siRNA-DAD1联合人参皂苷Rh 2对肝癌SMMC-7721细胞增殖和凋亡的影响

目的　探讨基因沉默DAD1 联合人参皂苷Rh2 对肝癌细胞增殖和凋亡的作用机制。方法　化学合成siRNADAD1,然后转染肝癌SMMC-7721 细胞,分别采用Q-PCR 和Western blot 检测DAD1 的mRNA 及蛋白表达水平变化,CCK-8 法检测肝癌SMMC-7721 细胞增殖能力的改变,DNA Ladder 法和流式细胞术检测肝癌SMMC-7721 细胞凋亡的变化。结果　siRNA-DAD1 可下调肝癌SMMC-7721 细胞中DAD1 的mRNA 及蛋白表达水平;siRNA-DAD1 与人参皂苷Rh2 均具有抑制SMMC-7721 细胞增殖的能力,并促使其发生凋亡,二者联合作用效果更好（P<0.05）。结论　siRNADAD1可下调肝癌SMMC-7721 细胞中DAD1 的mRNA 和蛋白表达水平,与人参皂苷Rh2 协同抑制肝癌细胞SMMC-7721的增殖并促进其凋亡。     

SUMO-1基因siRNA抑制肝癌细胞SMMC-7721生长的研究

目的:研究SUMO-1基因siRNA沉默人肝癌细胞SMMC-7721中SUMO-1基因表达的效果及对SMMC-7721生长的影响.方法:将人工合成的针对SUMO-1基因的siRNA片段转染体外培养的人肝癌细胞SMMC-7721,采用RT-PCR、Western blot方法在mRNA及蛋白水平检测siRNA沉默肝癌细胞中SUMO-1基因的效果.通过MTT、流式细胞仪及TUNEL试验检测SUMO-1基因沉默后对肝癌细胞生长的影响.结果:人工合成的针对SUMO-1基因的siRNA片段能显著地抑制SMMC-7721 中SUMO-1基因的表达,48 h抑制率可达73.43%.MTT结果显示肝癌细胞SMMC-7721转染SUMO-1 siRNA后生长明显受到抑制,流式细胞仪检测G_2期细胞明显增加,但TUNEL试验未发现凋亡细胞.结论:SUMO-1 siRNA沉默肝癌细胞SMMC-7721 中SUMO-1基因效果良好,SUMO-1基因控制SMMC-7721的生长,SUMO-1基因是肝癌生长的一个重要的调控因素.     

人剪切修复基因XPD对肝癌细胞生长及ERG基因的影响

目的探讨人剪切修复基因XPD转染人肝癌细胞SMMC-7721后对细胞自身生长及癌基因ERG表达的影响。方法将XPD基因通过LipofectamineTM2000转染入人肝癌细胞SMMC-7721。实验分为4组,分别为重组质粒转染细胞SMMC-7721-pEGFP-N2-XPD组(XPD组)、空载质粒转染细胞SMMC-7721-pEGFP-N2组(N2组)、脂质体转染细胞SMMC-7721组(脂质体组)、肝癌细胞SMMC-7721无转染空白对照组(空白对照组)。分别用逆转录聚合酶链反应(RT-PCR)和Westernblot法检测各组细胞中XPD、ERG的mRNA和蛋白质的表达量,用四甲基偶氮唑盐比色法(MTT)检测各组细胞的增殖活力及流式细胞仪检测各组细胞的凋亡情况。结果与其他3组比较,XPD组中的ERGmRNA及蛋白表达量显著降低,而XPDmRNA及蛋白表达量明显升高(P<0.01)。转染了XPD的肝癌细胞SMMC-7721,其细胞增殖活性(0.455±0.009)显著降低,细胞凋亡率(42.06±0.01)%明显升高(P<0.001)。结论 XPD基因可以抑制癌基因ERG的表达,明显降低肝癌细胞的增殖活力并提高肝癌细胞的凋亡率。     

