胆囊癌hTR反义RNA基因真核表达载体的构建

目的采用RT-PCR法,提取胆囊癌细胞内的端粒酶RNA(hTR)基因,并针对其序列设计反义RNA切割基因序列,并将其构建入真核表达载体pTriEx-4内。方法根据hTRcDNA序列,设计引物,经RT-PCR法从体外培养的胆囊癌细胞内调取hTR模板区基因,根据其测序结果,合成反义RNA基因,与经相应酶切的真核表达载体连接,酶切鉴定重组体的正确性。结果经RT-PCR从细胞内调出68bp序列,与hTRcDNA比较,与模板区域碱基序列一致。反义RNA基因重组子经酶切鉴定序列正确。结论RT-PCR法可调出胆囊癌hTR基因有效片段,成功构建了针对hTR模板区的反义RNA基因的真核表达载体,为胆囊癌的基因治疗奠定了良好的基础。     

MMP-24基因siRNA真核表达载体的构建及鉴定

目的:构建基质金属蛋白酶-24(matrix metalloproteinase-24,MMP-24)小干扰RNA(small interfering RNA,siRNA)表达质粒,以研究其对人卵巢浆液性囊腺癌细胞MMP-24基因表达的沉默效果,为探讨肿瘤的基因治疗奠定基础。方法:根据Gen-bank数据库提供的MMP-24基因核苷酸序列,选择设计能转录小发卡RNA(small hairpin RNA,shRNA)结构的DNA序列,克隆到空载体pIU6-B中,构建重组质粒,并进行酶切和PCR鉴定以及测序分析。结果:酶切和PCR鉴定以及测序证实重组质粒构建成功。结论:利用RNA干扰技术可成功构建siRNA表达载体,为进一步探索卵巢癌基因治疗奠定了基础。     

WWOX基因真核细胞表达载体的构建与鉴定

目的 构建人WWOX基因真核细胞表达载体.方法 采用RT-PCR方法 ,从人新鲜卵巢组织的总RNA中扩增出1245 bp的人WWOX cDNA片段, 然后用Hind III和Eco RI双酶切后定向克隆到真核细胞表达载体pcDNA 3.1中,用限制性内切酶酶切分析和DNA序列分析鉴定重组质粒.结果 WWOX基因的序列测定结果 与文献报道完全一致,其真核细胞表达载体被成功构建.结论 成功克隆出WWOX基因,并成功构建其真核细胞表达载体.     

Livinα特异性shRNA表达载体的构建与克隆的鉴定

目的:构建livin α的shRNA表达载体,验证livin靶向RNA干扰重组质粒是否构建成功并成功转化入大肠杆菌,方法:用DNA重组技术将人livnα基因及它对应的靶点 shRNA克隆到表达载体pGenesil-1中,构建livinα的shRNA表达载体,然后通过酶切电泳、测序对获得的克隆进行鉴定.结果:经限制性酶切...     

人Bcl—2基因短发夹样RNA真核表达载体的构建和鉴定

目的构建人Bcl-2基因序列特异性的短发夹样RNA（Short Hairpin RNA，shRNA）质粒载体。方法根据Bcl-2基因序列及shRNA设计原则，化学合成4段编码短发夹RNA的寡核苷酸序列，将其定向克隆到带有卡那霉素抗性和增强绿色荧光蛋白的真核表达载体pGPH1／GFP／Neo中H1启动子的下游，重组构建RNAi质粒，同时设立阴性对照，并对重组质粒进行酶切分析和DNA序列测定。结果限制性内切酶PstⅠ和BamH Ⅰ酶切显示设计合成的shRNA编码序列被成功插入pGPH1／GFP／Neo质粒中，测序结果证实插入片断与设计序列完全一致。结论针对人Bcl-2的shRNA真核表达载体成功构建及鉴定为进一步研究Bcl-2功能奠定了良好的基础。     

构建干扰RhoA的短发夹RNA真核表达载体

目的利用pGenesil-1质粒构建针对RhoA的短发夹RNA(shRNA)表达载体。方法设计2个shRNA结构的互补DNA序列,经退火成双链,胶回收酶切pGenesil-1大片段,T4DNALigase连接酶切大片段和DNA序列,得到质粒pGenesil-1-RhoA1和pGenesil-1-RhoA2.转化感受态细胞DH5a,扩增,提取重组质粒进行酶切鉴定。结果成功构建靶向RhoA的shRNA重组质粒载体.酶切鉴定和测序分析重组质粒,shRNA编码序列与设计的片段完全一致,经酶切凝胶电泳证实载体构建成功。结论表达靶向RhoA的shRNA表达框成功构建在重组质粒载体上。     

