帕金森恒河猴血液P53、Bax、Bcl-2、Caspase-3 mRNA的进程性变化
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摘要
帕金森(Parkinson's disease,PD)是一种以运动功能障碍为主要特征的神经退行性疾病,大量研究表明细胞凋亡参与了PD的发病机制。PD动物模型对于深入研究PD病因及发病机理、进行有效预防和提高治疗效果将起到重要作用。为探讨PD恒河猴(Macaca mulatta)外周血中肿瘤抑制基因P53、B细胞淋巴瘤-2(B cell lymphoma-2,Bcl-2)、B细胞淋巴瘤-2相关X蛋白(BCL-2-associated X protein,Bax)和半胱天冬氨酸蛋白酶-3(cysteinyl aspartate-specific proteinase-3,Caspase-3)mRNA表达水平与行为学变化之间的关系,本研究选取健康青年恒河猴6只,以0.2 mg/(kg·d)小剂量、多次重复肌内注射1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methy-4-pheny-l,2,3,6-tetrahydropyridine,MPTP)建立了PD恒河猴模型。于每次MPTP给药前后观察记录动物行为学表现30 min。分别于未注射MPTP(第0周),注射MPTP后第4、8、12、16、20和24周采集恒河猴前臂静脉血,采用qRT-PCR检测外周血P53、Bax、Bcl-2和Caspase-3 mRNA表达水平。行为学观察结果显示,第4周时,实验动物表现出温和的运动迟缓,随后症状逐渐加重,运动能力持续下降,到12周时实验动物均表现出活动量显著减少,姿势僵硬和严重的运动障碍。13周后临床症状基本稳定,实验动物表现出运动迟缓,姿势异常,肌强直和静止性震颤等。qRT-PCR结果显示,与0周相比,注射MPTP后第4、8、12周P53和Caspase-3 mRNA表达显著升高(P<0.05),而第16、20、24周差异不显著;与0周相比,Bcl-2/Bax比值在第4、8、12周显著降低(P<0.05),而第16、20、24周差异不显著。0~12周行为学评分与外周血P53和Caspase-3 mRNA表达水平呈正相关(r=0.975,r=0.956;P<0.05)。结果表明,当外周血P53和Caspase-3 mRNA表达水平显著升高时临床症状也处于逐渐加重的阶段,因此通过检测血液中凋亡调控基因的表达,能反应出PD恒河猴的疾病进程。研究结果可为PD诊断和疾病进程监测提供辅助参考。
Parkinson's disease(PD), the second most common neurodegenerative disease. It includes the major motor symptoms of akinesia,rigidity and tremor.A lot of study indicates that apoptosis is involved in the pathogenesis of PD.The present study is to investigate the mRNA expression of tumor suppressor gene P53,cysteinyl aspartate-specific proteinase-3(Caspase-3),B cell lymphoma-2(Bcl-2)and BCL-2-associated X protein(Bax)in peripheral blood before and after treatment in monkey(Macaca mulatta)with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP).Six healthy young rhesus monkeys were respectively daily injected a small dose(0.2 mg/(kg·d))of MPTP.The behavioral manifestations of all monkeys were evaluated before and after the injection for 30 min,respectively.The forearm venous blood of rhesus monkeys were took at 0 week(non-injected MPTP)and 4,8,12,16,20,24 week after injection MPTP.qRT-PCR detected P53,Bax,Bcl-2 and Caspase-3 mRNA expression in peripheral blood.The correlations between mRNA expression of P53,Caspase-3,Bcl-2 and Bax and the severity of behavior scores were analyzed.Behavioral observation indicated that from the 4 week,the experimental animals showed moderate bradykinesia,and then the symptoms gradually worsened,exercise capacity continued to decline,experimental animals showed a significant reduction in activity at 12 week,posture stiff and severe movement obstacle.After the 13 week of clinical symptoms was basically stable,the experimental animals showed slow movement,abnormal posture,muscle rigidity and resting tremor.qR T-PCR detected of apoptosis regulatory factors,compared with the 0 week,the expression of P53 and Caspase-3 significantly increased at the 4,8,12 week(P<0.05),but the difference was not significantly at the 16,20,24 week;compared with the 0 week,Bcl-2/Bax ratio at the 4,8,12 week significantly decreased(P<0.05),and the difference was not significantly at 16,20 and 24 week.The mRNA expression of P53 and Caspase-3 were positively correlated with behavior scores in 0~12 week(P<0.05).The above results suggested that when P53 and Casapse-3 significantly increased,the clinical symptoms of experimental animals also continued to intensify with the same period.The mR NA expression of P53 and Caspae-3 in peripheral blood can be used as one of the indicators for diagnosing PD,assessing the severity of PD.