RNAi-DNA稳定表达载体的构建与应用

目的:构建siRNA的DNA稳定表达载体,为研究RNAi在哺乳动物内持续、稳定抑制靶基因表达奠定基础。方法:合成含靶向hTERT基因的siRNA转录模板的发夹结构,将载体质粒pGenesil-1用BamH1 HindIII进行双酶切后,T4DNA连接酶连接成重组质粒,转染到肝癌SMMC-7721细胞中进行稳定筛选、表达,检测稳定筛选前后hTERT基因表达变化。结果:重组质粒在大肠杆菌菌株JM109内扩增。提纯、纯化后用HindIII、EcoRI酶切鉴定及测序鉴定证明hTERT-siRNA转录模板完整、正确的插入到pGenesil-1质粒中,建立了稳定抑制hTERT基因的SMMC-7721细胞株,并在mRNA水平抑制了肝癌SMMC-7721细胞hTERT基因表达。结论:成功构建了siRNA-DNA稳定表达载体,能在哺乳动物细胞中表达,并初步应用于靶基因的抑制。     

短发夹RNA沉默YAP表达对肝癌细胞SMMC-7721增殖和凋亡的影响

目的本实验通过短发夹RNA(shRNA)抑制Yes相关蛋白(YAP)基因的表达,并观察其对肝癌细胞SMMC-7721的增殖、凋亡和周期影响。方法以人YAP mRNA编码区中的4条序列作为RNA干扰靶点,分别构建4个真核表达载体质粒YAP-shRNA-1、YAP-shRNA-2、YAP-shRNA-3、YAP-shRNA-4,用阳离子聚合物转染法瞬时转染,筛选出2个干扰效果较好的质粒,将它们瞬时转染肝癌细胞SMMC-7721,应用实时荧光定量聚合酶链反应(PCR)和蛋白印迹法检测SMMC-7721细胞中YAP在mRNA和蛋白水平表达的变化。噻唑蓝(MTT)法检测转染后细胞增殖能力,流式细胞术检测细胞凋亡和周期的变化。结果转染后293T细胞中shRNA-YAP-2组与shRNA-YAP-4组YAP mRNA表达明显降低,在SMMC-7721中shRNA-YAP-2组与shRNAYAP-4组的YAP mRNA及蛋白表达水平分别为(0.871±0.081,0.106±0.013)和(1.010±0.097,0.192±0.013),与未转染组(2.399±0.148,0.372±0.007)比较差异有统计学意义(P<0.01)。MTT显示,shRNA-YAP-2、shRNA-YAP-4组细胞生长明显抑制,但未出现阻滞;流式细胞仪分析显示,shRNA-YAP-2组和shRNA-YAP-4组细胞凋亡率分别为(12.0±0.81)%和(5.03±1.01)%,明显高于空质粒组的(2.89±0.08)%(P<0.01)。细胞被阻滞在G2期,G0/G1期出现DNA亚二倍体峰。结论运用shRNA干扰技术,可以有效地干扰肝癌SMMC-7721细胞YAP的表达并能降低YAP的生成,抑制SMMC-7721细胞的增殖和促进细胞的凋亡。     

POLD1基因反义RNA对人肝癌细胞SMMC-7721的抑制作用北大核心CSCD

目的研究DNA聚合酶δ催化亚基基因1(POLD1)反义RNA对人肝癌细胞SMMC-7721的抑制作用及相应机制。方法将SMMC-7721细胞分为3组:实验组、阴性对照组、空白组。实验组及阴性对照组分别将POLD1基因反义RNA表达质粒及空质粒分别转染至SMMC-7721细胞,未经处理的SMMC-7721细胞为空白组。用聚合酶链反应法检测各组细胞POLD1基因表达情况;用细胞计数-8(CCK-8)法检测细胞增殖情况;用流式细胞术分析细胞凋亡率;用蛋白质印迹(Western blot)法检测p53、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)等凋亡相关蛋白表达量。结果实验组、阴性对照组和空白组肝癌细胞POLD1基因相对表达量分别为0.18±0.03,1.03±0.18和1.00±0.00;转染72 h增殖情况(OD_(450)值)分别为0.57±0.06,0.73±0.09和0.77±0.12;凋亡率分别为(23.43±4.12)%,(9.12±1.03)%,(1.24±0.14)%,实验组的上述指标与阴性对照组和空白组比较,差异均有统计学意义(均P<0.05)。结论POLD1基因反义RNA可显著抑制人肝癌细胞SMMC-7721的增殖,且能诱导人肝癌细胞SMMC-7721凋亡。     

