RNA干涉技术在胶质瘤基因治疗研究中的进展

RNA干涉(RNA interfernce,RNAi)机理为利用双链RNA(dsRNA)特异性地降解相应序列的mRNA成为siRNA,从而特异性地阻断靶基因的表达,最近对其在应用于胶质瘤基因治疗方面的研究也有进展。本文介绍了RNA干扰的分子机制、技术应用及在胶质瘤基因治疗方面的进展和广阔的发展前景。     

RNAi技术及其在胶质瘤中的研究进展

<正>RNA干扰(RNA interference,RNAi)是一种在动植物中广泛存在的,由双链RNA(double-stranded RNA,dsRNA)介导的序列特异性基因沉默现象。由于RNA干扰可以抑制特定基因的表达,至今短短十几年的时间,RNAi技术已迅速发展成为一种成熟的基因沉默工具,对生命科学研究产生了深远的影响。已广泛用于研究基因表达及调控,信号传导通路,药物的作用机制,肿瘤的临床治疗等诸多领域,现将RNAi技术及其在胶质瘤研究中的应用进展综述如下。     

RNA干扰技术在胶质瘤治疗中的应用

RNA干扰（RNA interference，RNAi）是生物体中普遍存在的、细胞本身固有的对抗外源基因侵害的一种自我保护现象。其作为新兴的基因表达阻断技术，目前已成功用于基因功能和肿瘤相关基因等方面的研究，为临床上特异性的基因干预治疗开辟了一条新的途径。     

RNA干扰在治疗脑胶质瘤中的研究

脑胶质瘤是颅内肿瘤中一类高发病率、高致残率、高死亡率的肿瘤,但目前,其临床治疗效果难以令人满意。如何提高疗效、改善预后是神经外科工作者研究的重点。RNA干扰(RNAi)技术作为一项新的基因治疗技术,因其独特的作用机制和优越性,正越来越受到研究者的重视。近几年,RNAi在治疗脑胶质瘤研究方面不断深入,对多种干扰策略中多个基因靶点的研究都取得了进展,并朝着联合干扰的方向发展,同时在载体和给药途径等技术环节上也有了很大改进。RNAi技术的发展将为脑胶质瘤的治疗带来新的希望。     

脊髓源星形胶质细胞GFAP表达抑制真核表达载体构建及最佳短发夹样RNA分子筛选

目的构建并筛选大鼠胶质原纤维酸性蛋白（GFAP）表达抑制短发夹样RNA（shRNA）真核表达载体。方法针对GFAP基因全编码序列设计并合成三对9bp茎环结构、19bp干扰序列特异性shRNA模板，体外定向克隆构建特异性重组质粒真核表达载体；通过体外大鼠脊髓源星形胶质细胞GFAP表达抑制模型，脂质体介导RNA干扰分子转染，实时荧光定量RT—PCR及Wesem blot技术观察RNA干扰后原代星形胶质细胞GFAP表达抑制效果．筛选最佳GFAP表达干扰抑制真核表达载体。结果序列测定证实GFAP—shRNA重组质粒真核表达载体构建成功，三对shRNA模板在mRNA及蛋白表达水平抑制靶基因表达效率分别为81％、63％、56％。结论高效率的GFAP—shRNA真核表达载体在大鼠原代星形胶质细胞GFAP表达抑制模型中能高效抑制GFAP基因表达，为后续多靶点RNA干扰技术在脊髓损伤胶质瘢痕抑制基因治疗中的应用奠定了前期基础。     

RNA干扰及其在神经变性性疾病研究中的应用

RNA干扰(RNAi)是指生物体内利用具有同源性的双链RNA(dsRNA)诱发序列特异的转录后基因沉默(PTGS)的现象，它可以通过抑制蛋白表达模拟基因敲除技术。RNAi主要通过dsRNA被核酸酶Dicer切割成21～25nt的小干扰RNA(siRNA)，由siRNA介导识别并靶向切割同源mRNA分子而实现。随着研究的不断深入，RNAi的作用机制将逐步被阐明，其技术也将日趋完善和成熟，并将得到广泛的应用。本文就RNAi技术的研究进展及其在阿尔茨海默病、帕金森病等神经变性性疾病研究中的应用作一综述。     

