RGMa特异性RNAi对MCAO/再灌注大鼠脑组织中RGMa表达、轴突生长及神经功能的影响
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摘要
目的缺血性卒中有着极高的发病率、致残率和死亡率,严重危害人类健康和生命安全,与之相关的神经康复问题一直是人们关注的热点和难点。由于大量轴突生长抑制因子的存在,中枢神经系统(CNS)损伤后神经元再生极为困难。然而在某些干预因素作用下,遭遇机械性损伤或缺血缺氧损害的神经元轴突可以再生,并促进原受损轴突支配区的功能恢复[1, 2]。RGMa(repulsive guidance molecule a)是一种轴突导向分子,同时也是迄今已知的轴突再生抑制因子中最有潜力的一种。研究发现,在大鼠脊髓损伤后给予特异性抗体中和RGMa表达,可显著改善损伤轴突生长情况并有效促进神经功能恢复[3]。本课题运用重组腺病毒rAd5-shRNA-RGMa对大脑中动脉阻塞(MCAO)/再灌注大鼠缺血区域皮质和海马进行RNA干扰,观察该特异性干预手段对急性局灶性缺血再灌注损伤大鼠脑皮质及海马中RGMa表达水平、轴突生长/再生情况以及神经功能恢复的影响,进一步阐明RGMa在CNS轴突再生抑制因子中的重要地位,为临床脑缺血治疗寻找有效靶点进行基因干预以促进神经功能恢复提供依据并奠定基础。
方法1.通过HEK 293细胞扩增得到大量重组腺病毒液并进行纯化,用50%组织培养感染剂量(TCID50)法测定滴度;再将三种不同滴度重组腺病毒rAd5-shRNA-RGMa及病毒保存液PBS(阴性对照)注射至线栓法MCAO/再灌注大鼠(n=32)缺血部位脑皮质和海马区,观察转染局部病理改变和炎症因子表达情况,评估毒性反应强弱,并计算转染效率,确定用于转染缺血脑组织的最佳滴度;
2.健康成年雄性SD大鼠66只,随机分为正常组,假手术2 d、7 d组,MCAO/再灌注模型2 d、7 d组,空白对照(PBS)2 d、7 d组,阴性对照(rAd5-HK)2 d、7 d组,RNAi干预(rAd5-shRNA-RGMa)2 d、7 d组共11组。分别取材用于RT-PCR和免疫组化检测缺血侧脑皮质和海马区RGMa mRNA和蛋白表达水平及轴突生长情况。
3. SD大鼠(n=42)随机分为正常组、模型组、阴性对照组和RNAi干预组,进行TTC染色和生物素葡聚糖胺(BDA)轴突示踪,观察脑梗死范围和轴突侧枝芽生数量,并通过行为学检查评估大鼠神经功能恢复情况。
结果1.纯化后的重组腺病毒滴度分别为:rAd5-shRNA-RGMa为5.01×1010 pfu/ml,rAd5-HK为3.16×1010 pfu/ml。三种不同滴度的重组腺病毒rAd5-shRNA-RGMa均可转染大鼠缺血部位脑皮质和海马,中、高滴度组的转染效率显著优于低滴度组(p<0.01),但该两组之间并无差异(p>0.05)。仅高滴度组大鼠腺病毒注射局部脑组织有血管周围炎性细胞浸润现象,且术后7 d该组大鼠脑组织炎症因子IL-1β的表达水平明显高于其他各组(p<0.01)。
2.与正常组和假手术组相比,其余各组大鼠缺血侧脑皮质和海马区的RGMa mRNA和蛋白水平均增高(p<0.01),MCAO/再灌注2 d、7 d组,空白对照2 d、7 d组和阴性对照2 d、7 d组尤为明显,同时伴有轴突形态紊乱、数目减少;而给予RGMa特异性RNAi干预后,缺血侧脑皮质和海马区RGMa mRNA和蛋白表达水平有所下调,轴突生长情况有所改善(p<0.01)。
3.和MCAO/再灌注组和阴性对照组相比,给予RGMa特异性RNAi干预的大鼠脑梗死体积没有显著差别(p>0.05),但在治疗6 w后行为学评分明显增高(p<0.01),健侧皮质红核束向病灶侧发出的侧枝数量亦明显增多(p<0.01)。
结论纯化后的重组腺病毒rAd5-shRNA-RGMa可稳定、低毒、高效地转染大鼠缺血脑组织,是用于缺血性脑损伤基因治疗研究的有利工具。大鼠脑皮质和海马区RGMa的mRNA和蛋白在急性局灶性脑缺血/再灌注后表达持续增高,给予RGMa特异性RNAi后可显著降低其表达水平,从而改善轴突生长情况,促进大鼠运动功能的康复。其途径可能是RGMa表达的降低促进了健侧皮质神经元轴突的芽生,大量延伸至病灶侧代偿其原有功能。
Objective Ischemic stroke is a great threat to human health and life safety and the related problem referring neurological rehabilitation has received extensive attentions. Regeneration of neurons is extremely difficult in adult central nervous system (CNS) after injury, due to the effections of numerous neurite growth inhibitors. But in some cases, axons damaged by mechanical factors or deprivation of blood and oxygen, could regrow and promote functional recovery as a result of special intervention. Repulsive guidance molecule a (RGMa) is a kind of axonal guidance molecule, one of the most potential reported neurite growth inhibitors to this day. Intrathecal administration of a neutralizing antibody to block function of RGMa could significantly enhance axonal growth and promote neurologic function recovery after spinal cord injury in rats. We planned to adopt a type of specific intervention approach, RNA interference (RNAi) mediated by recombinant adenovirus rAd5-shRNA-RGMa, to cure ischemic brain tissues, including cortex and hippocampus, of rats after middle cerebral artery occlusion (MCAO)/reperfusion. Then the important role of RGMa in all neurite inhibitors could be demonstrated by axonal growth and functional recovery due to the changes of RGMa expression brought by RNAi. It would provide an important clue for designing therapeutic strategies for clinical cerebral ischemia.