亚硝酸钠对人肝癌细胞增殖与凋亡的影响

目的探讨亚硝酸盐对人肝癌细胞增殖和凋亡作用。方法用不同浓度的亚硝酸钠处理人肝癌SMMC-7721细胞不同时间,用MTT分析细胞增殖,荧光显微镜下观察细胞分裂指数和凋亡率,RT-PCR分析c-myc mRNA变化,Westernblot分析缺氧诱导因子-1α(HIF-1α)蛋白表达。结果亚硝酸钠诱导SMMC-7721细胞增殖和凋亡呈双相剂量效应,亚硝酸钠在低浓度时(20～200 mg.L-1),刺激细胞增殖,抑制细胞凋亡,在高浓度时(>800 mg.L-1),抑制增殖,促进凋亡。20～200 mg.L-1亚硝酸钠处理细胞可以明显增加c-myc mRNA和HIF-1α蛋白表达。结论亚硝酸钠在一定浓度范围内刺激SMMC-7721细胞增殖,但是超过一定剂量则诱导细胞凋亡。     

p 16 基因对人肝癌细胞的生长抑制作用的初步研究

目的探讨抑癌基因p16对肝癌细胞生长的抑制作用。方法将p16 cDNA亚克隆至pcDNA3.1真核表达载体上,并经脂质体介导转染至人肝癌细胞株SMMC-7721。用MTT法和Western blot分析转染细胞的生长情况。结果成功构建重组表达质粒pcDNA3.1-p16,转染pcDNA3.1-p16的SMMC-7721细胞生长速度受到明显抑制;经Western blot证实,转染后有外源p16蛋白的表达,且伴随Bax上调,Bcl-2和cIAP2的下调。结论重组pcDNA3.1-p16质粒能在人肝癌细胞SMMC-7721内表达,且能抑制SMMC-7721的生长,其机理与诱导肿瘤细胞凋亡相关。     

没食子酸诱导人肝癌细胞SMMC-7721凋亡机制的探讨

目的探讨没食子酸(gallic acid,GA)抑制人肝癌细胞SMMC-7721增殖的作用,揭示其促凋亡的相关分子机制。方法体外培养人肝癌细胞SMMC-7721,MTT法观察细胞在GA作用24、48、72 h后的增殖情况;用倒置显微镜观察细胞形态学的变化;透视电镜观察细胞内部结构变化;Annexin V-FITC/PI检测细胞凋亡;采用RT-PCR技术研究p53 mRNA的变化;应用Western blot法检测p53蛋白水平的表达。探讨GA对人肝癌细胞SMMC-7721的诱导凋亡作用及机制。结果剂量为6.25⁵0μmol·L-1GA作用SMMC-7721细胞48 h有明显的增殖抑制活性,引起核固缩、凝聚、碎裂,诱导细胞凋亡,并呈剂量依赖性;RT-PCR和Western blot结果显示,GA能升高p53 mRNA水平和p53蛋白表达。结论 GA可抑制人肝癌细胞SMMC-7721的增殖,诱导凋亡,可能与其上调肿瘤相关抑癌基因p53有关。     

原花青素联合Apollon siRNA抑制肝癌HepG2细胞增殖及凋亡

目的研究原花青素联合凋亡抑制蛋白(inhibitor of apoptosis proteins,IAPs)家族成员Apollon小干扰RNA(small interfer-ence RNA,siRNA)对人肝癌(HepG2)细胞增殖、凋亡的影响。方法设计并合成Apollon siRNA的序列,将一定浓度的人工合成的Apollon siRNA经脂质体包裹转染肝癌(HepG2)细胞,运用实时荧光定量RT-PCR检测Apollon mRNA的表达水平,蛋白质免疫印记技术检测Apollon的蛋白表达水平,并筛选出Apollon siRNA的有效序列,并进一步联合不同浓度的原花青素作用于肝癌(HepG2)细胞48 h后,采用WST-8法检测单用Apollon siRNA、单用原花青素、及Apollon siRNA联合原花青素对肝癌细胞增殖抑制作用,流式细胞术检测细胞早期凋亡率;Hoechst 33258染色观察HepG2细胞凋亡形态。结果设计的三对Apollon siRNA序列,筛选出其中有一对(NO.2)能明显下调Apollon mRNA与蛋白的表达水平,Apollon siRNA联合原花青素作用于HepG2细胞48 h后,单用原花青素的IC50为25.24 mg.L-1;Apollon siRNA与原花青素联合使用,原花青素的IC50为9.99 mg.L-1,增敏倍数为2.53,表现为协同作用。流式细胞术检测结果显示:Apollon siRNA联合原花青素能促进HepG2细胞凋亡,其早期凋亡率达25.2%;经荧光显微镜观察,Apollon siRNA组可见核高强度荧光的细胞,并见凋亡小体。结论 Apollon siRNA可增强肝癌细胞对原花青素的敏感性,作用机制可能为共同促进肝癌细胞早期凋亡有关,为临床上肝癌的治疗提供理论基础。     

