CRMP-2的磷酸化对缺血再灌注脑损伤后神经再生的影响及机制研究

发布时间：2018-04-13 00:40

本文选题：CRMP-2 + RGMa　；参考：《重庆医科大学》2011年博士论文

【摘要】：目的排斥性导向分子(repulsive guidance molecule,RGMa)是中枢神经系统受损后过度表达的抑制轴突再生的蛋白之一,具有轴突排斥及诱导生长锥塌陷的作用。生长锥塌陷后,轴突的结构受到严重破坏,将导致神经突起间联系的中断,细胞间信息的传递受阻,最终导致神经功能的损害。在脑缺血/再灌注损伤动物模型中,观察到其明显抑制轴突再生、阻碍神经功能恢复的不良影响。同时发现在成年人局灶性缺血的脑片中,RGMa免疫阳性的细胞主要聚集在梗死区白质、出血区、梗死中心及梗死区周围。因此,通过特异性RNA干扰(RNA interference,RNAi),抑制其基因转录水平,进而下调蛋白表达,可能有助于轴突的再生及神经功能的恢复。此外,目前对于RGMa发挥抑制作用的细胞内信号途径并不清楚,可能与Rho激酶(Ras homologous kinase,Rho-kinase)下游分子塌陷反应介导蛋白-2(collapse response mediator protein-2,CRMP-2)有关。为了寻找到更合适的干预靶点,使缺血性损伤后的神经再生更为容易,神经功能的康复更为完全,对其作用机制的探索显得非常重要。本研究的结果,更为基础研究向临床应用转化提供了良好的理论依据。方法 1.成年雄性SD大鼠132只,随机分为:正常组,假手术组,MCAO/再灌注组,PBS注射组,rAd-HK组,rAd-shRGMa组,取手术后第2天、第7天为两个观察时间点,采用“线栓法”制作MCAO/再灌注模型,缺血侧皮质立体定向注射RGMa特异性重组腺病毒rAd-shRGMa及空载体,通过RT-PCR、免疫组织化学/荧光方法检测缺血侧脑皮质内RGMa与CRMP-2 mRNA、蛋白的表达水平; 2.成年雄性SD大鼠66只,分组同上,制作MCAO/再灌注模型,给予腺病毒干预,采用免疫组织化学方法检测缺血侧皮质内NF200的表达; 3.对各组大鼠进行神经功能评分; 4.成年雄性SD大鼠48只,随机分为:正常组,MCAO/再灌注组,rAd-HK组,rAd-shRGMa组,用Western blot检测RGMa、CRMP-2、pCRMP-2蛋白的表达,采用统计学方法对各分子间蛋白表达的关系进行相关性分析; 5.新生鼠皮质神经元原代培养,采用细胞免疫荧光方法进行神经元鉴定、纯度计算,以重组的RGMa蛋白体外诱导,光镜下观察神经元轴突形态、长度变化,用Western blot检测pCRMP-2随时间变化的规律; 6.新生鼠皮质神经元原代培养,于重组RGMa体外诱导前,进行Rho-kinase及GSK-3β特异性阻滞预处理,用Western blot检测细胞内pCRMP-2的表达水平。结果 1. MCAO/再灌注后,大鼠缺血侧皮质内RGMa mRNA及蛋白表达明显升高(p0.01),而CRMP-2表达显著降低(p0.01);用rAd-shRGMa干预后,大鼠在第2天时的RGMa水平较MCAO/再灌注组显著降低(p0.01),CRMP-2水平明显升高(p0.01),至第7天时基本接近正常水平。 2. MCAO/再灌注后,缺血侧皮质内pCRMP-2蛋白水平显著升高(p0.01),此时轴突破坏最为严重,NF200表达明显减少(p0.01),大鼠神经功能缺损明显(p0.01)。而rAd-shRGMa组大鼠的pCRMP-2蛋白水平明显降低(p0.01),部分轴突得到保存,但大鼠神经功能未见显著改善(p0.05);至第7天时,pCRMP-2水平仅稍高于正常水平(p0.01),NF200表达明显增多(p0.01),大鼠的神经功能缺损已不明显(p0.01)。 3. MCAO/再灌注组大鼠缺血侧皮质pCRMP-2蛋白表达与RGMa蛋白表达pCRMP-2呈直线正相关(r=0.994),与NF200呈直线负相关(r=-0.895)。 4.离体原代培养的皮质神经元存活状态良好,胞体丰满,轴突生长正常,纯度在90%以上。 5.加入重组RGMa培养后,各孔神经元轴突明显回缩(p0.01),细胞透光性欠佳,大部分神经元仅见胞体,而轴突基本消失;在Y-27632、GSK-3βinhibitor联合RGMa培养孔里,神经元轴突回缩现象较单纯RGMa培养孔有所改善(p0.01)。 6.随着RGMa诱导时间的延长,细胞内pCRMP-2的水平逐渐升高,6 h起即高于正常水平(p0.01),到24 h达到高峰(p0.01),以后开始下降,48 h的水平与6 h没有显著性差异(p0.05);经Rho-kinase及GSK-3β特异性抑制剂预处理后的神经元,对RGMa的诱导作用出现明显抵抗,pCRMP-2水平与正常时无统计学差异(p0.05)。结论脑缺血/再灌注后皮质内RGMa及pCRMP-2水平明显增高,腺病毒介导的RGMa特异性RNAi可以明显降低RGMa的表达,抑制CRMP-2的磷酸化,从而促进轴突再生及神经功能的恢复。RGMa可能是通过同时激活Rho-kinase及GSK-3β信号通路调控CRMP-2的磷酸化,介导神经元轴突缩短的。
[Abstract]:The purpose of repulsive guidance molecules (repulsive guidance molecule, RGMa) is one of the protein inhibits axonal damage of central nervous system after over expression of regeneration, with rejection and induced growth cone collapse. The role of axonal growth cone collapse after axonal structure has been severely damaged, will cause interruption of contact between neurites, blocked cell transfer information, resulting in neurological damage. In the animal model of cerebral ischemia / reperfusion injury, observe the inhibition of axon regeneration, hinder the adverse effects of nerve function recovery. At the same time found in the adult human brain slices of focal ischemia, RGMa positive cells mainly accumulated in white matter infarction area around the center of the bleeding area, infarction and cerebral infarction area. Therefore, the specific RNA interference (RNA interference, RNAi), the inhibition of gene transcription, and down-regulation of protein expression, may contribute to the axon The regeneration and functional recovery. In addition, the RGMa can inhibit the intracellular signaling pathways is not clear, may be related to Rho kinase (Ras homologous kinase, Rho-kinase) reaction mediated downstream molecular collapse protein -2 (collapse response mediator protein-2, CRMP-2). In order to find a more suitable target for intervention, so that nerve regeneration after ischemic injury is more easily, the rehabilitation of neurological function is more complete, to explore its mechanism of action is very important. The results of this study, more theoretical basis for the transformation of basic research to clinical application for good.
Method
1. adult male SD 132 rats were randomly divided into normal group, sham operation group, MCAO/ reperfusion group, PBS group, rAd-HK group, rAd-shRGMa group, second days after surgery, seventh days for the two observation time points, using the "suture method" MCAO/ reperfusion of the ischemic side. Cortex stereotactic injection of RGMa specific recombinant adenovirus vector and rAd-shRGMa, by RT-PCR, immunohistochemistry / fluorescence method to detect ischemic cortex in RGMa and CRMP-2 mRNA, the expression level of protein;
