【作者】 黄海潮；

【导师】 陈一岳； 臧林泉；

【作者基本信息】 广东药学院 ， 药剂学， 2008， 硕士

【摘要】 大脑动脉阻塞引起的中风和脑梗死,因其在老年人中致残高和易引起智能缺陷,成为严重影响人们健康的世界性难题。随着人类寿命的延长,遭受这种疾病的患者数量将进一步增加。最近,人们对局部缺血引起脑损伤的分子生物学机制研究取得了突破性进展,在基础药理的动物实验中报道了很多具有很强的大脑保护作用、颇有治疗前景的药物。然而,目前,针对脑缺血性中风的治疗药物仍然有限。在近几年的实验研究中,人们相继阐明由谷氨酸、Ca²⁺、局部脑缺血的基因差异表达、凋亡引起的神经元死亡,脑局部缺血后的炎症反应等介导的缺血性神经元死亡机制。Kitagawa等于1990年报道了短暂缺血预处理能诱导缺血性耐受,产生神经保护作用。有鉴于此,我们课题组的臧林泉等人研究发现急性局灶性脑缺血大鼠缺血组织与非缺血组织中存在基因差异表达,克隆出与脑缺血相关的基因KIAA0280,其在急性局灶性脑缺血大鼠中明显上调表达,该基因的功能在国内外均未见报道,其显著地差异表达提示其在脑缺血病理过程中具有重要作用。实验证实,缺血性神经元的死亡相当一部分是通过细胞凋亡的机制来实现。细胞凋亡过程涉及一系列基因和蛋白的表达,caspase,bcl-2等等,并在凋亡发生过程中会有新的物质来对凋亡作出调控。为此,我们研究KIAA0280在不同因素诱导神经元凋亡中的表达,将有助于明确其在凋亡中功能作用,为研究其在凋亡中的作用机制奠定基础,丰富细胞凋亡学说的内容,为临床治疗脑中风,心肌梗死,肾衰等与细胞凋亡相关的疾病提供新的治疗方法。目的通过建立原代神经元的体外培养技术和不同因素诱导的神经元凋亡模型;研究KIAA0280在不同因素诱导诱导神经元凋亡中表达情况;研究KIAA0280被特异性沉默后,基因的表达情况以及沉默KIAA0280后,对凋亡神经元的影响。方法（1）用原代分离、培养的方法,建立神经元的体外培养技术（2）将神经元放置于一密闭容器,然后抽真空,再充以95%N₂,5%CO₂,放入37℃培养箱中,分别缺氧不同时间,用Hoechst33258法,乳酸脱氢酶法, SRB法检测神经元的凋亡,建立缺氧诱导细胞凋亡模型（3）建立谷氨酸诱导神经元凋亡模型,用Hoechst33258法、乳酸脱氢酶法、SRB法检测神经元凋亡,建立脑缺血兴奋性氨基酸毒性细胞模型。（4）建立过氧化氢诱导神经元凋亡模型,用Hoechst33258法、乳酸脱氢酶法、SRB法检测神经元凋亡,建立脑缺血再灌注细胞模型（5）用RT-PCR特异性扩增出不同神经元凋亡模型中KIAA0280在神经元中的表达,观察不同因素引起的神经元凋亡中KIAA0280的表达变化。（6）用脂质体转染的方法,将KIAA0280基因特异性siRNA转染进神经元,观察转染前后神经元的生长变化,以及转染前后,凋亡神经元变化情况。结果（1）本实验成功建立原代神经元的体外培养技术,分离成功率高,神经元生长良好,存活率高,可存活30天以上。（2）用密闭容器抽真空的方法建立缺氧诱导神经元凋亡的模型。LDH,SRB的检测结果（P<0.05）表明本实验所建立细胞凋亡模型是成功的。RT-PCR结果表明在缺氧神经元中KIAA0280基因的表达明显上调,采用RNAi沉默KIAA0280的表达后,神经元的凋亡加重。（3）用谷氨酸诱导神经元凋亡,复制兴奋性氨基酸对神经系统的毒性作用细胞模型。结果显示,谷氨酸诱导神经元凋亡模型成功;RT-PCR结果显示,KIAA0280在谷氨酸诱导的凋亡神经元中没见明显表达,Hoechst33258和LDH结果显示,在转染KIAA0280特异性siRNA后,凋亡神经元的变化不明显。（4）用H₂O₂诱导神经元凋亡,复制脑缺血再灌氧自由基损伤的体外细胞模型。结果显示,H₂O₂诱导神经元凋亡模型成功;RT-PCR结果显示,KIAA0280在H₂O₂诱导的凋亡神经元中没有明显的表达,Hoechst33258和LDH结果显示,在转染KIAA0280特异性siRNA后,凋亡神经元的变化不明显。结论本实验成功建立原代神经元分离培养方法,建立不同因素诱导的神经元凋亡模型。从Hoechst33258、LDH、SRB以及RT-PCR结果,说明KIAA0280沉默后能促进缺氧引起的神经元凋亡,而在谷氨酸以及H₂O₂诱导的神经元凋亡模型中没有作用,提示KIAA0280跟缺氧诱导的神经元凋亡密切相关。更多还原