Methods 1. Recombinant adenoviruses rAd5-shRNA-RGMa and rAd5-HK were amplified through HEK-293 cells culture and purified by Sartorious Vivapure Adeno PACK 20. 50% tissue culture infective dose (TCID50) was used to estimate their titers. Three different titers of rAd5-shRNA-RGMa and PBS (negative control) were injected via microinjection needle into the cortex and hippocampus of ischemic side in rats (n=32) after MCAO/reperfusion by stereotactic surgery. Toxicity was detected by histopathology and immunohistochemistry approaches. Proportions of green fluorescent protein (GFP) positive cells in the recombinant adenovirus injected tissues were calculated by staining total nucleus by DAPI.
2. 66 heathy male adult Sprague Dawley (SD) rats were randomly divided into 11 groups, including normal group, sham 2d and 7d group, model (MCAO/reperfusion) 2 d and 7 d group, blank control (PBS) 2 d and 7 d group, negative control (rAd5-HK) 2 d and 7 d group, RNAi intervention (rAd5-shRNA-RGMa) 2 d and 7 d group. The mRNA and protein expressions of RGMa in the cortex and hippocampus of ischemic side were detected by reverse transcription polymerase chain reaction (RT-PCR) and immunehistochemistry respectively. And the axonal growth in corresponding regions was also estimated by immunehistochemistry.
3. Normal group, MCAO/reperfusion group, negative group and RNAi intervention group , divided from 42 SD rats, were set up to identify ischemic regions and newborn neurite branches by TTC staining and BDA neuronal tracing. Behavior tests were adopted to evaluate neurologic function recovery of the rats after focal cerebral ischemic injury and correlative interventions.
Results 1. Titers of purified rAd5-shRNA-RGMa and rAd5-HK were respectively 5.01×1010 pfu/ml and 3.16×1010 pfu/ml. All the three titers of rAd5-shRNA-RGMa could transfect cortex and hippocampus of ischemic side in rats’brain. Transfection efficacies of medium and high titer groups were similar (p >0.05), obviously better than low titer group. The phenomenon of perivascular inflammatory cell infiltration was only observed around the injection site in brain tissues of high titer group. The expression levels of IL-1βin brain tissues of ischemic side in this goup were also higher than other groups on 7 d post-operation (p <0.01).
2. Compared to normal and sham groups, both mRNA and protein expression levels of RGMa in cortex and hippocampus of ischemic side in other groups were significantly increased (p <0.01). The increase degrees of MCAO/reperfusion groups, blank control groups and negative groups were especially high, along with obviously reduction of axon numbers. Meanwhile the expression levels of RGMa were down-regulated and axonal growth was improved simultaneously in RNAi intervention groups (p <0.01).
3. The cerebral infarction volumes of rats in model group,negative group and RNAi intervention group were similar (p >0.05). Behavior test scores of rats in RNAi intervention group after 6 weeks were significantly better than other groups (p <0.01). And numbers of axonal sprouting of corticorubral tract from healthy side to opposite side in this group were also obviously increased (p <0.01).