SiRNA沉默 HBx对 HepG2.2.15细胞人端粒酶逆转录酶基因表达的影响

目的探讨应用RNA干扰技术沉默HBx对HepG2．2．15细胞中hTERT基因表达的影响。方法（1）将pSIHBV／X质粒转染HepG2．2．15细胞,RT-PCR法评估沉默效率。（2）MTT法检测转染24h、48h、72h后细胞增殖情况。（3）Real-timePCR和Westernblot检测hTERT表达情况。结果测序结果显示pSIHBV／X质粒构建正确;RT-PCR检测HBVX基因的沉默效率为53．6％;MTT检测结果显示转染24h、48h、72h后H印G2．2．15细胞的增殖受抑制,与对照组比较差异有统计学意义（P〈0．05）;Real-timePCR和Western blot检测结果均显示,转染pSIHBV／X质粒后HepG2．2．15细胞中hTERT基因的mRNA水平和蛋白水平表达分别有不同程度的下调,与对照组比较差异有统计学意义（P〈0．05）。结论siRNA沉默HBx基因可抑制HepG2．2．15细胞中癌症标志基因hTERT的表达。     

Gene Silencing Nucleic Acids Designed by Scanning Arrays: Anti-EGFR Activity of siRNA,Ribozyme and DNA Enzymes Targeting a Single Hybridization-accessible Region using the Same Delivery System

Gene silencing nucleic acids such as ribozymes, DNA enzymes (DNAzymes), antisense oligonucleotides (ODNs), and small interfering (si)RNA rely on hybridization to accessible sites within target mRNA for activity. However, the accurate prediction of hybridization accessible sites within mRNAs for design of effective gene silencing reagents has been problematic. Here we have evaluated the use of scanning arrays for the effective design of ribozymes, DNAzymes and siRNA sequences targeting the epidermal growth factor receptor (EGFR) mRNA. All three gene silencing nucleic acids designed to be complementary to the same array-defined hybridization accessible-site within EGFR mRNA were effective in inhibiting the growth of EGFR over-expressing A431 cancer cells in a dose dependent manner when delivered using the cationic lipid (Lipofectin) delivery system. Effects on cell growth were correlated in all cases with concomitant dose-dependent reduction in EGFR protein expression. The control sequences did not markedly alter cell growth or EGFR expression. The ribozyme and DNAzyme exhibited similar potency in inhibiting cell growth with IC₅₀ values of around 750 nM. In contrast, siRNA was significantly more potent with an IC₅₀ of about 100 nM when delivered with Lipofectin. The potency of siRNA was further enhanced when Oligofectamine was used to further improve both the cellular uptake and subcellular distribution of fluorescently labelled siRNA. Our studies show that active siRNAs can be designed using hybridization accessibility profiles on scanning arrays and that siRNAs targeting the same array-designed hybridization accessible site in EGFR mRNA and delivered using the same delivery system are more potent than ribozymes and DNAzymes in inhibiting EGFR expression in A431 cells.     

hTERT-siRNA对人肝癌细胞QGY增殖活性的影响

目的:利用siRNA在肝癌细胞内诱导RNAi,抑制hTERT基因表达,探讨RNAi对肝癌细胞增殖活性的影响.方法:构建靶向hTERT基因的siRNA(hTERT-siRNA),转导人肝癌细胞株QGY,采用3H-TdR掺入法检测转染质粒后细胞增殖活性.结果:成功构建hTERT-siRNA,其对肝癌细胞增殖具有明显抑制效应,抑制率达42%.结论:hTER-siRNA能明显抑制肝癌细胞的增殖.     

Gene silencing nucleic acids designed by scanning arrays: anti-EGFR activity of siRNA, ribozyme and DNA enzymes targeting a single hybridization-accessible region using the same delivery system

