备 ,应用基因芯片筛选老龄化相关基因 ,两组大鼠骨骼肌重复出现的差异表达基因 12 7个 ,下调基因涉及能量代谢、信号转导 ,上调基因涉及蛋白质分解、细胞凋亡     

Microarray-based gene expression profiles in rabbit retina due to negative pressure suction

We investigated a possible molecular pathogenesis involving retinal ganglion cell apoptosis following transient high intraocular pressure. Changes in the gene expression profiles of the retina were detected via gene chip methodology. Twelve New Zealand white rabbits were randomly assigned to control and 3-min negative pressure suction groups. The control group was treated only with a laser, and the experimental group was also treated with suction for 3 min, using a negative pressure generator. Total RNA was then extracted from the retinal tissue at different recovery stages to analyze gene expression profiles using the Agilent rabbit one-way gene chip. The groups were then compared. Immediately after negative pressure suction induction, 704 genes were differentially expressed. Among these, 485 genes were upregulated, and 219 were downregulated. Expression of the genes encoding CRYAA, CRYAB, and TLR3 genes, which are involved in apoptosis, was elevated. The KRT18 gene, which is involved in apoptosis, had reduced expression. Seven days after negative pressure suction, 482 genes were differentially expressed. Among these, 178 genes were upregulated, and 304 were downregulated. Expression of the genes encoding CRYAB, IL1-BETA and IL1R1, which are involved in apoptosis, was upregulated. Ten days after negative pressure suction, 402 genes were differentially expressed. Of these, 213 genes were upregulated, and 189 were downregulated. Apoptosis genes CRYAB, CRYBA3, CRYBB2, IL1- BETA, and IL1R1 showed higher expression levels. We concluded that negative pressure suction for long periods of time (for example, 3 min) results in changes in gene expression. Genes with higher fold changes help protect retinal ganglion cells from apoptosis. We suggest that promoting the expression of these genes should be considered as a new means for treating ischemic-hypoxic retinopathy.     

应用基因表达谱芯片研究黄药子对小鼠肝脏的毒性机制(简报)

黄药子为薯蓣科植物黄独(Dioscorea bulbifera L．)的块茎,临床常用于治疗甲状腺肿、抗肿瘤、抗炎、抗病毒等。近年来临床上关于黄药子的毒副作用,尤其是对肝、肾的不良反应屡有报道。当黄药子或其代谢物在肝细胞内累积时会直接干扰肝细     

椎间盘退变纤维环及髓核异常表达基因的比较及生物信息学分析

目的:通过对已公开发表的基因芯片表达谱数据进行研究,探究椎间盘退变过程中纤维环与髓核组织的基因表达差异,并采用生物信息学方法对差异进行分析。方法:经GEO数据库选取两组椎间盘退变相关的基因芯片表达谱数据GSE23130及GSE67567,GSE23130所研究标本来源于正常及退变纤维环组织,GSE67567标本来源于正常及退变髓核组织。对上述数据系列进行质量分析,GSE23130及GSE67567各有10例样本数据被纳入实验。采用Gene Spring 13.0软件对GSE23130正常及退变纤维环间差异表达基因及GSE67567正常及退变髓核间差异表达基因分别进行筛选,利用KEGG PATHWAY和DAVID功能注释簇集分析分别对GSE23130及GSE67567上调及下调基因进行生物信息学分析。结果:GSE23130及GSE67567各筛选出差异表达基因3182个和3017个,其中135个基因在上述两个基因表达谱数据中均存在差异表达。针对两组数据进行的KEGG PATHWAY分析发现TGF-beta signaling pathway和regulation of apoptosis等数个相同的生物学通路及DAVID功能注释簇集;此外,还发现了数个与GSE23130及GSE67567单独相关的DAVID功能注释簇集。结论:椎间盘退变过程中纤维环及髓核组织内基因表达情况存在差异,两种组织内发生的生物过程不尽相同。某些生物学过程在两种组织内均出现异常改变,这些生物学过程中的异常变化可能是椎间盘退变的关键环节,值得进行深入研究。