【作者】 刘靖；

【导师】 邹原；

【作者基本信息】 大连医科大学 ， 生理学， 2008， 硕士

【摘要】 目的:阿尔茨海默病(Alzheimer’s disease, AD)是一种常见的神经变性性疾病,其最显著的病理学特征是在脑区(皮质,海马)脑细胞外β淀粉样蛋白(amyloidβprotein, Aβ)沉积形成的老年斑,而Aβ的沉积可以引起神经元功能失调导致阿尔茨海默病的发生。近年来研究表明,Aβ是由40-42个氨基酸组成的肽段,由淀粉样前体蛋白(amyloid precursor protein, APP)经由酶切产生。当前研究发现,AD患者脑中APP的mRNA和蛋白的表达量较正常人高。APP基因转录后通过不同的剪接形式,产生几种不同的亚型,分别含有695到770个氨基酸,其中APP695主要存在于神经元细胞中。研究发现,某些家族性AD可能与APP基因发生错义突变有关,其突变后,更易使APP裂解产生Aβ。其中研究最多的是瑞典突变,它是发生于670和671位的串联双突变,发生错义突变后正好在Aβ的N末端前增加了一个酶切识别位点,结果导致Aβ产生增加。基于此,本试验拟通过HEK293细胞系建立高表达APP695sw(Swedish mutant APP695,APP695sw)和APP695wt(Wild-type APP695, APP695wt)的细胞模型,希望可以为进一步研究AD的发生、发展以及为探讨APP及Aβ表达的分子机制提供一个好的模型。方法:分别将质粒APP695sw/peak12,APP695wt/peak12与peak12转化大肠杆菌NovaBlue,以质粒提取试剂盒提取转化质粒。脂质体介导质粒APP695sw/peak12、APP695wt/peak12、peak12转染人胚肾细胞(human embryonic kidney epithelial cell, HEK293),转染48小时后,加入嘌呤霉素进行稳定筛选,获得稳定的具有嘌呤霉素抗性的HEK293细胞系。通过免疫细胞化学检测与鉴定转染后APP695sw,APP695wt的表达情况。结果:提取质粒后检测其A260/A280比值均小于2.0,大于1.8,浓度和纯度均能满足转染要求。HEK293细胞在含10%胎牛血清的DMEM(高糖)培养基中生长良好,经过多次传代换液,细胞数量和质量能充分满足试验要求。通过嘌呤霉素敏感试验确定了抑制HEK293细胞生长的最低浓度。用嘌呤霉素稳定筛选16天后,出现细胞克隆株。通过免疫细胞化学显示,稳定筛选后APP695sw的表达高于转染空载体组,而APP695wt的表达与转染空载体组没有差异。结论:通过脂质体成功将真核表达质粒APP695sw/peak12转染入HEK293细胞;通过免疫细胞化学试验证实转染后HEK293细胞中APP695sw基因的表达量增高。更多还原

【Abstract】 Objectives: Alzheimer’s disease (AD) is a widespread, neurode- generative disorder of the eldly. This disease is characterized pathologically by senile plaques in the cerebral cortex and the hippocampus containing the amyloidβ-pepide(Aβ), albeit largely outside the brain parenchyma. The accumulation and deposition of Aβin the brain over decades leads to neuron dysfunction and eventually clinical manifestation of the disease. Recent research demonstrates that Aβis 40 to 42 amino acids in length and is generated by proteolytic cleavage of the much larger amyloid precursor protein(APP).Current research demonstrates that there is an increasing expression of APP in the diseased-damaged brain. The transcripts of APP ranging in predicted size from 695 to 770 aa. APP695 is preferentially expressed in neuronal tissue, leading to the speculation that production of Aβ. The familial form of Alzheimer’s disease may be caused by APP mutations, which increase Aβproduction or lead to an increased proportion of Aβending at residue 42. A pathogenic mutation at codons 670/671 in APP(APP“Swedish”) leads to enhanced cleavage at theβ-secretase scissile bond and increased Aβformation. Mutations in the APP gene account for only a minority of familial AD cases. In this study, the eukaryotic expression of the Human APP695sw and APP695wt was established to provide a model for researching Alzheimer’s disease.Methods: The plasmid containing APP695sw/peak12 , APP695wt /peak12 and peak12 were introduced into NovaBlue E coli. Then, the plasmid was extracted through minibest plasmid purification kit after transformation. Cultured human embryonic kidney epithelial cells (HEK293) were transfected by LIPOFECTAMINE 2000. The HEK293 cells which stably expressed the APP of human would be survived in the further culture medium containing puromycin antibiotic. Last, the transfection result was demonstrated by immunocytochemistry.Result: The result showed that the specific value of plasmid was larger than 1.8 and less than 2.0. The density and purity can satisfied test requirement. HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and antibiotics at 37℃and 5% CO2. The cell population and quality can satisfied test requirement after high passage. The sensitivity test of puromycin defined the least concentration that resists cell growth. A cell clone appeared 16 days after screening. The level of APPsw increased in APPsw over-expressing cells with the methods of immunohistochemistry staining, but the level of APPwt had no remarkable difference.Conclusions: The plasmid APP695sw/peak12 had been introduced into HEK293 cells. Immunohistochemistry staining indicated that the level of APP695sw was increased in APP over-expressing cells.更多还原