bcr／abl特异性siRNA真核表达载体的构建及鉴定

目的:构建ber/abl的特异性siRNA真核细胞表达载体,并初步探索对K562细胞bcr/abl mRNA和P210蛋白的影响.方法:根据GenBank数据库提供的bcr/abl基因核苷酸序列,按照Tusch Ⅰ设计原则,选择设计双链小干扰RNA(siRNA),再转化为能表达其小发卡结构RNA(shRNA)的DNA序列,并与pTER质粒定向连接,构建受控于人RNA聚合酶Ⅲ启动子H1的真核表达载体pTER117,经限制性内切酶酶切和DNA测序进行鉴定;在脂质体的介导下转染K562细胞,用RT-PCR分析bcr/abl mRNA的表达,细胞化学染色检测P210蛋白的表达.结果:构建bcr/abl融合基因siRNA真核表达载体pTER117经限制性内切酶酶切和DNA测序证实与设计完全一致,转染K562细胞24 h后,pTER117使bcr/abl mRNA的相对水平下降50%,使P210蛋白下降47%.结论:bcr/abl融合基因siR-NA真核细胞表达载体构建成功,并有效干扰K562细胞bcr/abl的表达.     

大鼠脑源性神经营养因子基因克隆及其真核表达载体的构建

目的 克隆大鼠脑源性神经营养因子(BDNF)基因的表达序列,构建大鼠BDNF基因真核表达载体.方法 以RT-PCR从大鼠脑组织总RNA中扩增BDNF cDNA,将其克隆到真核表达载体pcDNA3中构建重组质粒pcDNA3-BN,以限制酶酶切鉴定和DNA序列分析鉴定重组质粒.结果 RT-PCR产物为783bp特异片段,重组质粒酶切后产生783bp和5.2 kb的片段,DNA测序证实783bp片段的碱基序列与大鼠BDNF基因序列完全一致.结论 成功克隆大鼠BDNF基因表达序列并构建了BDNF基因真核表达栽体pcDNA3-BN.     

ATX短发夹shRNA表达载体的构建和测序

目的 构建ATX基因重组表达载体,并进行测序鉴定,为下一步探索肿瘤基因治疗的途径打下基础.方法 设计有小发夹结构的两条DNA序列,经退火成互补双链,再克隆至Psilencer2.1-U6 neo质粒载体中构建重组表达载体,转化DH5a菌株,提取质粒行酶切鉴定后,进行序列鉴定.结果 成功构建了ATXshRNA重组表达载体.结论 ATX shRNA质粒表达载体的成功构建为研究ATX靶向RNA干扰抗肿瘤的作用打下基础.     

乙型肝炎病毒preS基因小分子干扰RNA真核表达载体的构建和鉴定

目的以乙型肝炎病毒（HBV）preS基因区为靶位，构建表达siRNA的质粒载体。方法根据Genbank数据库提供的乙型肝炎病毒preS基因核苷酸序列，根据siRNA设计原则和程序，设计2个靶向preS基因的发夹状siRNA，克隆到pSUPER．puro载体中，构建重组质粒，并进行鉴定分析。结果酶切、PCR鉴定以及测序证实重组质粒构建成功。结论利用RNA干扰技术可成功构建siRNA表达载体，为进一步探索卵巢癌基因治疗奠定了基础。     

CD137-CD137L信号通路通过活化TNF受体相关因子6促进小鼠动脉粥样硬化斑块内血管新生#br#

目的　探讨CD137-CD137L信号通路是否通过活化TNF受体相关因子6（TRAF6）促进粥样硬化斑块内血管新生。方法　将小鼠内皮细胞（bEnd.3）和小鼠主动脉环分别分为对照组（培养基中加入10 μg/L TNF-α）、IgG同型对照组（培养基中加入10 μg/L TNF-α+5 mg/L IgG2b）和CD137刺激组（培养基中加入10 μg/L TNF-α+5 mg/L CD137抗体）。利用转染小干扰RNA（siRNA）技术抑制内皮细胞和主动脉环TRAF6基因表达,将内皮细胞和主动脉环分别分为对照siRNA组（转染对照siRNA）和TRAF6 siRNA组（转染TRAF6 siRNA）。分别采用Western blot和实时荧光定量PCR（qPCR）检测内皮细胞和主动脉环TRAF6、血管内皮生长因子（VEGF）、Smad1/5蛋白和mRNA的表达;采用Transwell迁移实验检测内皮细胞的迁移能力;内皮细胞管腔形成实验检测内皮细胞的管腔形成能力;主动脉环血管新生实验检测主动脉环新生微血管形成能力。结果　CD137刺激组内皮细胞和主动脉环TRAF6、VEGF蛋白和mRNA表达水平均高于对照组和IgG同型对照组（均P＜0.05）。与对照siRNA组比较,TRAF6 siRNA组内皮细胞和主动脉环TRAF6、Smad1/5蛋白和mRNA表达水平明显降低,内皮细胞迁移数量减少,内皮细胞小管长度和分支数量降低,主动脉环新生微血管数量减少（均P＜0.05）。结论　CD137-CD137L信号通路可能通过TRAF6促进小鼠动脉粥样硬化斑块内血管新生。     

