【作者】 刘荣芳；

【导师】 俞昌喜；

【作者基本信息】 福建医科大学 ， 药理学， 2007， 硕士

【摘要】 目的:以细胞形态改变和tau蛋白过度磷酸化为反映AD(Alzheimer’s Disease)发生发展的关键指标,初步探明实验性AD诱导产生过程中的PTEN变化状况及其与tau蛋白过度磷酸化的关系,以深化理解AD的发病机制。方法:①采用冈田酸(Okadaic acid,OA)诱导SH-SY5Y细胞和NG108-15细胞,以细胞形态改变和tau蛋白过度磷酸化为观察指标,建立用于AD研究的细胞模型。②倒置显微镜观察细胞形态的变化。③Western blot法测定SH-SY5Y细胞和NG108-15细胞Ser396位点磷酸化tau蛋白水平和PTEN蛋白水平。④RT-PCR方法观测SH-SY5Y细胞PTEN mRNA水平。结果:1.冈田酸诱导SH-SY5Y细胞AD样发生发展中PTEN的变化:①OA孵育SH-SY5Y细胞可显著改变细胞的形态,随OA孵育时间的延长,6 h后细胞开始变圆,突触缩回,18 h后细胞大部分变圆,突触消失,48 h后细胞漂浮生长,部分细胞呈死亡状态;40 nmol/L OA作用于细胞,Ser396位点磷酸化tau蛋白水平在OA孵育后12 h显著升高, 18 h、24 h达高峰,之后下降,48 h显著降低。提示OA可诱导SH-SY5Y细胞进行性损伤变性和tau蛋白过度磷酸化。②40 nmol/L OA孵育SH-SY5Y细胞,同时观测细胞中Ser396位点磷酸化tau蛋白水平和PTEN蛋白水平随OA作用时间的变化,可见Ser396位点磷酸化tau蛋白水平变化同上,而PTEN蛋白水平则于3 h、6 h显著升高,6 h达高峰,之后下降,48 h显著降低。提示在OA诱导SH-SY5Y细胞AD样发生发展过程中,PTEN蛋白水平呈先升高后降低的变化。③40nmol/L OA作用于SH-SY5Y细胞,PTEN mRNA水平于12 h、18 h、24 h显著升高,其它时间点未见明显变化,提示在OA诱导SH-SY5Y细胞AD样发生发展过程中,PTEN mRNA水平存在着变化。2.冈田酸诱导NG108-15细胞AD样发生发展中PTEN的变化:①OA孵育NG108-15细胞,随OA孵育时间的延长,6 h后细胞未见明显变化,18 h后细胞变圆,开始贴壁不牢,48 h后细胞呈悬浮生长,但胞壁完整;20 nmol/L OA作用于细胞,Ser396位点磷酸化tau蛋白水平在OA孵育后6 h开始显著升高,18 h达高峰,48 h仍然显著升高。提示随OA孵育时间的延长,NG108-15细胞可发生进行性损伤变性,但由于孵育时间不够长,细胞并没有开始坏死;OA可诱导NG108-15细胞tau蛋白过度磷酸化。②20 nmol/L OA孵育NG108-15细胞,同时观测细胞中Ser396位点磷酸化tau蛋白水平和PTEN蛋白水平。随OA作用时间的变化,可见Ser396位点磷酸化tau蛋白水平变化同上, PTEN蛋白水平则于12 h、18 h、24 h显著升高,18 h达高峰,其余时间点未见显著变化,提示在OA诱导NG108-15细胞AD样发生过程中,PTEN蛋白水平升高。结论1. OA可诱导SH-SY5Y细胞、NG108-15细胞进行性损伤变性和tau蛋白过度磷酸化,这些细胞模型可用于AD研究。2.在OA诱导SH-SY5Y细胞以及NG108-15细胞AD样发生过程中,PTEN蛋白水平升高,提示PTEN在实验性AD诱导产生中可能存在促进作用。更多还原

【Abstract】 Objective: With morphology of cells and tau protein hyperphosphorylation as indexes of AD(Alzheimer’s Disease) development, the present study was designed to explore initially the expression of PTEN in experimental AD and its relationship with tau protein hyperphosphorylation in order to comprehend the pathogeneses of AD.Methods:①. SH-SY5Y cells and NG108-15 cells were treated with OA (Okadaic acid),taking cell morphology and tau protein hyperphosphorylation as indexes, to establish two types of cell model for research on AD;②Inverted microscope was used to observe the morphology of cells;③Western Blotting was used to measure the levels of phosphorylated tau protein at site of Ser396 and PTEN;④RT-PCR was used to measure the levels of PTEN mRNA in SH-SY5Y cells.Results:1. Expression of PTEN in SH-SY5Y cells in which Tau protein was induced hyperphosphorylation by okadaic acid①SH-SY5Y cells changed gradually after the treatment with 40 nmol/L OA and appeared typical morphology of AD; cells were treated with 40 nmol/L OA for various periods. The levels of phosphorylated tau at site Ser396 was increased after the exposure for 12 h and reached the maximum level after 18 h, then declined to the level below normal after 48 h, indicating that OA can induce decrease of cell viabilities and tau protein hyperphosphorylation in SH-SY5Y cells.②With treatment of SH-SY5Y cells with 40 nmol/L OA for various periods, the level of PTEN and phosphorylated tau protein were detected simultaneously. The levels of phosphorylated tau protein were the same as above; the levels of PTEN was increased after the exposure for 3 h and reached the maximum level after 6 h then decreased to the level below normal after 48 h, indicating that in the proceeding of tau protein hyperphosphorylation induced by OA in SH-SH5Y cells, the expression of PTEN was altered.③With treatment of SH-SY5Y cells with 40 nmol/L OA for various periods, the levels of PTEN mRNA was increased after the exposure for 12 h and reached the maximum level after 18 h. 2. Expression of PTEN in NG108-15 cells in which Tau protein was induced hyperphosphorylation by okadaic acid①NG108-15 cells changed gradually after the treatment with 20 nmol/L OA and appeared typical morphology of AD; cells were treated with 20 nmol/L OA for various periods. The levels of phosphorylated tau at site Ser396 was increased after exposure of 6 h and reached the maximum level after 18 h, indicating that OA can induce decrease of cell viabilities and tau protein hyperphosphorylation in NG108-15 cells.②With treatment of NG108-15 cells with 20 nmol/L OA for various periods, the level of PTEN and phosphorylated tau protein were detected at the same time. The levels of phosphorylated tau protein were the same as above; the levels of PTEN was increased after exposure of 12 h and reached the maximum level after 18 h, indicating that in the proceeding of tau protein hyperphosphorylation induced by OA in NG108-15 cells, the expression of PTEN was increased.Conclusion:1. OA can induce tau protein hyperphosphorylation in SH-SY5Y cells and NG108-15 cells, and these cell models can be used on AD research. 2. In the proceeding of tau protein hyperphosphorylation induced by OA, the expression of PTEN was altered; indicating PTEN may contribute to the pathogenesis of AD.更多还原