RNA干扰技术及其应用

RNA干扰(RNA in terference,RNA i)技术是抑制和逆转基因表达的新型生物学研究工具.论述了RNA i的作用机制及其在基因组学、基因治疗、药物探查等方面的应用情况.d sRNA在酶切作用下产生siRNA,后者与RNA诱导沉默复合物R ISC结合,共同降解靶mRNA,从而达到阻断特异基因表达的目的.该技术已对基因功能、基因治疗和药物筛选等方面的体外实验产生了深刻的影响,但由于存在一些尚未解决的问题,这项技术尚未进行大规模临床应用.     

D-sn-GPHEEDA的制备及其脂质纳米粒的评价

优化以二油酰基卵磷脂为原料的1,2-二油酰-sn-甘油-3-磷脂酰羟乙基乙二胺合成工艺,并评价脂质纳米粒的理化特性、siRNA结合力、血清稳定性和细胞毒性。通过HPLC、LC-MS、1H-NMR、13C-NMR表征确证其结构,考察了酶的用量、反应时间、反应物用量、两相反应体系比对1,2-二油酰-sn-甘油-3-磷脂酰羟乙基乙二胺合成的影响。结果表明,最佳反应条件为酶用量150 IU、最佳反应时间72 h、二油酰基卵磷脂与乙二胺的投料比1∶15、乙酸乙酯与乙酸钠缓冲溶液的体积比2∶1时,产率达68.72%。所得脂质纳米粒的粒径为（102.100 ± 2.495） nm,平均多分散指数为0.183 ± 0.023,Zeta电位为+（28.90 ± 2.46） mV,外观呈圆形,稳定性良好;在N/P比为12∶1时可与 siRNA 完全结合,可显著提高 siRNA 的血清稳定性;MTT 结果表明所合成磷脂及其 LNPs 对293T细胞活力无显著影响。该研究为其他磷脂酰化合物的合成及纳米递送载体的应用提供参考。     

可抑制 I 型胶原表达的siRNA 重组腺病毒的构建

滑膜间质干细胞(SMSCs)与其他来源的间质干细胞相比,具有更强的生长能力和更大的软骨分化潜力．但由于 SMSCs 固有地表达 I 型胶原,这将会大大损害再生软骨的整体质量．为解决此问题,文中首先设计了可抑制 I 型胶原表达的 shRNA,并将其构建到腺病毒载体上．接着通过 Pac I 酶切反应,使重组腺病毒载体线性化并通过脂质体转染293 细胞进行病毒包装．收集的病毒初液进行滴度测定后,再次通过 293 细胞进行病毒扩增,经过纯化后,最终获得高滴度重组腺病毒．将重组腺病毒分别感染成纤维细胞和成骨细胞,通过荧光显微镜观察,检测转染效率,利用实时定量 PCR 实验验证 siRNA 的干扰效果．实验结果表明,包装后的重组腺病毒具有高的转染效率,能成功编译 siRNA,并具有抑制 I 型胶原表达的能力．     

在Hep G2细胞中利用siRNA沉默GFP基因的实验探讨

目的:通过探讨小分子双链RNA(siRNA)通过RNA干扰(RNAi)机制沉默肝癌细胞株Hep G2荧光素报告基因GFP的各项生物学指标,为进一步实验和临床研究提供依据.方法:通过体外实验,对siRNA和siRNA脂质体的稳定性和细胞毒性进行评估;通过荧光标记技术,研究不同时间siRNA脂质体的转染效率;通过荧光显微镜下观察转染细胞和RT-PCR检测靶基因mRNA表达,确定不同种类、形式、剂量的siRNA对靶基因的沉默效能.结果:在空白培养液中,siRNA和siRNA脂质体可稳定至4h,在5%血清中2～4h后明显降解;100nM的siRNA和siRNA脂质体无明显细胞毒性;siRNA脂质体转染细胞株的效率随时间不同而不同,4h可达90%;非特异性siRNA/脂质体无沉默靶基因表达作用,特异性siRNA/脂质体对靶基因的沉默作用在一定范围内随剂量升高(20nM、50nM、100nM)而升高.结论:siRNA的稳定性可满足实验需要,且无明显细胞毒性,特异性siRNA转染肝癌细胞株能有效抑制靶基因表达,用脂质体转染可提高转染效率.siRNA通过RNAi机制沉默靶基因表达,为肿瘤的治疗和基础研究提供了新的工具和思路.     