Quantitative and temporal analysis of gene silencing in tumor cells induced by small interfering RNA or short hairpin RNA expressed from plasmid vectors

Vector-based RNA interference (RNAi) has attracted great interest, because of its more prolonged gene silencing effect compared with small interfering RNA (siRNA). However, the intensity and duration of vector-based RNAi effect has received little attention. In this study, the gene silencing kinetics of short hairpin RNA (shRNA)-expressing plasmid DNA (pDNA) driven by U6, H1 or tRNA promoter (pU6-shLuc, pH1-shLuc, and ptRNA-shLuc) was studied in melanoma cells expressing firefly luciferase. A bootstrap method-based moment analysis was performed to statistically and quantitatively evaluate the profile of gene silencing. The analysis showed that pU6-shLuc induced a significantly greater and longer gene silencing than that produced by other promoter-driven shRNA expression vectors. In addition, it was found that pU6-shLuc was at least 100-fold more potent in gene silencing than siRNA targeting the same gene on a numerical basis. These statistical considerations demonstrated that U6 promoter-driven shRNA expressing pDNA is the most effective in inducing gene silencing effect as far as the intensity and duration of RNAi effect is concerned.     

Future promise of siRNA and other nucleic acid based therapeutics for the treatment of chronic HCV

RNA interference (RNAi) is gaining favor as a potential therapeutic option for the treatment of Hepatitis C virus infections. RNAi, first discovered in plants, induces sequence specific degradation of messenger RNA following the introduction of short interference RNA (siRNA). RNAi is a natural defense mechanism used by plants to combat viral infections, and the discovery of RNAi activity in mammalian cells has prompted several drug companies to investigate and exploit RNAi based drugs as a potential therapy against HCV infections. A number of research groups have demonstrated that strong RNAi activity can be induced against HCV using synthetic siRNA duplexes as triggers, or by expressing short hairpin RNAs from plasmid or viral vectors. However, much work remains to improve delivery, maintain specificity and limit the development of virus resistance. HCV is capable of evading RNAi activity through the incorporation escape mutations within the siRNA target sequence, highlighting the importance of implementing strategies to limit the development of resistance. Other nucleic acid based therapies such as antisense oligonucleotides, RNA aptamers and ribozymes have also been considered for use as HCV therapeutics, and we will outline the potential opportunities and obstacles to their use as well as RNAi.     

Interference of porcine reproductive and respiratory syndrome virus replication on MARC-145 cells using DNA-based short interfering RNAs

Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease in swine-producing areas of the world. Many vaccine strategies developed to control the disease are not yet completely successful. The objective of this study was to determine if RNA interference (RNAi) could be utilized to inhibit PRRSV replication on MARC-145 cells. Four short interfering RNA (siRNA) sequences (N95, N179, N218 and N294) directed against a well-conserved region of PRRSV genome ORF7 gene were selected. Sense and antisense siRNA encode sequences separated by a hairpin loop sequence were designed as short hairpin RNA (shRNA) expression cassettes driven by mouse U6 promoter. Using a polymerase chain reaction (PCR)-based approach, shRNAs were generated from shRNA expression cassettes. The PCR products were cloned into pEGFP-N1 vector and shRNA expression vectors were constructed. When MARC-145 cells were transfected with shRNA expression vectors and then infected with PRRSV, N179 was found to be the most effective inhibition site in decreasing cytopathic effect (CPE) induced by PRRSV. Western blot, indirect immunofluorescence and fluorescence quantitative PCR (FQ-PCR) confirmed that the expression of ORF7 was reduced both at protein and RNA levels comparing to controls. The results presented here indicated that DNA-based siRNA could effectively inhibit the replication of PRRS virus (approximately 681-fold reduction of viral titers) on MARC-145 cells.     

Nonviral vector-mediated RNA interference: Its gene silencing characteristics and important factors to achieve RNAi-based gene therapy

RNA interference (RNAi) is a potent and specific gene silencing event in which small interfering RNA (siRNA) degrades target mRNA. Therefore, RNAi is of potential use as a therapeutic approach for the treatment of a variety of diseases in which aberrant expression of mRNA causes a problem. RNAi can be achieved by delivering siRNA or vectors that transcribe siRNA or short-hairpin RNA (shRNA). The aim of this review is to examine the potential of nonviral vector-mediated RNAi technology in treating diseases. The characteristics of plasmid DNA expressing shRNA were compared with those of siRNA, focusing on the duration of gene silencing, delivery to target cells and target specificity. Recent progresses in prolonging the RNAi effect, improving the delivery to target cells and increasing the specificity of RNAi in vivo are also reviewed.     