Parkinson's disease(PD), the second most common neurodegenerative disease. It includes the major motor symptoms of akinesia,rigidity and tremor.A lot of study indicates that apoptosis is involved in the pathogenesis of PD.The present study is to investigate the mRNA expression of tumor suppressor gene P53,cysteinyl aspartate-specific proteinase-3(Caspase-3),B cell lymphoma-2(Bcl-2)and BCL-2-associated X protein(Bax)in peripheral blood before and after treatment in monkey(Macaca mulatta)with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP).Six healthy young rhesus monkeys were respectively daily injected a small dose(0.2 mg/(kg·d))of MPTP.The behavioral manifestations of all monkeys were evaluated before and after the injection for 30 min,respectively.The forearm venous blood of rhesus monkeys were took at 0 week(non-injected MPTP)and 4,8,12,16,20,24 week after injection MPTP.qRT-PCR detected P53,Bax,Bcl-2 and Caspase-3 mRNA expression in peripheral blood.The correlations between mRNA expression of P53,Caspase-3,Bcl-2 and Bax and the severity of behavior scores were analyzed.Behavioral observation indicated that from the 4 week,the experimental animals showed moderate bradykinesia,and then the symptoms gradually worsened,exercise capacity continued to decline,experimental animals showed a significant reduction in activity at 12 week,posture stiff and severe movement obstacle.After the 13 week of clinical symptoms was basically stable,the experimental animals showed slow movement,abnormal posture,muscle rigidity and resting tremor.qR T-PCR detected of apoptosis regulatory factors,compared with the 0 week,the expression of P53 and Caspase-3 significantly increased at the 4,8,12 week(P<0.05),but the difference was not significantly at the 16,20,24 week;compared with the 0 week,Bcl-2/Bax ratio at the 4,8,12 week significantly decreased(P<0.05),and the difference was not significantly at 16,20 and 24 week.The mRNA expression of P53 and Caspase-3 were positively correlated with behavior scores in 0~12 week(P<0.05).The above results suggested that when P53 and Casapse-3 significantly increased,the clinical symptoms of experimental animals also continued to intensify with the same period.The mR NA expression of P53 and Caspae-3 in peripheral blood can be used as one of the indicators for diagnosing PD,assessing the severity of PD.
引文
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Cersosimo M G,Benarroch E E.2012.Autonomic involvement in Parkinson's disease:Pathology,pathophysiology,clinical features and possible peripheral biomarkers[J].Journal of the Neurological Sciences,313(1-2):57-63.
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Chen Z,Wang E,Hu R,et al.2015.Tetranectin gene deletion induces Parkinson's disease by enhancing neuronal apoptosis[J].Biochem Biophys Res Commun,468(1-2):400-407.
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Eslamboli A.2005.Marmoset monkey models of Parkinson's disease:Which model,when and why?[J].Brain research bulletin,68(3):140-149.
Frohlich M,Jaeger A,Weiss D G,et al.2016.Inhibition of BCL-2 leads to increased apoptosis and delayed neuronal differentiation in human Re Ncell VM cells in vitro[J].International Journal of Developmental Neuroscience,48:9-17.
Ghavami S,Shojaei S,Yeganeh B,et al.2014.Autophagy and apoptosis dysfunction in neurodegenerative disorders[J].Progress in Neurobiology,112:24-49.