【Abstract】 Stroke and cerebral infarction as a result of occlusion of cerebral arteries have long been a major health problem worldwide because they are major causes of disability and intellectual impairment in elderly. The number of patients suffering from these disorders will increase further in the future, as the life span of man is becoming longer. Recently, considerable advances have been made in our understanding of the molecular and biochemical mechanisms of ischemia-induced brain damage and a growing number of promising drugs with powerful cerebroprotective effects have been reported in basic pharmacology using experimental animals. However, the therapeutic efficiency of the drugs for patients with ischemic stroke is still limited at present. During the past years, a considerable amount of experimental studies have been devoted to elucidate the mechanism of ischemic neuronal death mediated by glutamate, calcium, altered gene expression after ischemia, neuronal death by apoptosis, inflammatory reactions induced in postischemic brain. In 1990 Kitagawa reported that transient ischemia preconditioning （IPC） could induce ischemic tolerance, which might produce neuroprotective effect.Therefore LinQ, Zang screened the differential expressed gene between acute focal ischemia and non-ischemia region in rat brain, and cloned the novel ischemia associated gene KIAA0280. And found that it over-expressed in acute focal ischemia. The function of this gene has not been reported based on literatures. It obviously over-expressed in ischemia region demonstrated that KIAA0280 had a critical function in the ischemia pathology.Many experiments showed that nerve cells death in brain occurred partly by apoptosis. The process of apoptosis was related to the expression of many genes and proteins and regulated by some genes, such as: caspase, bcl-2 et al. It can produce some new genes to regulate apoptosis in the process of apoptosis. Therefore we studied the expression of KIAA0280 in the apoptotic neurons induced by different factor, and to understand the function of KIAA0280, to contribute foundation for the study of mechanism of action in apoptosis and apoptotic theory, to provide new method for apoptotic disease such as stroke, Myocardial Infarction, renal failure and so on.ObjectiveTo establish the separation and culture of primary neuron in vitro; To establish the model of apoptotic neurons induced by different factor; To study the KIAA0280 expression in the apoptotic neurons; To study the KIAA0280 expression after silencing KIAA0280 using the technology of RNAi, and to observe the effect on the normal neurons after silencing KIAA0280; To observe the effect of apoptotic neurons after silencing KIAA0280.Methods（1） We adopted the methods of primary separation and culture to establish neuron culture in vitro.（2） We established the model of neuronal apoptosis induced by hypoxia applied airtight container to evacuate and fill with 95%N₂, 5%CO₂, and then put in the cell incubator at 37℃, hypoxia at different time, then detected apoptotic neurons with Hoechst33258, LDH leakage, SRB.（3） Established cell model of the toxic effect of excitatory amino acid in cerebral ischemia with glutamic-induced apoptotic neurons, and then we detected apoptotic neurons with Hoechst33258, LDH leakage, SRB.（4） We established the cell model of cerebral ischemia-reperfusion with H₂O₂ inducing neuronal apoptosis, and then detected apoptotic neurons with Hoechst33258, LDH leakage, SRB. （5） Adopted RT-PCR to specificity amplify the expression of KIAA0280 in the apoptotic neurons induced by different factor, and then we observed the expression of KIAA0280 in the different apoptotic neurons.（6） Transfered KIAA0280 specificity siRNA into neurons with liposome lipofectamine2000, then we observed the differences of normal and apoptotic neurons after transfection.Results（1） We have successfully established the technology of primary neuron culture. The results showed that the neurodendrons and neuraxons, the feature of neuron shape, is obvious and the neurons can survive over 30d.（2） We adopted airtight container to evacuate and filled with 95%N₂, 5%CO₂ can reduce interference factor in the experiment, and make the result more confidence and reproducibility. The results of Hoechst33258staining, SRB, and LDH approved the neuronal apoptotic model induced by hypoxia is success. The result of RT-PCR revealed that KIAA0280 obviously over-expressed in the hypoxia-induced apoptotic neurons. And the result showed that the apoptotic neurons were aggravated after silencing KIAA0280.（3） We established the cell model of excitatory amino acid toxity effect of cerebral ischemia with glutamate-induced neuronal apoptosis, and the results showed that the model is success. The result of RT-PCR showed that KIAA0280 was not obviously expressed in the apoptotic neurons induced by glutamate. And the result showed that the apoptotic neurons after transferring KIAA0280 specificity siRNA were not different from those after transferring negative control siRNA.（4） We added H₂O₂ into medium to induce neuronal apoptosis, and established the cell model of the oxygen free radical injury in cerebral ischemia-reperfusion. The results showed that the model is success. The results of RT-PCR showed that KIAA0280 was not obviously expressed in apoptotic neurons induced by H₂O₂. And the result showed that the apoptotic neurons after transferring KIAA0280 specificity siRNA were not different from those after transferring negative control siRNA. ConclusionWe successfully established the methods of primary neuron separation and culture in vitro and the model of neuronal apoptosis induced by different factor. The result from Hoechst33258, LDH, SRB and RT-PCR revealed that KIAA0280 had support effect on the normal neurons growth and played a positive role in protecting hypoxia-induced apoptosis neurons, rather than in the glutamic acid and H₂O₂ induced apoptotic neurons.更多还原