稳定整合靶向EGFL7基因的shRNA载体的胶质瘤细胞系的建立

目的构建针对EGFL7基因的特异性RNA干扰载体,建立稳定转染该干扰载体的人胶质瘤细胞系。方法根据EGFL7基因的编码序列设计并合成针对EGFL7基因的特异性shRNA,将其克隆入pSilencer3.1-H1neo载体中,重组载体经脂质体LipofectamineTM2000介导转染胶质瘤细胞株U251;转染细胞经G418筛选,采用Westernblot方法在蛋白水平检测筛选出的G418抗性的克隆细胞。结果酶切鉴定和测序证实,成功构建了靶向EGFL7基因的shRNA真核表达载体pSilencer-shEGFL7,并获得了稳定表达靶向EGFL7基因的shRNA的胶质瘤细胞株。结论 EGFL7基因特异性RNA干扰载体能够显著抑制EGFL7基因在U251细胞中的表达,这为进一步研究EGFL7在胶质瘤细胞系U251中的生物学功能和作用机制奠定了基础。     

IRAK1/TRAF6通路在急性髓系白血病发生发展中的作用机制研究

目的　探讨白细胞介素-1受体相关激酶（IRAK1）/肿瘤坏死因子受体相关因子6（TRAF6）通路影响急性髓系白血病（AML）发生发展的作用机制。方法　选取54例AML患者为研究对象（AML组）,30例于本院治疗的缺铁性贫血患者作为对照组,分离所有受试者骨髓单核细胞;体外常规培养人AML细胞系HL-60、KG-1、U937和人正常单核细胞系THP-1;将HL-60细胞分为空白组（转染Lipofectamine 3000试剂）、阴性对照（NC）组（转染空质粒）、IRAK1短发夹RNA（IRAK1 shRNA）组（转染IRAK1 shRNA）;实时荧光定量PCR（qPCR）法检测单核细胞及不同细胞系各组中IRAK1、TRAF6 mRNA表达水平;CCK-8法检测各组细胞增殖抑制率;流式细胞术检测各组细胞凋亡率;蛋白免疫印迹法检测各组细胞β-连环蛋白（β-catenin）、细胞周期蛋白D1（CyclinD1）、B淋巴细胞瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）及IRAK1/TRAF6通路蛋白表达水平。结果　与对照组比较,AML组单核细胞中IRAK1、TRAF6 mRNA表达水平显著升高（P＜0.05）。与THP-1细胞比较,KG-1、U937、HL-60细胞中IRAK1、TRAF6 mRNA表达水平显著升高,且HL-60细胞最高（P＜0.05）。与空白组和NC组比较,IRAK1 shRNA组细胞增殖抑制率、凋亡率及Bax蛋白表达显著升高,IRAK1 mRNA、TRAF6 mRNA、β-catenin、CyclinD1、Bcl-2、磷酸化-IRAK1（p-IRAK1）/IRAK1、TRAF6、核因子-кB（NF-кB）蛋白表达显著降低（P＜0.05）。结论　IRAK1/TRAF6通路与AML的发生发展有关,抑制IRAK1/TRAF6通路可抑制人AML细胞系HL-60增殖,促进其凋亡,IRAK1/TRAF6可能是AML治疗的潜在靶点。     

大鼠VR1基因siRNA表达载体的构建及体内研究

目的探讨辣椒素受体(vanilloid receptor subtype1,VR1)在慢性疼痛发生机制中的作用,构建携带VR1siRNA的慢病毒载体并检测其对大鼠DRG神经元VR1基因的干扰作用。方法设计靶向大鼠VR1基因的发夹状siRNA,合成两对互补的寡核苷酸序列,退火后克隆到酶切的pRNAT-U6.2/Lenti siRNA载体,通过PCR和DNA测序鉴定重组质粒。空载体及重组质粒分别与慢病毒包装质粒共转染293T细胞生成慢病毒载体。通过蛛网膜下腔将慢病毒载体注射到大鼠体内,采用RT-PCR方法检测L4-L6DRG神经元内VR1的沉默效果。结果测序鉴定证实目的寡核苷酸片段被克隆到pRNAT-U6.2/Lenti载体中;经蛛网膜下腔注射后,pRNAT-U6.2/Lenti-siVR1可下调正常大鼠L4-L6DRG神经元内VR1mRNA的表达。结论成功构建了靶向VR1基因的siRNA载体,可有效抑制VR1mRNA的表达,为深入研究和治疗慢性痛提供了有力的工具。