【摘要】：帕金森(Parkinson's disease,PD)是一種以運(yùn)動(dòng)功能障礙為主要特征的神經(jīng)退行性疾病,大量研究表明細(xì)胞凋亡參與了PD的發(fā)病機(jī)制。PD動(dòng)物模型對(duì)于深入研究PD病因及發(fā)病機(jī)理、進(jìn)行有效預(yù)防和提高治療效果將起到重要作用。為探討PD恒河猴(Macaca mulatta)外周血中腫瘤抑制基因P53、B細(xì)胞淋巴瘤-2(B cell lymphoma-2,Bcl-2)、B細(xì)胞淋巴瘤-2相關(guān)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表達(dá)水平與行為學(xué)變化之間的關(guān)系,本研究選取健康青年恒河猴6只,以0.2 mg/(kg·d)小劑量、多次重復(fù)肌內(nèi)注射1-甲基-4-苯基-1,2,3,6-四氫吡啶(1-methy-4-pheny-l,2,3,6-tetrahydropyridine,MPTP)建立了PD恒河猴模型。于每次MPTP給藥前后觀察記錄動(dòng)物行為學(xué)表現(xiàn)30 min。分別于未注射MPTP(第0周),注射MPTP后第4、8、12、16、20和24周采集恒河猴前臂靜脈血,采用qRT-PCR檢測(cè)外周血P53、Bax、Bcl-2和Caspase-3 mRNA表達(dá)水平。行為學(xué)觀察結(jié)果顯示,第4周時(shí),實(shí)驗(yàn)動(dòng)物表現(xiàn)出溫和的運(yùn)動(dòng)遲緩,隨后癥狀逐漸加重,運(yùn)動(dòng)能力持續(xù)下降,到12周時(shí)實(shí)驗(yàn)動(dòng)物均表現(xiàn)出活動(dòng)量顯著減少,姿勢(shì)僵硬和嚴(yán)重的運(yùn)動(dòng)障礙。13周后臨床癥狀基本穩(wěn)定,實(shí)驗(yàn)動(dòng)物表現(xiàn)出運(yùn)動(dòng)遲緩,姿勢(shì)異常,肌強(qiáng)直和靜止性震顫等。qRT-PCR結(jié)果顯示,與0周相比,注射MPTP后第4、8、12周P53和Caspase-3 mRNA表達(dá)顯著升高(P0.05),而第16、20、24周差異不顯著;與0周相比,Bcl-2/Bax比值在第4、8、12周顯著降低(P0.05),而第16、20、24周差異不顯著。0~12周行為學(xué)評(píng)分與外周血P53和Caspase-3 mRNA表達(dá)水平呈正相關(guān)(r=0.975,r=0.956;P0.05)。結(jié)果表明,當(dāng)外周血P53和Caspase-3 mRNA表達(dá)水平顯著升高時(shí)臨床癥狀也處于逐漸加重的階段,因此通過(guò)檢測(cè)血液中凋亡調(diào)控基因的表達(dá),能反應(yīng)出PD恒河猴的疾病進(jìn)程。研究結(jié)果可為PD診斷和疾病進(jìn)程監(jiān)測(cè)提供輔助參考。
[Abstract]:Parkinson's disease (Parkinson's disease,PD) is a neurodegenerative disease characterized by motor dysfunction. A large number of studies have shown that apoptosis is involved in the pathogenesis of PD. PD animal model for in-depth study of the etiology and pathogenesis of PD, Effective prevention and improvement of therapeutic effects will play an important role. To investigate the tumor suppressor gene P53, B cell lymphoma-2 (B cell lymphoma-2,Bcl-2), B cell lymphoma-2-associated X protein (BCL-2-associated X protein,) in peripheral blood of PD rhesus monkey (Macaca mulatta) The relationship between the expression level of Bax and caspase-3 (cysteinyl aspartate-specific proteinase-3,Caspase-3) mRNA and behavioral changes was studied in this study. Six healthy young rhesus monkeys were selected and given a low dose of 0.2 mg/ (kg 路d). PD rhesus monkey model was established by repeated intramuscular injection of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). The behavior of animals was observed and recorded for 30 min. before and after each MPTP administration. The blood samples of forearm vein of rhesus monkeys were collected at 4,8,12,16,20 and 24 weeks after injection of MPTP (0 weeks), respectively. The expression levels of P53, Bax, Bcl-2 and Caspase-3 mRNA in peripheral blood were detected by qRT-PCR. Behavioral observation showed that at the 4th week, the animals showed mild slow movement, then the symptoms became more severe and the motor ability continued to decrease, and at the 12th week, all the animals showed a significant decrease in the amount of activity, and the experimental animals showed a significant decrease in activity at the end of the 12th week. The clinical symptoms were basically stable after 13 weeks, the experimental animals showed slow movement, abnormal posture, muscular rigidity and quivering tremor, etc. The results of qRT-PCR showed that compared with 0 weeks, 4,8,8 after MPTP injection, the clinical symptoms were basically stable, and the experimental animals showed slow movement, abnormal posture, muscular stiffness and quivering tremor. The expression of p53 and Caspase-3 mRNA increased significantly at the 12th week (P0.05), but there was no significant difference at the 16th, 20th, 24th week. Compared with 0 weeks, the ratio of Bcl-2/Bax decreased significantly at 4,8,12 weeks (P0.05), but there was no significant difference at 16,20,24 weeks (P 0.05). There was a positive correlation between the behavioral score and the expression of p53 and Caspase-3 mRNA in peripheral blood (r = 0.975, r = 0.956) at week 16, week 20 and week 24 (r = 0.975, r = 0.956). (P0.05). The results showed that when the expression level of p53 and Caspase-3 mRNA in peripheral blood was significantly increased, the clinical symptoms were in the stage of gradual aggravation. Therefore, the expression of apoptosis-regulated genes could reflect the disease progression of PD rhesus monkeys by detecting the expression of apoptosis-regulated genes in blood. The results can be used as a reference for the diagnosis of PD and the monitoring of disease progression.
【作者單位】： 四川農(nóng)業(yè)大學(xué)動(dòng)物醫(yī)學(xué)院/動(dòng)物疾病模型實(shí)驗(yàn)室;四川農(nóng)業(yè)大學(xué)預(yù)防獸醫(yī)研究所;四川普萊美生物科技有限公司/國(guó)家實(shí)驗(yàn)獼猴種源基地;
【基金】：國(guó)家科技支撐計(jì)劃課題(No.2014BAI03B01) 國(guó)家重大科學(xué)儀器設(shè)備開(kāi)發(fā)專項(xiàng)(No.2013YQ49085906)
【分類號(hào)】：R742.5

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