Can we increase the speed and efficacy of antidepressant treatments? Part II. Glutamatergic and RNA interference strategies

In the second part we focus on two treatment strategies that may overcome the main limitations of current antidepressant drugs. First, we review the experimental and clinical evidence supporting the use of glutamatergic drugs as fast-acting antidepressants. Secondly, we review the involvement of microRNAs (miRNAs) in the pathophysiology of major depressive disorder (MDD) and the use of small RNAs (e.g.., small interfering RNAs or siRNAs) to knockdown genes in monoaminergic and non-monoaminergic neurons and induce antidepressant-like responses in experimental animals.The development of glutamatergic agents is a promising venue for antidepressant drug development, given the antidepressant properties of the non-competitive NMDA receptor antagonist ketamine. Its unique properties appear to result from the activation of AMPA receptors by a metabolite [(2 S,6 S;2 R,6 R)-hydroxynorketamine (HNK)] and mTOR signaling. These effects increase synaptogenesis in prefrontal cortical pyramidal neurons and enhance serotonergic neurotransmission via descending inputs to the raphe nuclei. This view is supported by the cancellation of ketamine's antidepressant-like effects by inhibition of serotonin synthesis.We also review existing evidence supporting the involvement of miRNAs in MDD and the preclinical use of RNA interference (RNAi) strategies to target genes involved in antidepressant response. Many miRNAs have been associated to MDD, some of which e.g., miR-135 targets genes involved in antidepressant actions. Likewise, SSRI-conjugated siRNA evokes faster and/or more effective antidepressant-like responses. Intranasal application of sertraline-conjugated siRNAs directed to 5-HT_1A receptors and SERT evoked much faster changes of pre- and postsynaptic antidepressant markers than those produced by fluoxetine.     

靶向人端粒酶反转录酶RNA干扰载体质粒的构建与筛选

目的:设计靶向人端粒酶反转录酶的RNAi序列,构建shRNA表达载体质粒和hTERT真核表达质粒,采用共转染工具细胞筛选出有效靶点.方法:首先以EASYMsiRNA软件针对hTERT设计5个靶点siRNA序列和1条通用阴性对照序列,合成shRNA oligo表达框架,将其分别连接至经酶切消化的载体质粒、pGCsi-U6/Neo/GFP、pGCL-GFP,重组质粒转化1)H5α,阳性克隆进行PCR与测序鉴定;然后从cDNA文库中利用PCR钓取hTERT基因片段,与表达载体pEGFP-C1分别进行双酶切、纯化及定向连接、重组、转化,阳性克隆行PCR与测序鉴定,荧光镜检GFP表达;最后上述2个质粒系统共转染293T细胞,Western印迹检测hTERT蛋白表达水平,筛选最有效序列.应用SPSS16.0软件包对所得数据进行单因素方差分析.结果:BLAST检索5条hTERT siRNA序列均能100%命中,且不与其他基因同源:PCR鉴定及阳性克隆测序验证shRNA oligo与载体质粒成功连接、重组.hTERT基因PCR钓取片段产物大小1.4 Kp,经酶切与质粒连接,形成pEGFP-C1-hTERT表达载体,阳性克隆PCR、测序鉴定确定成功重组;转染293T细胞48h时荧光镜F可观察到GFP标记的hTERT融合蛋白.hTERT表达质粒和shRNA载体质粒共转染293T细胞,48h时Western印迹榆测结果显示:实验各组hTERT蛋白量均有不同程度减少,而内参对照B-aetin和GAPDH尤明显变化.经β-actin校正,得到各组hTERT蛋向相对表达量:KD No.1-5实验组与NC组相比,hTERT基因表达均得到不同程度的敲减(54.61%～76.84%.P＜0.05),其中No.4敲减效能最高.结论:5条RNAi设计序列均可有效、特异性地沉默hTERT基因的转录和表达水平,其中以5'-GCAAGTTGCAAAGCArrGGAA-3'敲减效能最优;构建外源基因的过表达载体质粒转染工具细胞,可作为RNAi敲减效率筛选的验绩标靶.     

RNA干扰Survivin对腺样囊性癌细胞株ACC-M生长的抑制作用

目的:探讨RNA干扰Survivin基因对腺样囊性癌细胞生长的抑制效应.方法:设计、合成siRNA(small interfering RNA),构建真核表达载体pGenesil-shRNA-Survivin,将重组质粒导入人腺样囊性癌ACC-M细胞株.采用逆转录-聚合酶链反应(RT-PcR)、Western-blot法分别检测转染后的ACC-M细胞中Survivin mRNA和蛋白表达的变化,用噻唑蓝(MTT)法检测细胞的增殖,结果:测序证实真核表达载体构建成功,在pGenesil-shRNA-Survivin的ACCM细胞株中,Survivin mRNA及蛋白表达明显降低,ACC-M细胞生长受到抑制.结论:pGenesil-shRNA-Survivin重组质粒能有效抑制腺样囊性癌细胞生长.     