Conclusions Purified RGMa specific RNAi recombinant adenovirus rAd5-shRNA-RGMa, which could transfect cortex and hippocampus of ischemic side in rats’brain with high stability, high efficacy and low toxicity,is a very effective tool in gene therapeutic research of cerebral ischemia. RGMa mRNA and protein expressions were persistently induced in cortex and hippocampus of ischemic side in rats after middle cerebral artery occlusion/reperfusion,which could be prominently reduced by RGMa-specific RNAi intervention and lead to improved axonal growth and neural anatomy plasticity, as well as neural function recovery.
方法1.通过HEK 293细胞扩增得到大量重组腺病毒液并进行纯化,用50%组织培养感染剂量(TCID50)法测定滴度;再将三种不同滴度重组腺病毒rAd5-shRNA-RGMa及病毒保存液PBS(阴性对照)注射至线栓法MCAO/再灌注大鼠(n=32)缺血部位脑皮质和海马区,观察转染局部病理改变和炎症因子表达情况,评估毒性反应强弱,并计算转染效率,确定用于转染缺血脑组织的最佳滴度;
2.健康成年雄性SD大鼠66只,随机分为正常组,假手术2 d、7 d组,MCAO/再灌注模型2 d、7 d组,空白对照(PBS)2 d、7 d组,阴性对照(rAd5-HK)2 d、7 d组,RNAi干预(rAd5-shRNA-RGMa)2 d、7 d组共11组。分别取材用于RT-PCR和免疫组化检测缺血侧脑皮质和海马区RGMa mRNA和蛋白表达水平及轴突生长情况。
3. SD大鼠(n=42)随机分为正常组、模型组、阴性对照组和RNAi干预组,进行TTC染色和生物素葡聚糖胺(BDA)轴突示踪,观察脑梗死范围和轴突侧枝芽生数量,并通过行为学检查评估大鼠神经功能恢复情况。
结果1.纯化后的重组腺病毒滴度分别为:rAd5-shRNA-RGMa为5.01×1010 pfu/ml,rAd5-HK为3.16×1010 pfu/ml。三种不同滴度的重组腺病毒rAd5-shRNA-RGMa均可转染大鼠缺血部位脑皮质和海马,中、高滴度组的转染效率显著优于低滴度组(p<0.01),但该两组之间并无差异(p>0.05)。仅高滴度组大鼠腺病毒注射局部脑组织有血管周围炎性细胞浸润现象,且术后7 d该组大鼠脑组织炎症因子IL-1β的表达水平明显高于其他各组(p<0.01)。
2.与正常组和假手术组相比,其余各组大鼠缺血侧脑皮质和海马区的RGMa mRNA和蛋白水平均增高(p<0.01),MCAO/再灌注2 d、7 d组,空白对照2 d、7 d组和阴性对照2 d、7 d组尤为明显,同时伴有轴突形态紊乱、数目减少;而给予RGMa特异性RNAi干预后,缺血侧脑皮质和海马区RGMa mRNA和蛋白表达水平有所下调,轴突生长情况有所改善(p<0.01)。
3.和MCAO/再灌注组和阴性对照组相比,给予RGMa特异性RNAi干预的大鼠脑梗死体积没有显著差别(p>0.05),但在治疗6 w后行为学评分明显增高(p<0.01),健侧皮质红核束向病灶侧发出的侧枝数量亦明显增多(p<0.01)。
结论纯化后的重组腺病毒rAd5-shRNA-RGMa可稳定、低毒、高效地转染大鼠缺血脑组织,是用于缺血性脑损伤基因治疗研究的有利工具。大鼠脑皮质和海马区RGMa的mRNA和蛋白在急性局灶性脑缺血/再灌注后表达持续增高,给予RGMa特异性RNAi后可显著降低其表达水平,从而改善轴突生长情况,促进大鼠运动功能的康复。其途径可能是RGMa表达的降低促进了健侧皮质神经元轴突的芽生,大量延伸至病灶侧代偿其原有功能。
Objective Ischemic stroke is a great threat to human health and life safety and the related problem referring neurological rehabilitation has received extensive attentions. Regeneration of neurons is extremely difficult in adult central nervous system (CNS) after injury, due to the effections of numerous neurite growth inhibitors. But in some cases, axons damaged by mechanical factors or deprivation of blood and oxygen, could regrow and promote functional recovery as a result of special intervention. Repulsive guidance molecule a (RGMa) is a kind of axonal guidance molecule, one of the most potential reported neurite growth inhibitors to this day. Intrathecal administration of a neutralizing antibody to block function of RGMa could significantly enhance axonal growth and promote neurologic function recovery after spinal cord injury in rats. We planned to adopt a type of specific intervention approach, RNA interference (RNAi) mediated by recombinant adenovirus rAd5-shRNA-RGMa, to cure ischemic brain tissues, including cortex and hippocampus, of rats after middle cerebral artery occlusion (MCAO)/reperfusion. Then the important role of RGMa in all neurite inhibitors could be demonstrated by axonal growth and functional recovery due to the changes of RGMa expression brought by RNAi. It would provide an important clue for designing therapeutic strategies for clinical cerebral ischemia.