RNA干扰技术及其在神经科学研究中的应用

RNA干扰(RNAi)是指生物体内由双链RNA介导同源序列mRNA的特异性降解，从而导致基因沉默的现象。近年来，随着对RNAi研究的不断深入，其作用机制正在逐步被阐明；同时作为阻断基因表达的新手段，RNAi技术也日趋完善和成熟。以其高效性和特异性，RNAi为反向遗传学在神经科学研究中的应用提供了有力的工具。本文仅就RNAi技术的有关研究进展及其在神经科学研究中的应用作一概述。     

RNA干扰技术原理及其在脑胶质瘤治疗中的应用

RNA干扰是dsRNA在转录后水平特异性降解目的基因mRNA,导致该基因沉默的现象。脑胶质瘤是颅内最常见的恶性肿瘤,传统的手术、放、化疗均不能达到彻底治愈的目的。RNA干扰在脑胶质瘤治疗中的应用,可能会取得更好的效果。本文就RNA干扰技术原理及其在脑胶质瘤治疗中的应用的研究进展作一综述。     

RNAi及其基因治疗研究进展

RNAi(RNA inter ference)即RNA干扰,是外源双链RNA(double-stranded RNA,dsRNA)分子在mRNA水平关闭相应序列基因表达所致的细胞内有效、特异性基因封闭,是广泛存在于生物界的古老现象,是生物体抵御病毒或其它外来核酸入侵以及保持自身遗传稳定的保护性机制,以其高效率,高特异性等特性,较反义核酸、核酶、三链RNA等基因沉默技术显示了独特优势及广泛的应用前景,本文就其特点、分子机制及其在基因治疗等方面的探索进行综述.     

RNA干扰与肿瘤的基因治疗

RNA干扰(RNAi)是指双链RNA(dsRNA)在细胞内特异性诱导同源互补的mRNA降解,从而阻断相应基因表达的现象.随着哺乳动物RNA干扰的研究不断取得进展,RNA干扰显示出作为一种新型肿瘤基因治疗方法的巨大优越性.初步的实验结果表明,用RNA干扰治疗癌症的设想有可能变成现实.     

匹罗卡品致(癎)大鼠海马γ-氨基丁酸能中间神经元生长抑素和微清蛋白mRNA表达研究

目的 观察匹罗卡品致(癎)大鼠海马γ-氨基丁酸能中间神经元生长抑素(SS)mRNA和微清蛋白(PV)mRNA表达水平变化,拟从基因水平探讨其表达阳性γ-氮基丁酸能中间神经元在颞叶癫(癎)发生发展中的作用.方法 建立匹罗卡品致(癎)大鼠模型,采用原位杂交法检测各观察时间点海马SSmRNA和PVmRNA表达阳性神经元数目.结果 模型组大鼠海马各区γ-氨基丁酸能中间神经元SSmRNA表达水平均于出现癫(癎)持续状态后3d降低最为显著(均P=0.000),随后逐渐升高;至发病后60d,海马CA3区SSmRNA表达水平高于对照组(t=1.021,P=0.005),海马门区(t=3.211,P=0.009)和CA1区(t=1.902,P=0.048)则仍低于对照组.模型组大鼠海马门区γ-氨基丁酸能中间神经元PVmRNA表达水平于出现癫(癎)持续状态后6h开始降低,至发病后60d降低最为显著(均P=0.000);海马CA1区PVmRNA表达水平于发病后3d降低最为显著(均P=0.000),随后逐渐升高但仍低于对照组(江2.216,P=0.048);癫(癎)持续状态早期,海马CA3区PVmRNA表达水平无明显变化,至发病后7d逐渐升高且高于对照组(t=1.021,P=0.005).结论 γ-氨基丁酸能中间神经元SSmRNA和PVmRNA表达水平的下调可能在颞叶癫(癎)的发生中起重要作用,至慢性期γ-氨基丁酸能中间神经元SSmRNA和PVmRNA表达水平的恢复或上调可能与颞叶癫(癎)的发展或修复有关.γ-氨基丁酸能中间神经元数目的 变化,部分是由于其标志物mRNA表达水平的调节所致,并非神经元数目变化的唯一因素.     

