本文選題：CRMP-2 + RGMa�。� 參考：《重慶醫(yī)科大學》2011年博士論文

【摘要】：目的排斥性導向分子(repulsive guidance molecule,RGMa)是中樞神經(jīng)系統(tǒng)受損后過度表達的抑制軸突再生的蛋白之一,具有軸突排斥及誘導生長錐塌陷的作用。生長錐塌陷后,軸突的結構受到嚴重破壞,將導致神經(jīng)突起間聯(lián)系的中斷,細胞間信息的傳遞受阻,最終導致神經(jīng)功能的損害。在腦缺血/再灌注損傷動物模型中,觀察到其明顯抑制軸突再生、阻礙神經(jīng)功能恢復的不良影響。同時發(fā)現(xiàn)在成年人局灶性缺血的腦片中,RGMa免疫陽性的細胞主要聚集在梗死區(qū)白質、出血區(qū)、梗死中心及梗死區(qū)周圍。因此,通過特異性RNA干擾(RNA interference,RNAi),抑制其基因轉錄水平,進而下調(diào)蛋白表達,可能有助于軸突的再生及神經(jīng)功能的恢復。此外,目前對于RGMa發(fā)揮抑制作用的細胞內(nèi)信號途徑并不清楚,可能與Rho激酶(Ras homologous kinase,Rho-kinase)下游分子塌陷反應介導蛋白-2(collapse response mediator protein-2,CRMP-2)有關。為了尋找到更合適的干預靶點,使缺血性損傷后的神經(jīng)再生更為容易,神經(jīng)功能的康復更為完全,對其作用機制的探索顯得非常重要。本研究的結果,更為基礎研究向臨床應用轉化提供了良好的理論依據(jù)。 方法 1.成年雄性SD大鼠132只,隨機分為:正常組,假手術組,MCAO/再灌注組,PBS注射組,rAd-HK組,rAd-shRGMa組,取手術后第2天、第7天為兩個觀察時間點,采用“線栓法”制作MCAO/再灌注模型,缺血側皮質立體定向注射RGMa特異性重組腺病毒rAd-shRGMa及空載體,通過RT-PCR、免疫組織化學/熒光方法檢測缺血側腦皮質內(nèi)RGMa與CRMP-2 mRNA、蛋白的表達水平; 2.成年雄性SD大鼠66只,分組同上,制作MCAO/再灌注模型,給予腺病毒干預,采用免疫組織化學方法檢測缺血側皮質內(nèi)NF200的表達; 3.對各組大鼠進行神經(jīng)功能評分; 4.成年雄性SD大鼠48只,隨機分為:正常組,MCAO/再灌注組,rAd-HK組,rAd-shRGMa組,用Western blot檢測RGMa、CRMP-2、pCRMP-2蛋白的表達,采用統(tǒng)計學方法對各分子間蛋白表達的關系進行相關性分析; 5.新生鼠皮質神經(jīng)元原代培養(yǎng),采用細胞免疫熒光方法進行神經(jīng)元鑒定、純度計算,以重組的RGMa蛋白體外誘導,光鏡下觀察神經(jīng)元軸突形態(tài)、長度變化,用Western blot檢測pCRMP-2隨時間變化的規(guī)律; 6.新生鼠皮質神經(jīng)元原代培養(yǎng),于重組RGMa體外誘導前,進行Rho-kinase及GSK-3β特異性阻滯預處理,用Western blot檢測細胞內(nèi)pCRMP-2的表達水平。 結果 1. MCAO/再灌注后,大鼠缺血側皮質內(nèi)RGMa mRNA及蛋白表達明顯升高(p0.01),而CRMP-2表達顯著降低(p0.01);用rAd-shRGMa干預后,大鼠在第2天時的RGMa水平較MCAO/再灌注組顯著降低(p0.01),CRMP-2水平明顯升高(p0.01),至第7天時基本接近正常水平。 2. MCAO/再灌注后,缺血側皮質內(nèi)pCRMP-2蛋白水平顯著升高(p0.01),此時軸突破壞最為嚴重,NF200表達明顯減少(p0.01),大鼠神經(jīng)功能缺損明顯(p0.01)。而rAd-shRGMa組大鼠的pCRMP-2蛋白水平明顯降低(p0.01),部分軸突得到保存,但大鼠神經(jīng)功能未見顯著改善(p0.05);至第7天時,pCRMP-2水平僅稍高于正常水平(p0.01),NF200表達明顯增多(p0.01),大鼠的神經(jīng)功能缺損已不明顯(p0.01)。 3. MCAO/再灌注組大鼠缺血側皮質pCRMP-2蛋白表達與RGMa蛋白表達pCRMP-2呈直線正相關(r=0.994),與NF200呈直線負相關(r=-0.895)。 4.離體原代培養(yǎng)的皮質神經(jīng)元存活狀態(tài)良好,胞體豐滿,軸突生長正常,純度在90%以上。 5.加入重組RGMa培養(yǎng)后,各孔神經(jīng)元軸突明顯回縮(p0.01),細胞透光性欠佳,大部分神經(jīng)元僅見胞體,而軸突基本消失;在Y-27632、GSK-3βinhibitor聯(lián)合RGMa培養(yǎng)孔里,神經(jīng)元軸突回縮現(xiàn)象較單純RGMa培養(yǎng)孔有所改善(p0.01)。 6.隨著RGMa誘導時間的延長,細胞內(nèi)pCRMP-2的水平逐漸升高,6 h起即高于正常水平(p0.01),到24 h達到高峰(p0.01),以后開始下降,48 h的水平與6 h沒有顯著性差異(p0.05);經(jīng)Rho-kinase及GSK-3β特異性抑制劑預處理后的神經(jīng)元,對RGMa的誘導作用出現(xiàn)明顯抵抗,pCRMP-2水平與正常時無統(tǒng)計學差異(p0.05)。 結論 腦缺血/再灌注后皮質內(nèi)RGMa及pCRMP-2水平明顯增高,腺病毒介導的RGMa特異性RNAi可以明顯降低RGMa的表達,抑制CRMP-2的磷酸化,從而促進軸突再生及神經(jīng)功能的恢復。RGMa可能是通過同時激活Rho-kinase及GSK-3β信號通路調(diào)控CRMP-2的磷酸化,介導神經(jīng)元軸突縮短的。
[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.

【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R363

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本文編號：1742195