【作者】 李晨晨；

【导师】 阮迪云；

【作者基本信息】 中国科学技术大学 ， 生物物理， 2008， 博士

【摘要】 本文应用免疫荧光、免疫沉淀、海马脑片场电位记录等技术研究了细胞周期蛋白cyclinD1/CDK4在不同发育期小鼠海马CA1区突触可塑性中的不同作用,以及cyclinD1/CDK4/p—Rb信号参与了铅诱导的培养海马神经元死亡过程,并探讨了其可能涉及的上游信号通路。技术和方法:脑片场电位技术,免疫荧光,免疫沉淀,原代海马神经元培养实验结果:1)细胞周期蛋白cyclinD1/CDK4在不同发育期小鼠海马CA1区突触可塑性中的不同作用在本研究中,我们发现在出生10天的小鼠海马CA1区,CDK4在锥体细胞的核中和胞质中都有分布,但核中分布较多;而在28天的小鼠海马CA1区,CDK4主要定位于胞质,甚至在树突中也有分布。用CDK4抑制剂处理的出生8-15天及21-35天的小鼠海马脑片中,都发现短时程突触可塑性(STP)的削弱。然而,在CDK4抑制剂处理的出生21-35天的小鼠海马脑片上,双脉冲异化(PPF)却没有明显改变。在小鼠海马CA1区用强直刺激诱导的长时程增强(LTP)没有受到CDK4抑制剂的影响。我们还发现,在分别用CDK4抑制剂预处理两个不同发育阶段的小鼠海马脑片上,由代谢型谷氨酸一型受体的激动剂或低频双脉冲刺激诱导的长时程抑制(mGiuR-LTD)都受到了损伤。但是在21-35天的小鼠海马上,CDK4抑制剂引起的mGluR-LTD的损伤,没有在8-15天的小鼠海马脑片上引起的损伤显著。我们的这些结果表明:cyclinD1-CDK4复合物的表达和活化参与了小鼠海马CA1区短时程突触可塑性和代谢型谷氨酸受体依赖的LTD的维持过程,并且提出,在发育的不同阶段,cyclinD1-CDK4在小鼠海马CA1区突触传递功能中可能起不同的作用。2)细胞周期蛋白cyclinD1/CDK4及其下游的p-Rb参与了铅诱导的培养海马神经元死亡过程,并且可能是通过PI3K/AKT通道介导的铅的神经毒性其中一个重要的表现就是会导致神经元死亡。并且铅是如何导致神经元死亡的机制目前还不清楚。我们应用免疫荧光以及免疫沉淀等方法在原代培养的大鼠海马神经元上,研究了铅暴露引起的神经元死亡过程中,细胞周期蛋白cyclinD1/CDK4,它的下游底物pRb,以及其特定的上游信号phosphoinositide 3-kinase(PI3K)/AKT通路的作用。结果表明,铅暴露处理原代培养海马神经元会导致剂量依赖的神经元死亡。抑制CDK4的活性,会显著的降低铅诱导的这种类型的神经元死亡,但是这种保护作用是不完全的。另外,铅暴露处理后,cyclinD1的表达水平和pRb/p107的磷酸化水平与对照组相比都显著升高。这种升高能够持续48小时,并且在72h后会恢复到对照组的水平。为了明确CDK4在神经元内的分布和表达,我们采用CDK4与神经元标记物微管相关蛋白-2(MAP-2)双标记的方法。在对照组中,在神经元的胞质和胞核均有CDK4存在;在用铅离子孵育的神经元中,CDK4只分布于胞核中。并且多数CDK4和p-Rb阳性细胞与TUNEL阳性细胞共分布。加入PI3K的抑制剂LY294002(30μM)或wortmannin(100nm)能够显著的提高神经元的存活率。另外,阻断PI3K/AKT信号通路的活性后,抑制了铅暴露诱导的cyclinD1的表达水平和部分p-Rb/p107的表达水平的升高。以上结果表明,cyclinD1/CDK4/pRb信号通路参与了铅诱导的神经元死亡过程,并且铅离子通过激活PI3K/AKT通路从而诱导cyclinD1和部分p-Rb的表达。更多还原