不同保存方法对脑胶质瘤和其周边组织RNA保存的研究

目的：探讨不同保存方法对脑胶质瘤和其周边组织RNA保存的影响,为脑胶质瘤组织库规范化建设提供理论依据。方法：选择复旦大学附属华山医院脑胶质瘤组织库中采用新（RNALater法组5组）、旧（速冻法组4组）两种方法保存的脑胶质瘤组织共9组27份样本（包含肿瘤组织及其配对的瘤周组织）,分别抽提总RNA,测定其A260/280比值,凝胶电泳判断其完整性,比较不同保存方法对脑胶质瘤和其周边组织RNA保存的影响。结果：RNA质检合格率RNALater法组为100％,速冻法组为33．3％,两者差异有统计学意义（P〈0．05）。质检结果为速冻法组中3份肿瘤组织合格,1份近瘤周组织合格,无远瘤周组织合格。结论：RNALater在保存RNA、预防RNA降解方面优于速冻法组。     

A study on postmortem stability of vasopressin messenger RNA in rat brain compared with those in total RNA and ribosomal RNA

Summary Postmortem stability of arginine-vasopressin (AVP) messenger RNA (mRNA) in the rat brain was studied comparing changes with those in the recovered amounts of total RNA and ribosomal RNA (rRNA). The amount of AVP mRNA and rRNA showed a decrease with increasing time interval after death (postmortem time), whereas the amount of total RNA did not alter with postmortem time. The half-life of AVP mRNA in the rat postmortem seemed to be approximately 16 hrs. The analysis of the ratio of AVP mRNA to 18S-rRNA suggested that AVP mRNA was degraded postmortem more rapidly than rRNA. These results suggest that autopsied human brains should be used for AVP mRNA study within a short postmortem time.     

A rapid microprocedure for isolating RNA from multiple samples of human and rat brain

In order to establish a routine procedure for isolating undegraded RNA from small amounts of rat and human brain tissue, several techniques were investigated. Initial studies demonstrated that undegraded RNA could not be reproducibly isolated from milligram amounts of brain tissue homogenized in an aqueous medium. Several isolation techniques utilizing tissue homogenization in the denaturing agent guanidinium chloride were compared. This method of homogenization, followed by sedimentation of RNA through cesium chloride, resulted in good yields of undegraded translationally active RNA. A maximum of 6 RNA samples could be processed simultaneously. In contrast, when homogenization in guanidinium chloride was followed by repeated guanidinium chloride-ethanol precipitations many samples could be processed simultaneously. The resulting RNA yields were low. The introduction of several modifications in the guanidinium chloride-ethanol precipitation technique resulted in a high yield of undegraded translationally active RNA. DNA was removed by two guanidinium-ethanol precipitations. Residual protein was digested with proteinase K. RNA was precipitated after extraction with phenol-chloroform-isoamyl alcohol. This refined procedure allows the recovery, in high yields, of translationally active undegraded RNA which is both DNA and protein free. Thirty-six samples can be processed in one day.     