Methods 1. Recombinant adenoviruses rAd5-shRNA-RGMa and rAd5-HK were amplified through HEK-293 cells culture and purified by Sartorious Vivapure Adeno PACK 20. 50% tissue culture infective dose (TCID50) was used to estimate their titers. Three different titers of rAd5-shRNA-RGMa and PBS (negative control) were injected via microinjection needle into the cortex and hippocampus of ischemic side in rats (n=32) after MCAO/reperfusion by stereotactic surgery. Toxicity was detected by histopathology and immunohistochemistry approaches. Proportions of green fluorescent protein (GFP) positive cells in the recombinant adenovirus injected tissues were calculated by staining total nucleus by DAPI.
2. 66 heathy male adult Sprague Dawley (SD) rats were randomly divided into 11 groups, including normal group, sham 2d and 7d group, model (MCAO/reperfusion) 2 d and 7 d group, blank control (PBS) 2 d and 7 d group, negative control (rAd5-HK) 2 d and 7 d group, RNAi intervention (rAd5-shRNA-RGMa) 2 d and 7 d group. The mRNA and protein expressions of RGMa in the cortex and hippocampus of ischemic side were detected by reverse transcription polymerase chain reaction (RT-PCR) and immunehistochemistry respectively. And the axonal growth in corresponding regions was also estimated by immunehistochemistry.
3. Normal group, MCAO/reperfusion group, negative group and RNAi intervention group , divided from 42 SD rats, were set up to identify ischemic regions and newborn neurite branches by TTC staining and BDA neuronal tracing. Behavior tests were adopted to evaluate neurologic function recovery of the rats after focal cerebral ischemic injury and correlative interventions.
Results 1. Titers of purified rAd5-shRNA-RGMa and rAd5-HK were respectively 5.01×1010 pfu/ml and 3.16×1010 pfu/ml. All the three titers of rAd5-shRNA-RGMa could transfect cortex and hippocampus of ischemic side in rats’brain. Transfection efficacies of medium and high titer groups were similar (p >0.05), obviously better than low titer group. The phenomenon of perivascular inflammatory cell infiltration was only observed around the injection site in brain tissues of high titer group. The expression levels of IL-1βin brain tissues of ischemic side in this goup were also higher than other groups on 7 d post-operation (p <0.01).
2. Compared to normal and sham groups, both mRNA and protein expression levels of RGMa in cortex and hippocampus of ischemic side in other groups were significantly increased (p <0.01). The increase degrees of MCAO/reperfusion groups, blank control groups and negative groups were especially high, along with obviously reduction of axon numbers. Meanwhile the expression levels of RGMa were down-regulated and axonal growth was improved simultaneously in RNAi intervention groups (p <0.01).
3. The cerebral infarction volumes of rats in model group,negative group and RNAi intervention group were similar (p >0.05). Behavior test scores of rats in RNAi intervention group after 6 weeks were significantly better than other groups (p <0.01). And numbers of axonal sprouting of corticorubral tract from healthy side to opposite side in this group were also obviously increased (p <0.01).
Conclusions Purified RGMa specific RNAi recombinant adenovirus rAd5-shRNA-RGMa, which could transfect cortex and hippocampus of ischemic side in rats’brain with high stability, high efficacy and low toxicity,is a very effective tool in gene therapeutic research of cerebral ischemia. RGMa mRNA and protein expressions were persistently induced in cortex and hippocampus of ischemic side in rats after middle cerebral artery occlusion/reperfusion,which could be prominently reduced by RGMa-specific RNAi intervention and lead to improved axonal growth and neural anatomy plasticity, as well as neural function recovery.
