【作者】 魏佳军；

【导师】 张苏明；

【作者基本信息】 华中科技大学 ， 神经病学， 2008， 博士

【摘要】 第一部分利用Helper Free系统构建携带cdc2-siRNA重组腺相关病毒及功能鉴定目的周期素依赖性蛋白激酶(cyclin dependent kinases, cdks)在多种神经变性疾病的发病中扮演着重要的角色。实验目的是为了探讨与cdks相关的基因治疗C型尼曼-皮克病的可能性,我们包装携带cdc2-siRNA的重组腺相关病毒(recombinant adeno-associated virus, rAAV)。方法应用分子生物学技术构建生成pAAV-EGFP-U6-cdc2-siRNA表达质粒。用磷酸钙将该质粒和p-RC、p-Helper质粒共转染于AAV-293细胞中,包装生成携带cdc2-siRNA的rAAV (rAAV-EGFP-U6-cdc2-siRNA)。行Western Blot检测cdc2-siRNA对AAV-293细胞cdc2基因的沉默效果。结果DNA测序证明U6-cdc2-siRNA已成功构建到质粒pAAV-MCS-EGFP中。经72小时的包装,AAV-293细胞中可见EGFP的表达。rAAV-EGFP-U6-cdc2-siRNA感染AAV-293细胞后,经Western Blot检测显示AAV-293细胞中cdc2基因表达显著下调,这证明该重组腺相关病毒具有令人满意的基因沉默作用。结论这些结果表明该携带cdc2-siRNA重组腺相关病毒的包装获得成功。它具有有效沉默cdc2基因的作用,这将可能为C型尼曼-皮克病提供一种新的治疗手段。第二部分携带cdc2-siRNA重组腺相关病毒对C型尼曼-皮克病小鼠行为学的影响目的观察小脑注射携带cdc2-小干扰RNA(cdc2-siRNA)重组腺相关病毒(rAAV)对C型尼曼-皮克病(NPC)小鼠行为学的影响。方法2周龄npc-/-小鼠经小脑注射携带cdc2-siRNA的rAAV,动态测量小鼠体重,并进行衣架悬挂试验及足印试验评估小鼠4到8周的运动能力。结果①cdc2-siRNA组小鼠体重减轻明显延缓。②衣架悬挂试验显示cdc2-siRNA组小鼠运动缺陷明显改善。③足印试验显示cdc2-siRNA组相对步距(75.1%±8.6%)与对照病毒组及非手术组相比明显增加。结论小脑注射携带cdc2-siRNA的rAAV改善了npc-/-小鼠的行为学,有可能为NPC的治疗提供新的有效途径。第三部分携带cdc2-siRNA重组腺相关病毒对C型尼曼-皮克病小鼠神经病理的影响目的评估小脑注射携带cdc2-siRNA的重组腺相关病毒(rAAV)对C型尼曼-皮克病(NPC)小鼠神经病理的影响。方法①经小脑途径给2周龄npc-/-小鼠注射携带cdc2-siRNA的rAAV。②通过免疫组织化学、HE染色及免疫印迹方法评估注射携带cdc2-siRNA rAAV npc-/-小鼠的病理改变。结果①携带cdc2-siRNA的rAAV在npc-/-小鼠脑中广泛表达。②携带cdc2-siRNA的rAAV明显减少npc-/-小鼠轴突球状体的数量、延缓浦肯野细胞的脱失。③携带cdc2-siRNA的rAAV能有效抑制cdc2、磷酸化的神经丝(由SMI31识别)、磷酸化的有丝分裂相关蛋白(由TG-3和MPM-2识别)及磷酸化的Tau蛋白(由PHF-1识别)的表达。结论小脑注射携带cdc2-siRNA的rAAV对npc-/-小鼠神经元细胞骨架病理具有保护作用,cdc2有可能成为NPC治疗的新靶点。更多还原

【Abstract】 Part One Packaging and functional identification of recombinant adeno-associated virus encoding cdc2-siRNA by Helper Free systemObjectiveCyclin dependent kinases(cdks) play an important role in the pathogenesis of multiple neurodegenerative diseases. As the first step toward exploring the possibility of cdks related gene therapy for Niemann-pick disease type C, we packed recombinant adeno- associated virus (rAAV) encoding cdc2-siRNA.MethodsThe expressing plasmid pAAV-EGFP-U6-cdc2-siRNA was constructed by molecular biological techniques. The rAAV encoding cdc2-siRNA (rAAV-EGFP-U6-cdc2-siRNA) was packed by calcium phosphate mediated cotransfection of pAAV-EGFP-U6-cdc2- siRNA, p-RC and p-Helper into AAV-293 cells.ResultsDNA sequencing proved the successful construction of U6-cdc2-siRNA in pAAV- MCS-EGFP. After 72 hours’packaging, the expression of EGFP could be detected in AAV-293 cells. Cdc2 gene expression in AAV-293 cells assessed by Western Blot was down-regulated markedly after infection of rAAV-EGFP-U6-cdc2-siRNA, which evidenced the satisfactory silencing effect of this virus. ConclusionOur data indicate that the packaging of rAAV encoding cdc2-siRNA is successful. It can silence cdc2 gene effectively, which might offer a novel means for treating Niemann- Pick disease type C.Part Two Effect of recombinant adeno-associated virus encoding cdc2-siRNA on the behavior of Niemann-Pick disease type C miceObjectiveTo observe the effect of cerebellar injection of recombinant adeno-associated virus (rAAV) encoding cdc2-siRNA on the behavior of Niemann-Pick disease type C (NPC) mice.MethodsThe rAAV encoding cdc2-siRNA was injected into the cerebellum of 2 weeks old npc-/- mice. The mice were weighed dynamically and coat hanger test and footprint test were performed to assess their motor ability from 4 to 8 weeks age.Results①Weight loss was significantly delayed in cdc2-siRNA group.②Coat hanger test showed that the motor defect was remarkably ameliorated in cdc2-siRNA group. ③Footprint test showed that the relative stride in cdc2-siRNA group (75.1%±8.6%) was markedly longer than that in the controlled viral group and the non-surgical group.ConclusionThe cerebellar injection of rAAV encoding cdc2-siRNA improves the behavior of npc-/- mice, which may offer a novel means for treating NPC.Part Three Effect of recombinant adeno-associated virus encoding cdc2-siRNA on the neuropathology of Niemann-Pick disease type C miceObjectiveTo assess the effect of cerebellar injection of recombinant adeno-associated virus (rAAV) encoding cdc2-siRNA on the neuropathology of Niemann-Pick disease type C (NPC) mice.MethodsRAAV encoding cdc2-siRNA was injected into the cerebellum of 2 weeks old npc-/- mice. The neuropathology in the treated mice was evaluated by immunohistology, HE staining and immunoblotting technology.Results①rAAV encoding cdc2-siRNA was widely expressed in npc-/- mice brain.②rAAV encoding cdc2-siRNA remarkably reduced the number of axonal spheroids, and ameliorated the deletion of purkinje cells in npc-/- mice. ③rAAV encoding cdc2-siRNA markedly inhibited the expressions of cdc2, phospho- neurofilament (recognized by SMI31), phospho-mitotic associated proteins (recognized by TG-3 and MPM-2), and phospho-Tau (recognized by PHF-1).ConclusionThe rAAV encoding cdc2-siRNA injected into the cerebellum of npc-/- mice can protect the neuronal cytoskeletal pathology, which implys that cdc2 might be a novel therapeutic target in NPC.更多还原