微小RNA155经Rheb/mTOR通路在缺血性脑损伤中的作用及其机制研究

With the current limitation of therapeutic approach and efficiency,ischemic stroke is a disvasting disease causing disability and mortality wordwide，whose injury mechanism is not fully understood. Our previous study in rat MCAO/R model demonstrated that during brain ischemia/reperfusion imjury,the activity of mTOR signaling pathway was inhibited and autophage activated. Recent studies demonstrated that microRNA155(miR155) potently induce autophage under hypoxia condition by targeting multiple mRNAs molecules of mammalian target of rapamycin(mTOR) signaling pathway,leading to cell apoptosis, suggesting that miR155 is likely invoved in the ischemic cerebral injury mediated by mTOR signaling pathway.Thus,we propose to use the rat MCAO/R model and the OGD/R model of rat primary cortical neurons.First,use qRT-PCR and stereotaxic injection of recombined lentivirus technique to investigate the role of miR155 in brain ischemia/reperfusion injury and its regulation on mTOR signaling pathway;Second,using point-mutation of target mRNA technique to explore the molecular mechanism of miR155 regulating mTOR signaling pathway in the OGD/R model of rat primary cortical neurons; Finally, using immunohistochemistry(IHC) and molecular neurobiological techniques to further explore the mechanism of the function of miR155 in ischemia/reperfusion injury from the standpoints of autophage and apopotosis.The present study will provide new theoretical evidence for better understanding the mechanism of ischemia/reperfusion injury and thus lay the foundation for seeking acurrate and effective intervention targets for neuroprotective therapies of ischemic stroke.

缺血性脑卒中危害广、疗效局限，损伤机制尚不十分清楚。既往研究提示mTOR通路可能在其中发挥重要作用。我们预实验表明，大鼠MCAO/R模型中缺血区脑组织内mTOR通路活性受到抑制、自噬活化。最近研究发现，缺氧状态下miR155通过抑制mTOR通路活性强力诱导自噬、促进细胞凋亡。据此推测：miR155可能通过抑制mTOR通路活性介导缺血性脑损伤。因此本项目拟用大鼠MCAO/R模型和大鼠原代皮层神经元OGD/R模型：1.通过立体定位颅内注射重组慢病毒等技术体内观察miR155在脑缺血中的作用及其对mTOR通路的调控；2.通过靶mRNA定点突变技术体外探讨miR155在脑缺血中调控mTOR通路的分子机制；3.用免疫组化和分子生物学技术在体外从自噬和细胞凋亡两个角度深入探讨miR155在脑缺血中的作用机制。本项目将为脑缺血/再灌注损伤提供新的理论依据，为寻求精确、有效的脑保护治疗的干预靶点奠定基础。

缺血性脑卒中危害广、疗效局限，损伤机制尚不十分清楚。既往研究提示mTOR通路可能在其中发挥重要作用。我们预实验表明，大鼠MCAO/R模型中缺血区脑组织内mTOR通路活性受到抑制、自噬活化。最近研究发现，缺氧状态下miR155通过抑制mTOR通路活性强力诱导自噬、促进 细胞凋亡。据此推测:miR155可能通过抑制mTOR通路活性介导缺血性脑损伤。本项目成功建立大鼠MCAO/R模型，通过qRT-PCR技术体内观察了miR155及Rheb/mTOR信号通路相关分子mRNA在脑缺血中的时空表达特点，初步探讨miR155在脑缺血中的作用机制。另外，本项目成功构建出有生物学效应的miR-155海绵载体，并已包装成慢病毒，将为后续miR155在脑缺血中的机制研究奠定基础。本项目将为脑缺血/再灌注损伤提供新的理论依据，为寻求精确、有效的脑保护治疗的干预靶点奠定基础。另外本项目外延性研究发现，miR-183-96-182 簇在体内通过靶向FOXO1可部分缓解氧化型LDL诱导的人脐静脉内皮细胞(HUVECs) 和人动脉内皮细胞 (HAECs) 的凋亡。另外我们还研究发现选择素1通过激活PI3k/Akt 信号通路而抑制氧糖剥夺状态下的人脐静脉内皮细胞(HUVECs)的凋亡，并促进血管生成。在临床研究方面，我们通过病例对照研究，发现阿司匹林联合氯吡格雷短期应用可有效降低急性脑梗死并2型糖尿病患者的脑梗死复发率，促进神经功能恢复。长期联合应用阿司匹林及氯吡格雷对预防及治疗脑梗死合并2型糖尿病患者有临床治疗意义，但会增加胃肠道出血的风险。