Gene silencing nucleic acids such as ribozymes, DNA enzymes (DNAzymes), antisense oligonucleotides (ODNs), and small interfering (si)RNA rely on hybridization to accessible sites within target mRNA for activity. However, the accurate prediction of hybridization accessible sites within mRNAs for design of effective gene silencing reagents has been problematic. Here we have evaluated the use of scanning arrays for the effective design of ribozymes, DNAzymes and siRNA sequences targeting the epidermal growth factor receptor (EGFR) mRNA. All three gene silencing nucleic acids designed to be complementary to the same array-defined hybridization accessible-site within EGFR mRNA were effective in inhibiting the growth of EGFR over-expressing A431 cancer cells in a dose dependent manner when delivered using the cationic lipid (Lipofectin) delivery system. Effects on cell growth were correlated in all cases with concomitant dose-dependent reduction in EGFR protein expression. The control sequences did not markedly alter cell growth or EGFR expression. The ribozyme and DNAzyme exhibited similar potency in inhibiting cell growth with IC50 values of around 750 nM. In contrast, siRNA was significantly more potent with an IC50 of about 100 nM when delivered with Lipofectin. The potency of siRNA was further enhanced when Oligofectamine was used to further improve both the cellular uptake and subcellular distribution of fluorescently labelled siRNA. Our studies show that active siRNAs can be designed using hybridization accessibility profiles on scanning arrays and that siRNAs targeting the same array-designed hybridization accessible site in EGFR mRNA and delivered using the same delivery system are more potent than ribozymes and DNAzymes in inhibiting EGFR expression in A431 cells.     

Synthesis andin vitro antitumor activity of 2-alkyl, 2-aryl, and 2-piperazinyl benzimidazole-4,7-dione derivatives

A series of 2-alkyl, 2-aryl, and 2-piperazinyl benzimidazole-4,7-dione derivatives (7a-h) and 16m-o) were prepared, and their cytotoxicities were tested against three cancer cell lines (mouse lymphocytic leukemia cell line P388, and human gastric carcinoma cell lines SNU-1 and SNU-16). These compounds showed potent cytotoxicity against all of three cell lines tested, and especially SNU-16 was sensitive to them. 2-Aryl (7g,h) and 2-piperazinyl benzimidazole-4,7-dione derivative (16 m) were more potent than mitomycin C against P388 and SNU-16. Among benzimidazole-4,7-dione derivatives with alkyl group at position 2, 7a had the most potent cytotoxicity against all of the cell lines tested.     

Arylalkyl ketones, benzophenones, desoxybenzoins and chalcones inhibit TNF-α induced expression of ICAM-1: structure-activity analysis

The interaction between leukocytes and the vascular endothelial cells (EC) via cellular adhesion molecules plays an important role in the pathogenesis of various inflammatory and autoimmune diseases. Small molecules that block these interactions have been targeted as potential therapeutic agents against acute and chronic inflammatory diseases. In an effort to identify potent intercellular cell adhesion molecule-1 (ICAM-1) inhibitors, a large number of arylalkyl ketones, benzophenones, desoxybenzoins and chalcones and their analogs (54 in total) have been synthesized and screened for their ICAM-1 inhibitory activity. The structure-activity relationship studies of these compounds identified three potent chalcone derivatives and also demonstrated the possible mechanism for their ICAM-1 inhibitory activities. The most active compound was found to be 79.     

双启动子shRNA 表达载体对hTERT和bcl-2基因表达的抑制作用

目的: 将已构建好的双启动子shRNA表达载体pdPRO-TB、单启动子对照pdPRO-T、pdPRO-B和阴性对照质粒pdPRO转染hela细胞,研究双启动子shRNA表达载体可否同时抑制双基因的表达,并且通过建立这种模式化工具载体,为今后的实验研究开拓新的思路。方法: 分别从构建好的大杆菌DH5α菌体中提取含有双启动子shRNA表达载体pdPRO-TB、单启动子对照pdPRO-T、pdPRO-B和阴性对照质粒pdPRO,通过脂质体介导瞬时转染hela细胞,镜下观察细胞形态。实时荧光定量RCR检测hTERT和bcl-2基因的表达水平。结果: 转染pdPRO-T和pdPRO-B的hela细胞生长受到抑制,转染pdPRO-TB的hela细胞生长抑制更为明显。转染pdPRO-T的hela细胞hTERT基因表达明显抑制,bcl-2基因无明显抑制;转染pdPRO-B的hela细胞bcl-2基因表达明显抑制, hTERT基因无明显抑制;而转染pdPRO-TB的hela细胞hTERT基因和bcl-2基因均受到明显抑制。。结论: 双启动子shRNA表达载体构建成功,可以同时抑制双基因的表达。该方法可作为新的研究手段运用于基因治疗等领域,构建双启动子shRNA表达载体,使其在细胞中表达两个不同的siRNA,同时诱导两个基因的表达沉默,达到双基因治疗的目的。