Knockdown of <Emphasis Type=Italic>MRP4</Emphasis> by lentivirus-mediated siRNA improves sensitivity to adriamycin in adriamycin-resistant acute myeloid leukemia cells

Chemotherapy remains the standard treatment for acute myeloid leukemia;however,the emergence of drug resistance is a major hurdle in the successful treatment of leukemia.The expression of multidrug resistance-associated protein 4(MRP4)induces re- sistance in the adriamycin-resistant acute myeloid leukemia cell line,K562/ADR.The aim of this study was to investigate whether knockdown of MRP4 by lentivirus-mediated siRNA could improve the sensitivity of K562/ADR cells to adriamycin.Five lenti- virus-mediated short hairpin RNAs(lv-shRNAs-MRP4)were designed to trigger the gene silencing RNA interference(RNAi) pathway.The efficiency of lentivirus-mediated siRNA infection into K562/ADR cells was determined using fluorescence mi- croscopy to observe lentivirus-mediated GFP expression.MRP4 expression in infected K562/ADR cells was evaluated by real- time PCR and Western blot analysis.The MTS assay was used to measure cell viability and flow cytometry was used to measure apoptosis.The transfection efficiency of K562/ADR cells was over 80 percent.The gene silencing efficacy of lv-shRNA1-MRP4 was superior to the other constructs.Infection of K562/ADR cells with lv-shRNA1-MRP4 led to strong inhibition of MRP4 mRNA and protein expression.Combined treatment with lv-shRNA1-MRP4 and adriamycin decreased cell growth and increased apoptosis compared to treatment with lv-shRNA1-MRP4 or adriamycin alone.These data indicate that in K562/ADR cells MRP4 is involved in drug resistance mechanisms and that lentivirus-mediated knockdown of MRP4 may enhance sensitivity to adriamycin.     

Delayed resistance to Cucumber mosaic virus mediated by 3’UTR-derived hairpin RNA

RNA silencing has been shown to function in the plant antivirus defense response, leading to viral RNA degradation induced by vsiRNA-containing RISC cleavage activity. Cucumber mosaic virus （CMV） 3′UTR sequences share a high conservation of nucleotide sequence and secondary structures that are important for CMV replication. Here, in an attempt to simultaneously target the multiple genomic and subgenomic RNAs of CMV for degradation, CMV 3′UTR were used to design hairpin RNA （hpRNA） to transform tobacco （Xanthi. nc） so as to constitutively produce viral siRNAs. Most of the transgenic plants expressing CMV Q strain （Q-CMV, subgroup Ⅱ strain） RNA3 3′UTR-derived hpRNA showed delayed resistance to Q-CMV infection and exhibited recovery phenotypes. Compared with Q-CMV-inoculated leaves, the upper leaves showed weak or no disease symptoms and a reduced accumulation level of viral RNAs. Together with transient assays, our results indicate that the 3′UTR-derived siRNAs were biologically active in targeting viral RNA for degradation. Recovery resistance in transgenic plants was also observed against subgroup IB strain SD-CMV infection, indicating a broad-spectrum anti-CMV effect of the 3′UTR-based antiviral silencing. Northern blot assays indicated that there was no strong correlation between the degree of resistance and the accumulation level of 3′UTR-derived siRNAs, suggesting that to target a highly structured RNA, such as the CMV 3′UTR, the quantity of siRNAs may not be the only determinant of silencing efficiency. Target RNA secondary structures may also affect target accessibility, siRNA-containing RISC-target recognition and the consequent antiviral effect.     

大鼠血管内皮细胞siRNA转染条件的优化

目的:旨在优化siRNA转染大鼠血管内皮细胞的转染条件.方法:将0.75μL和1.5μL的脂质体LipofectamineTM3000分别与20、40、60、80nmol带荧光标记的FAMsiRNA组合,转染6、12、18、24h后,用荧光显微镜计数阳性细胞率、MTT法检测各浓度条件下内皮细胞的存活率,筛选最优转染条件.结果:(1)转染12h后,用荧光显微镜检测20、40、60、80nmol各组,均可观察到绿色荧光(2)siRNA浓度为60nmol/L,脂质体为1.5μL的组合转染效率最高,继续增加siRNA的浓度,转染效率提高不明显.(3)转染时间超过24h,各组细胞荧光减弱,细胞死亡率显著增加.结论:结果表明,以1.5μL LipofectamineTM3000与60nmol/L的siRNA浓度组成转染混合物转染12h可以实现对大鼠血管内皮细胞高效转染并保持较高的细胞活性.     

小分子RNA家族中的新成员--microRNA

近来,人们在多种生物中发现了一类新的小分子RNA——microRNA(miRNA)．miRNA是20～24nt的单链RNA,在进化上具有高度的保守性,它通过与靶mRNA不完全互补配对,抑制蛋白翻译,调节内源基因表达,在基因调控中扮演了重要的角色．miRNA和siRNA(small interferenceRNA)在生成机制、作用途径等方面关系密切,它们既有区别又相互联系．小分子RNA的研究将是今后分子生物学的研究热点之一．     

RNAi与siRNA在医学中应用的新进展

RNAi（RNA interference,RNAi）是序列特异的双链RNA（dsRNA）使细胞内同源性mRNA降解,产生特异的基因沉默的过程．RNAi最早是由Fire在线虫实验中发现并提出的．随后,在其他动物包括哺乳动物中也发现了RNAi．RNAi技术是一种发展很快并有应用前景的基因控制技术,目前已广泛地应用于多学科的研究,尤以医学领域的研究最为突出,它不仅可用于基础研究,而且为病毒感染性疾病和肿瘤的防治开辟了更为广阔的前景．