Kalia L V,Lang A E.2015.Parkinson's disease[J].The Lancet,386(9996):896-912.
Kavitha M,Nataraj J,Essa M M,et al.2013.Mangiferin attenuates MPTP induced dopaminergic neurodegeneration and improves motor impairment,redox balance and Bcl-2/Bax expression in experimental Parkinson's disease mice[J].Chemico-biological Interactions,206(2):239-247.
Liu K,Xu H,Xiang H,et al.2015.Protective effects of Ndfip1 on MPP(+)-induced apoptosis in MES23.5 cells and its underlying mechanisms[J].Experimental Neurology,273:215-224.
Yalcinkaya N,Haytural H,Bilgic B,et al.2016.Expression changes of genes associated with apoptosis and survival processes in Parkinson's disease[J].Neuroscience letters,615:72-77.
Antonelli F,Strafella A P.2014.Behavioral disorders in Parkinson's disease:The role of dopamine[J].Parkinsonism&Related Disorders,20:10-12.
Asakawa T,Fang H,Sugiyama K J,et al.2016.Animal behavioral assessments in current research of Parkinson's disease[J].Neuroscience and Biobehavioral Reviews,65:63-94.
Bernstein A I,O'Malley K L.2013.MPP+-induces PUMA-and p53-dependent,but ATF3-independent cell death[J].Toxicology Letters,219(2):93-98.
Bove J,Perier C.2012.Neurotoxin-based models of Parkinson's disease[J].Neuroscience,211:51-76.
Calopa M,Bas J,Callen A,et al.2010.Apoptosis of peripheral blood lymphocytes in Parkinson patients[J].Neurobiology of Disease,38(1):1-7.
Cersosimo M G,Benarroch E E.2012.Autonomic involvement in Parkinson's disease:Pathology,pathophysiology,clinical features and possible peripheral biomarkers[J].Journal of the Neurological Sciences,313(1-2):57-63.
Checler F,Alves da Costa C.2014.p53 in neurodegenerative diseases and brain cancers[J].Pharmacology Therapeutics,142(1):99-113.
Chen Z,Wang E,Hu R,et al.2015.Tetranectin gene deletion induces Parkinson's disease by enhancing neuronal apoptosis[J].Biochem Biophys Res Commun,468(1-2):400-407.
Delenclos M,Jones D R,Mc Lean P J,et al.2016.Biomarkers in Parkinson's disease:Advances and strategies[J].Parkinsonism Relat Disord,22(1):106-110.
Dexter D T,Jenner P.2013.Parkinson disease:From pathology to molecular disease mechanisms[J].Free Radical Biology and Medicine,62:132-144.
Eslamboli A.2005.Marmoset monkey models of Parkinson's disease:Which model,when and why?[J].Brain research bulletin,68(3):140-149.
Frohlich M,Jaeger A,Weiss D G,et al.2016.Inhibition of BCL-2 leads to increased apoptosis and delayed neuronal differentiation in human Re Ncell VM cells in vitro[J].International Journal of Developmental Neuroscience,48:9-17.
Ghavami S,Shojaei S,Yeganeh B,et al.2014.Autophagy and apoptosis dysfunction in neurodegenerative disorders[J].Progress in Neurobiology,112:24-49.
Kalia L V,Lang A E.2015.Parkinson's disease[J].The Lancet,386(9996):896-912.
Kavitha M,Nataraj J,Essa M M,et al.2013.Mangiferin attenuates MPTP induced dopaminergic neurodegeneration and improves motor impairment,redox balance and Bcl-2/Bax expression in experimental Parkinson's disease mice[J].Chemico-biological Interactions,206(2):239-247.
Liu K,Xu H,Xiang H,et al.2015.Protective effects of Ndfip1 on MPP(+)-induced apoptosis in MES23.5 cells and its underlying mechanisms[J].Experimental Neurology,273:215-224.
Yalcinkaya N,Haytural H,Bilgic B,et al.2016.Expression changes of genes associated with apoptosis and survival processes in Parkinson's disease[J].Neuroscience letters,615:72-77.