【Abstract】 In this paper, using immunofluorescence, western blot, immunohistochemical techniques and electrophysiology techniques of brain slices and cultured hippocampal neurons, we studied the roles of CDK4/cyclinD1 in synaptic pasticity during the postnatal development in mice hippocampus area CA1 and lead evoked cultured hippocampal neuronal death. Moreover, we investigated the possible upstream signal pathway involved.Techniques: field potential recording in brain slices, western blot and immunohistochemistryResults:1) The different roles of cyclinD1-CDK4 in STP and mGluR-LTD during thepostnatal development in mice hippocampus area CA1The expression and translocation of cyclinD1-CDK4 in post-mitotic neurons indicate that they may have supplementary functions in differentiated neurons that might be associated with neuronal plasticity. In the present study, our findings showed that the expression of CDK4 was localized mostly in nuclei and cytoplasm of pyramidal cells of CA1 at postnatal day 10 （P10）; whereas at P28 staining of CDK4 could be detected predominantly in the cytoplasm but not nuclei. Basal synaptic transmission was normal in the presence of CDK4 inhibitor. Short-term synaptic plasticity （STP） was impaired in CDK4 inhibitor pre-treated slices both from neonatal （P8-15） and adolescent （P21-35） animals; however there was no significant change in paired-pulse facilitation （PPF） in slices pre-incubated with the CDK4 inhibitor from adolescent animals. By the treatment of CDK4 inhibitor, the induction or the maintenance of Long-term potentiation （LTP） in response to a strong tetanus and NMDA receptor-dependent long-term depression （LTD） were normal in hippocampus. However, long-term depression （LTD） induced either by group I metabotropic glutamate receptors （mGluRs） agonist or by paired-pulse low-frequency stimulation （PP-LFS） was impaired in CDK4 inhibitor pretreated slices both from neonatal and adolescent animals. But the effects of the CDK4 inhibitor at slices from adolescent animals were not as robust as at slices from neonatal animals. Our results indicated that the activation of cyclinD1-CDK4 is required for short-term synaptic plasticity and mGluR-dependent LTD, and suggested that this cyclin-dependent kinase may have different roles during the postnatal development in mice hippocampus area CA1.2) Involvement of cyclinD1/CDK4 and pRb mediated by PI3K/AKT pathway activation in Pb2+-induced neuronal death in cultured hippocampal neuronsThe present study sought to examine the obligate nature of cyclinD1CDK4, phosphorylation of its substrate retinoblastoma protein （pRb） and its select upstream signal phosphoinositide 3-kinase （PI3K）/AKT pathway in the death of primary cultured rat hippocampal neurons evoked by Pb²⁺. Our data showed that lead treatment of primary hippocampal cultures results in dose-dependent cell death. Inhibition of CDK4 prevented Pb2+-induced neuronal death significantly but incomplete. In addition, we demonstrated that the levels of cyclinD1 and pRb/p107 were increased during Pb2+ treatment. These elevated expression persisted up to 48h, returning to control levels after 72h. We also presented pharmacological and morphological evidences that cyclinD1/CDK4 and pRb/p107 were required for such kind of neuronal death. Addition of the PI3K inhibitor LY294002（30μM） or wortmannin （100nM） significantly rescued the cultured hippocampal neurons from death caused by Pb2+. And that Pb2+-elicited phospho-AKT （Ser473） participated in the induction of cyclinD1 and partial pRb/p107 expression. These results provide evidences that cell cycle elements play a required role in death of neurons evoked by Pb²⁺ and suggest that certain signaling elements upstream of cyclinD1/CDK4 are modified and/or required for this form of neuronal death.更多还原