Quantitative cytochemical studies of RNA in experimental mercury poisoning

Summary Adult male rats were intoxicated with methylmercuric chloride (CH₃HgCl) or mercuric bichloride (HgCl₂) at a daily dosage of 1.0 mg/kg body weight for various length of periods. Single neurons were dissected out from the dorsal root ganglia with the aid of a de Fonbrune micromanipulator. The RNA of these neurons was extracted and the base composition of the RNA was analyzed by the microphoretic technique of Edström.Although methylmercury and short-termed mercuric bichloride intoxication induced a marked reduction of the total RNA content in these neurons, there was no change in the base values under these conditions. Change in the RNA base composition and ratios were detected after prolonged mercuric bichloride intoxication (11 weeks). The guanine value was increased from 32 to 37 and the cytosine value was decreased from 28 to 21 with a consequent shift of the G/C ratio from 1.16 to 1.73 and the A+G/C+U ratio from 1.12 to 1.33. The change in the RNA base composition occurred at the same period where there was an increased activity of RNA production. Since some animals showed signs of increasing tolerance and recovery from the mercury toxicity at the same time, it can be speculated that the newly produced RNA may be responsible for these phenomena.     

特异性shRNA干扰人慢病毒载体抑制U251细胞株Chk1、Chk2基因的表达

目的构建针对细胞周期检测点激酶1和2(Chk1和Chk2)基因的短发夹RNA(shRNA)表达载体,包装成慢病毒,建立稳定转染的细胞株,为探讨抑制Chk1和Chk2基因表达对脑胶质瘤细胞生物学行为调控的研究奠定基础。方法根据GenBank数据库提供的Chk1和Chk2基因核苷酸序列,选择设计2条能转录短发夹RNA(shRNA)的DNA序列,命名为Chk1-shRNA和Chk2-shRNA,同时设计1条非特异性序列作为阴性对照,命名为blank-shRNA。并与pLKO.1-TRC质粒载体连接,转化感受态大肠杆菌,挑取阳性克隆,抽取重组质粒,使用限制性内切酶EcoRⅠ、NcoⅠ酶切电泳,DNA测序鉴定,包装慢病毒。3组重组表达慢病毒载体转染胶质瘤细胞系U251,用嘌呤霉素筛选后挑选单克隆并扩增获得稳定株。逆转录酶-聚合酶连反应(RT-PCR)和Western blot分别在mRNA和蛋白水平上检测Chk1和Chk2的表达。结果重组质粒成功转化感受态大肠杆菌,经酶切琼脂糖凝胶电泳分析,结果表明寡核苷酸成功插入到预计位点,经测序鉴定,序列完全正确。嘌呤霉素对U251细胞的筛选浓度为4ug/ml,筛选出稳定转染三种质粒的U251细胞,Chk1-shRNA和Chk2-shRNA组细胞各自Chk1和Chk2的mRNA和蛋白表达水平明显低于blank-shRNA组。结论成功构建了针对Chk1和Chk2基因的shRNA慢病毒表达载体,转染后可抑制ChK1和Chk2基因的表达,为进一步研究Chk1和Chk2基因在脑胶质瘤细胞中的作用奠定了基础。     

RNA synthesis and processing in the gerbil brain after transient hindbrain ischaemia

Ribonucleic acid (RNA) synthesis was investigated in gerbils subjected to 15 min transient hindbrain ischaemia using [2-¹⁴C]uridine autoradiography. Distribution of synthesized RNA in the subcellular fraction of the tissue was detected by differential centrifugation and density gradient separation using Whittaker's method. In [2-¹⁴C]uridine autoradiography, uptake of the tracer into the RNA fraction was not reduced after transient ischaemia. Distributional analysis of [2-¹⁴C]uridine in the subcellular fractions revealed that tracer activity in the P₃ (microsomes) fraction decreased in the ischaemic regions and tended to decrease in the P₄ (ribosomes) fraction, although not significantly. Tracer activity in the P₁ (nuclei and cell debris) and P₂ (mitochondria, myelin and nerve ending particles) fractions did not decrease. These results indicate that RNA synthesis in the nuclei is not inhibited by ischaemia, but RNA processing is disturbed by the level of the transport. Modification of RNA synthesis and processing by transient ischaemia may influence protein synthesis.