引文
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[1] Matsunaga, E. and A. Chedotal, Repulsive guidance molecule/neogenin: a novel ligand-receptor system playing multiple roles in neural development. Dev Growth Differ, 2004. 46(6): 481-6. Stahl, B., et al.,
[2] Stahl, B., et al., Biochemical characterization of a putative axonal guidance molecule of the chick visual system. Neuron, 1990. 5(5): 735-43.
[3] Monnier, P.P., et al., RGM is a repulsive guidance molecule for retinal axons. Nature, 2002. 419(6905): 392-5.
[4] Schmidtmer, J. and D. Engelkamp, Isolation and expression pattern of three mouse homologues of chick Rgm. Gene Expr Patterns, 2004. 4(1): 105-10.
[5] Matsunaga, E., H. Nakamura, and A. Chedotal, Repulsive guidance molecule plays multiple roles in neuronal differentiation and axon guidance. J Neurosci, 2006. 26(22): 6082-8.
[6] Severyn, C.J., U. Shinde, and P. Rotwein, Molecular biology, genetics and biochemistry of the repulsive guidance molecule family. Biochem J, 2009. 422(3): 93-403.
[7] De Vries, M. and H.M. Cooper, Emerging roles for neogenin and its ligands in CNS development. J Neurochem, 2008. 106(4):1483-92.
[8] Tassew, N.G., et al., Intraretinal RGMa is involved in retino-tectal mapping. Mol Cell Neurosci, 2008. 37(4): 761-9.
[9] Wilson, N.H. and B. Key, Neogenin interacts with RGMa and netrin-1 to guide axons within the embryonic vertebrate forebrain. Dev Biol, 2006. 296(2): 485-98.
[10] Brinks, H., et al., The repulsive guidance molecule RGMa is involved in the formation of afferent connections in the dentate gyrus. J Neurosci, 2004. 24(15): 3862-9.
[11] Shoemaker, L.D., et al., Identification of differentially expressed proteins in murine embryonic and postnatal cortical neural progenitors. PLoS One. 5(2): e9121.
[12] Shin, G.J. and N.H. Wilson, Overexpression of repulsive guidance molecule (RGM) a induces cell death through Neogenin in early vertebrate development. J MolHistol, 2008. 39(1): 105-13.
[13] Yoshida, J., T. Kubo, and T. Yamashita, Inhibition of branching and spine maturation by repulsive guidance molecule in cultured cortical neurons. Biochem Biophys Res Commun, 2008. 372(4): 725-9.
[14] Metzger, M., et al., RGMa inhibits neurite outgrowth of neuronal progenitors from murine enteric nervous system via the neogenin receptor in vitro. J Neurochem, 2007.
[15] Kee, N., et al., Neogenin and RGMa control neural tube closure and neuroepithelial morphology by regulating cell polarity. J Neurosci, 2008. 28(48): 12643-53.
[16] Niederkofler, V., et al., Repulsive guidance molecule (RGM) gene function is required for neural tube closure but not retinal topography in the mouse visual system. J Neurosci, 2004. 24(4): 808-18.
[17] Hunt, D., R.S. Coffin, and P.N. Anderson, The Nogo receptor, its ligands and axonal regeneration in the spinal cord; a review. J Neurocytol, 2002. 31(2): 93-120.
[18] Schwab, J.M., et al., Spinal cord injury-induced lesional expression of the repulsive guidance molecule (RGM). Eur J Neurosci, 2005. 21(6): p. 1569-76.
[19] Hata, K., et al., RGMa inhibition promotes axonal growth and recovery after spinal cord injury. J Cell Biol, 2006. 173(1): 47-58.
[20] Schwab, J.M., et al., Central nervous system injury-induced repulsive guidance molecule expression in the adult human brain. Arch Neurol, 2005. 62(10): 1561-8.
[21] Shifman, M.I., et al., Expression of the repulsive guidance molecule RGM and its receptor neogenin after spinal cord injury in sea lamprey. Exp Neurol, 2009. 217(2): 242-51.
[22] Doya, H., et al., Induction of repulsive guidance molecule in neurons following sciatic nerve injury. J Chem Neuroanat, 2006. 32(1): 74-7.
[23] Nohra, R., et al., RGMA and IL21R show association with experimental inflammation and multiple sclerosis. Genes Immun, 2010.
[24] Li, V.S., et al., Frequent inactivation of axon guidance molecule RGMA in human colon cancer through genetic and epigenetic mechanisms. Gastroenterology, 2009. 137(1): 176-87.