2. adult male SD rats were divided into 66 groups. The rats were divided into groups. The MCAO/ reperfusion model was made, and adenovirus was used to intervene. Immunohistochemistry was used to detect the expression of NF200 in the ischemic cortex.
3. the nerve function score of rats in each group was evaluated.
4. adult male SD rats were randomly divided into 48 groups: normal group, MCAO/ reperfusion group, rAd-HK group and rAd-shRGMa group. The expressions of RGMa, CRMP-2 and pCRMP-2 protein were detected by Western blot, and the correlation between protein expression among different molecules was analyzed by statistical method.
5. neonatal rat cortical neurons were primarily cultured. Cell immunofluorescence was used to identify neurons. The purity was calculated. The recombinant RGMa protein was induced in vitro. The axonal morphology and length of neurons were observed under light microscope. Western blot was used to detect the change rule of pCRMP-2 with time.
6. the primary culture of neonatal rat cortical neurons was carried out before induction of recombinant RGMa in vitro. Rho-kinase and GSK-3 GSK-3 were specifically pretreated, and the expression level of pCRMP-2 was detected by Western blot.
Result
1. MCAO/ after reperfusion in rat ischemic cortex in the expression of RGMa and mRNA protein increased significantly (P0.01), and the expression of CRMP-2 was significantly reduced (P0.01); rAd-shRGMa intervention, RGMa level on the second day rats compared with MCAO/ reperfusion group decreased significantly (P0.01), the level of CRMP-2 increased significantly (P0.01) the seventh day, to close to the normal level.
2. MCAO/ after reperfusion, pCRMP-2 protein levels in the ischemic cortex was significantly increased (P0.01), the axons are severely damaged, the expression of NF200 decreased significantly (P0.01), the neurological deficit in rats obviously (P0.01). The pCRMP-2 and protein levels of rAd-shRGMa rats decreased significantly (P0.01), part of the axon to be preserved, but there was no significant improvement of neurological function in rats (P0.05); to the seventh day, the level of pCRMP-2 was only slightly higher than the normal level (P0.01), the expression of NF200 increased significantly (P0.01), neurological deficit of rats was not significant (P0.01).
In 3. MCAO/ reperfusion group, there was a positive linear correlation between the expression of pCRMP-2 protein and pCRMP-2 expression of RGMa protein (r=0.994), and negatively correlated with NF200 (r=-0.895).
The primary cultured cortical neurons of 4. in vitro survived well, the cell body was plump, the axon growth was normal and the purity was above 90%.
5. after incubation with recombinant RGMa, the axonal retraction obvious hole (P0.01), and thediaphaneity were poor, most of the neurons cell bodies and axons were observed, disappeared; in Y-27632, GSK-3 beta inhibitor combined with RGMa culture hole, axonal retraction phenomenon is relatively simple RGMa training improved hole (P0.01).
6. with prolonged induction time of RGMa, the intracellular pCRMP-2 level increased gradually, h 6 is higher than the normal level (P0.01), peak at 24 h (P0.01), began to decline after 48 h level had no significant difference with 6 h (P0.05); the Rho-kinase and GSK-3 specific inhibitor of pre beta after the treatment of neurons induced by RGMa showed obvious resistance, pCRMP-2 level and normal no significant difference (P0.05).
conclusion
Cerebral ischemia / RGMa and pCRMP-2 levels in cortex increased significantly after reperfusion, RNAi specific RGMa mediated by adenovirus can significantly reduce the expression of RGMa, inhibit the phosphorylation of CRMP-2, so as to promote the recovery of.RGMa axon regeneration and nerve function may be through the activation of Rho-kinase and GSK-3 and beta signaling pathways regulate the phosphorylation of CRMP-2. Mediated neuronal axon shortening.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363

【参考文献】