沉默Mcpr1促进大鼠牙乳头细胞增殖

目的:观察用RNAi技术沉默Mcpr1对大鼠牙乳头细胞增殖能力的影响。方法:分离培养原代大鼠牙乳头细胞,用LipofectamineTM 2000将具有沉默Mcpr1作用的质粒pGenesil-shRNA、阴性对照的空载体转染大鼠牙乳头细胞,荧光显微镜观察转染效率,半定量PCR检测沉默效率,噻唑蓝(MTT)检测沉默Mcpr1后牙乳头细胞增殖能力的改变,流式细胞仪检测沉默Mcpr1后细胞周期的变化。结果:转染pGenesil-shRNA48 h后,Mcpr1的表达明显受到抑制,MTT结果显示转染pGenesil-shRNA后沉默组较未沉默组光吸收值明显增高,流式细胞仪检测沉默后的细胞周期在G0/G1期细胞比例明显下降。结论:pGensil-Mcpr1 shRNA载体能够抑制目的基因的表达。Mcpr1基因对牙乳头细胞的增殖具有明显的抑制作用。     

RNA干涉CD44基因抑制脑胶质瘤细胞U251的增殖与侵袭性研究

目的通过合成靶向分化抗原簇44(CD44)基因的siRNA片段,下调CD44在脑胶质瘤细胞U251中的表达,揭示其在胶质瘤增殖、迁移和侵袭中所起的作用。方法根据CD44基因序列设计并合成的siRNA片段转染U251细胞,利用荧光实时定量(qRT-PCR)检测细胞中CD44基因mRNA水平的变化;蛋白印迹实验(Western blot)检测CD44基因蛋白水平的变化;四甲基偶氮唑蓝染色(methyhhiazolyl tetrazolium,MTT)法检测U251细胞增殖;单细胞划痕愈合实验、Transwell小室侵袭实验检测U251细胞的迁移与侵袭能力变化。结果体外合成的CD44-siRNA能有效抑制U251细胞中CD44基因在mRNA水平与蛋白水平的表达(P0.05),并抑制细胞的增殖(P0.05)和迁移侵袭能力(P0.05)。结论 CD44-siRNA能够有效抑制U251脑胶质瘤细胞的增殖及其迁移侵袭力,为以CD44为靶点的肿瘤基因治疗奠定了基础。     

The Tribolium chitin synthase genes TcCHS1 and TcCHS2 are specialized for synthesis of epidermal cuticle and midgut peritrophic matrix

Functional analysis of the two chitin synthase genes, TcCHS1 and TcCHS2, in the red flour beetle, Tribolium castaneum, revealed unique and complementary roles for each gene. TcCHS1-specific RNA interference (RNAi) disrupted all three types of moult (larval-larval, larval-pupal and pupal-adult) and greatly reduced whole-body chitin content. Exon-specific RNAi showed that splice variant 8a of TcCHS1 was required for both the larval-pupal and pupal-adult moults, whereas splice variant 8b was required only for the latter. TcCHS2-specific RNAi had no effect on metamorphosis or on total body chitin content. However, RNAi-mediated down-regulation of TcCHS2, but not TcCHS1, led to cessation of feeding, a dramatic shrinkage in larval size and reduced chitin content in the midgut.     

促红细胞生成素对破骨细胞调控机理的研究

目的研究小鼠单核巨噬细胞在体外向破骨细胞分化的过程中,促红细胞生成素（erythropoietin,EPO）与破骨细胞表面受体Epor结合发挥作用的机理。方法 EPO作用于Epor受体沉默的经诱导的小鼠单核巨噬细胞,观察TRAP阳性多核细胞数目变化。Real-time PCR检测RAW264.7向破骨细胞分化过程中相关基因Epor、Nfatc1、Mmp9、EphrinB2基因的表达。结果与对照组比较,4天后,Epor沉默组的Nfatc1、EphrinB2 mRNA的表达明显升高（P〈0.01）,Ctsk Mmp9 mRNA的表达明显降低（P〈0.01）。结论 EPO通过与破骨细胞表面受体Epor结合,引发破骨细胞内相关基因表达改变,进而影响破骨细胞的分化和活化。     

埃及伊蚊多巴脱羧酶在蚊卵产出、孵化和抗干燥能力中的功能

目的 为了探究埃及伊蚊多巴脱羧酶（dopa decarboxylase, DDC）基因在蚊卵发育过程中所发挥的功能。 方法 首先通过荧光定量PCR技术构建DDC基因在埃及伊蚊中的时空表达谱,然后利用RNA干扰（RNAi）技术沉默埃及伊蚊成蚊DDC基因的表达,比较分析实验组与对照组的产卵量、卵孵化率和卵抗干燥能力。结果 DDC基因在卵时期表达量高于其他发育时期幼虫,在不同组织的相对表达量中,DDC基因在卵巢中转录本表达量水平约为胸部的5.35倍,是表皮和中肠的6.63倍和38.9倍。两个对照组间的产卵量和卵孵化率均无显著性差异（DEPC组产卵量和卵孵化率分别为122个/只蚊和95.1%;dsGUS组产卵量和卵孵化率分别为120个/只蚊和92.9%）。但实验组（dsDDC注射组）的产卵量（39个/只蚊）和孵化率（10%）均显著低于对照组（t=49.68,P<0.01）。在低湿度条件下（室温26 ℃,0~5%湿度）,0~10 min之内,对照组蚊卵壳结构和饱和度基本无变化,而dsDDC组蚊卵壳结构出现坍塌,卵饱和度明显下降;30 min之后,dsDDC组蚊卵已脱水干瘪,而对照组蚊卵仍保持较高的饱和度。结论 基于以上结果,本研究初步推断DDC基因从埃及伊蚊卵的形成到发育过程起到重要的调控作用。