基因沉默氨基酰-tRNA合成酶- - 一种新的脑缺血耐受保护机制？

The humans life and health was threated by ischemic stroke,the therapeutic effect was not satisfied despite of lots of treatment methods. Cerebral ischemic tolerance is a phenomena of powerful endogenous self-protection of body. Any how,the exact molecular mechanism is not very clear.Aminoacyl-tRNA synthetase (AARS)，is a kind of key enzymes in the process of biological protein synthesis, The AARS substrates are amino acids、tRNA and adenosine triphosphate (ATP) , AARS catalyze amino acylation reaction of corresponding tRNA, amino acylation tRNA connect amino acids in proper order to synthesize protein on ribosome through codonm in mRNA pair to anticodon in tRNA.Research by team of Lori L. Anderson et al.found that inactivation of arginal-tRNA synthetase in c.elegant by means of RNAi technology can leads to significant reduction in protein translation. Compared to control group, hypoxia tolerance of c. elegant in RNAi group was significantly enhanced, they supposed that inactivation of arginal-tRNA synthetase can result in translation suppression and lead to secondary change of biologic behavior of the cell such as decrease in ATP consumption. Energy consumption reduction caused by translation repression is a well-established mechanism of survival for hibernating animals such as arctic ground squirrel in a prolonged hypoxic environment.they strongly suggest that AARS Gene silencing is possibly a new mechanism of ischemic tolerance in acute cerebral ischemia. Up to now, there is no experimental report about the relationship between cerebral ischemic tolerance and AARS Gene silencing in mammal on a global scale.we suppose that under condition of acute ischemia and hypoxia, endougenous AARS gene silensing is triggered by one kind of mechanism unknown,thus lead to protein translation decreasing in cerebral tissue,and a series of physiological activity decreased.So, oxygen and energy demand decreases sharply,the brain cells suffered from ischemia fell in condition of hibernation,finally protect the cerebral tissue from ischemia insult.To confirm this assumption, the experiment is designed to observe if AARS gene knock-down can play a key role in brain protection in cerebral ischemia. if any, the extent of brain protection is aimed to find out through AARS gene knock-down in vitro and in vivo by means of RNAi technology,the experiment also try to outline the features and mechanism of brain protection of AARS gene knock-out in cerebral ischemia in rats by RNAi technology and lay a good basis for the further studying and throwing light on the mechanism of brain ischemic tolerance as well.

脑缺血耐受是机体自身一种强大的内源性保护现象,但其确切的分子机制仍然不明.最新研究表明:抑制氨基酰-tRNA合成酶（AARS）基因能够减少能量消耗,极大增强线虫的缺氧耐受能力,与冬眠动物北极地松鼠缺糖、缺氧条件下神经元能量代谢率急剧下降、能量消耗迅速减少有着极其相似。研究强烈提示AARS基因沉默极有可能是一种新的脑缺血耐受保护机制,即:在急性缺血缺氧状态下,启动内源性AARS基因抑制，脑细胞蛋白质翻译减少,一系列生理活动降低,由此导致细胞氧耗和能耗急剧减少,细胞处于能量需求的相对冬眠期,从而保护缺血缺氧状态下的脑细胞.为验证这一设想,本课题拟采用RNAi技术,体外和体内抑制大鼠AARS基因，观察基因沉默后该基因和蛋白的表达以及大鼠各项生理和监测指标的变化，判断AARS基因沉默能否在大鼠急性脑缺血中起脑保护作用,试图初步勾勒出大鼠AARS基因沉默在脑缺血耐受中的作用机制，为下一步研究打下基础

脑缺血耐受是机体自身一种强大的内源性保护现象,但其确切的分子机制仍然不明. 氨基酰-tRNA 合成酶(AARS)为蛋白质生物合成过程中的一类关键酶。最新研究表明:抑制AARS基因能够减少能量消耗,极大增强线虫的缺氧耐受能力。提示AARS基因沉默极有可能是一种新的脑缺血耐受保护机制,即:在急性缺血缺氧状态下，通过某种机制启动内源性AARS基因抑制，使脑细胞蛋白质翻译减少,一系列生理活动降低,由此导致细胞氧耗和能耗急剧减少,细胞处于能量需求的相对冬眠期,从而保护缺血缺氧状态下的脑细胞。.我们首先采用改良Longa线栓法制作大鼠局灶脑缺血模型，观察缺血预处理所产生的脑缺血耐受大鼠脑梗塞缺血半暗带ArgRS基因和蛋白的表达变化。将大鼠随机分为正常组、缺血预处理后脑梗塞组、脑梗塞组，在相应时间点进行神经功能缺损评分，根据评分筛查模型，手术显微镜下取梗塞半暗带的皮质脑组织标本进行 RT-PCR 及 Western-blot 测定。结果表明，缺血预处理后脑梗塞组在不同时间点的ArgRS基因及蛋白表达均显著低于脑梗塞组（P<0.05）。正常组，缺血预处理后脑梗塞假手术组及脑梗塞假手术组间 ArgRS 基因及蛋白表达差异无统计学意义。这说明在大鼠体内脑缺血预处理后所产生的脑缺血耐受现象早期可能存在通过抑制ArgRS 基因及蛋白表达来发挥其神经保护作用。.在细胞层面，我们观察了氧糖剥夺模型预处理的原代神经元其缺血耐受性的改变，并研究预处理前后细胞中精氨酰-tRNA合成酶（ArgRS）基因Rars的活性及蛋白表达的变化。结果表明缺血预处理可以明显提高体外培养的神经元细胞对于持续缺血损伤的耐受性。并且缺血预处理后遭持续缺血损伤的神经元各时间点的Rars转录活性及ArgRS表达均显著低于单纯缺血组（P< 0.05）。这说明缺血预处理后神经元Rars转录活性抑制及ArgRS表达下调可能参与诱导细胞缺血耐受性的发生。.我们进而构建并鉴定了针对大鼠 Rars基因的 siRNA重组腺病毒，以构建好的腺病毒载体转染大鼠皮层原代神经元，随后检测该载体转染后不同时间点 ArgRS 基因和蛋白表达。结果表明ArgRS RNAi抑制后可通过抑制蛋白翻译，减少能量消耗参与诱导细胞缺血耐受性的发生。