【作者】 付书贞；

【导师】 岳保红；

【作者基本信息】 郑州大学 ， 临床检验诊断学， 2012， 硕士

【摘要】 目的：Nucleostemin是2002年发现并被命名的一种基因,其表达产物NS蛋白参与维持干细胞和肿瘤细胞的增殖状态,广泛表达于干细胞和肿瘤细胞的胞核中,可以通过与P53蛋白相结合影响细胞的生物学效应。人为下调NS的表达可以改变细胞的生物学性状。先前的研究证实NS在多种白血病细胞系以及急性白血病中都存在高表达现象,也通过一系列分析推测NS除了与细胞内的P53相结合外,还有可能存在另外一条信号转导通路发挥作用。有关资料显示NS与人蛋白磷酸酶2A(PP2A)上的调节亚基的α异构体PPP2R5A之间有着相互作用。PP2A是哺乳动物中含量最为丰富的丝/苏氨酸特异性蛋白磷酸酶,约占细胞总蛋白的0.3%,被认为在许多生命活动中扮演了重要的角色,尤其是细胞的分化、增殖和凋亡,通过对蛋白激酶的反馈调节,在许多信号通路中起着重要的作用,在细胞膜、质和核中都行使着重要的功能,同时也参与了肿瘤的发生与发展。先前的课题研究证实PPP2R5A在急性白血病细胞中也存在表达并通过免疫组化和免疫共沉淀技术证实两者之间有着相互结合,此现象提示了PPP2R5A有可能是NS除P53外的另一条信号转导通路。这也解释了对于大部分急性白血病细胞都是p53缺失或者突变,不能产生有正常功能的P53蛋白的同时,NS是通过何种途径发挥其维持细胞增殖状态作用的。导师课题的总目标是探讨是否存在非P53依赖性的NS信号传导途径,如果存在,此通路是否与PPP2R5A介导的信号通路有关?本课题为其中一个小子课题,旨在验证PPP2R5A是否和细胞的增殖、分化、凋亡相关,人为抑制PPP2R5A的表达,细胞的生物学状态是否有所改变,为寻找非P53依赖性信号转导通路奠定基础。方法：(1)利用脂质体转染技术将针对PPP2R5A的siRNA转染到白血病细胞HL-60、THP-1和Raji细胞内,沉默PPP2R5A基因。(2) RT-PCR法和免疫细胞化学法检测siRNA的转染效果并找出最佳转染浓度和时间。(3)倒置显微镜观察转染组和对照组细胞的生长状态并绘制细胞生长曲线。(4)瑞-吉染色观察转染组和对照组细胞的胞核、染色质及形态改变。(5)流式细胞术Annexin V/PI双染法进行细胞凋亡分析,PI DNA染色法进行细胞周期测定。(6)数据结果用SPSS17.0统计软件处理,对定量资料的统计学均数用均数士标准差来表示,对于定性资料用卡方检验,多样本均数比较采用方差分析,如果方差不齐则进行变量变换。显著性检验水准为a=0.05。结果：(1) RT-PCR法和免疫细胞化学染色显示1×105/ml的细胞浓度,siRNA-PPP2R5A终浓度为65nM时转染后细胞PPP2R5A表达量显著降低。(2)倒置显微镜观察显示转染48h后细胞出现大小不均,聚团性变差,细胞散在或不聚团,细胞形状不一,部分细胞胞体不完整,细胞碎片增多。(3)瑞-吉染色显示转染后细胞形态变的不规则,胞体大小不一,胞体不完整,碎片增多,核趋于聚集,并有凋亡小体形成。(4)细胞凋亡分析和细胞周期测定显示转染组凋亡细胞百分比率增高,G1期和G2/M期细胞百分比上升,而S期细胞百分比减低,表明细胞增殖减弱。结论(1)本实验成功转染急性白血病细胞,显著干预了PPP2R5A基因的表达,细胞呈现出一系列趋于凋亡和细胞周期改变的迹象。(2)验证了PPP2R5A和细胞的凋亡分化有一定关系,为验证NS非P53途径的信号通路是否与PPP2R5A有关奠定了基础。更多还原

【Abstract】 Objective:NS is a protein that discovered and named in2002, which is very important and essential to the stem cell and tumor cell proliferation condition. This protein widely presents in the stem cells and tumor cells in the nucleus, and can play the biological effects of cells by combining with a protein named P53. The pathological properties can be changed by cutting the expression of intracellular NS artificially. The study before confirmed that NS can highly express in many leukemia cell lines and acute leukemia cells. Through the analysis of a series of speculation, we confirmed that NS can play the biological effects of cells by another signal transduction pathway independently on P53. According to some data, NS can combine with PPP2R5A, which is α isomers of regulatory subunit of the PP2A. PP2A is the most abundant content serine/threonine specific protein phosphatase in mammals, accounts for about0.3%of total protein cells. PP2A is considered being important to many life activities, especially to cell differentiation, proliferation and apoptosis, and participate in the development of tumors simultaneously. It can play an important role to cell membrane, cytoplasm and nucleus in many signal transduction pathways passed feedback adjustment to protein kinase. Our earlier study confirmed that PPP2R5A can express in acute leukemia cells and can combine with the NS protein by immunocytochemistry and coimmunoprecipitation. This reveals that the PPP2R5A can play the important role by another signal transduction pathway independently on P53. This appearance also can explain why the NS protein can maintain cell proliferation condition in many acute leukemia cells, which are deficient or mutational with p53and can’t produce effective P53protein. The tutor’s study is to explore whether it has another signal transduction pathway of NS independent with P53, and whether the pathway relates to PPP2R5A. This topic is a small unit of this study, to test and verify the relevance between the PPP2R5A and cell differentiation, proliferation and apoptosis. Whether the cell biology state can be changed by regulating down the expression of PPP2R5A, laying a foundation of finding the signal transduction pathway independently on P53.Method:(1) Transfected siRN A-PPP2R5A of to the leukemia cells HL-60. THP-1and Raji by liposome transfect technology to silent the PPP2R5A(2) Test the transfect effect by RT-PCR and immunocytochemistry to find the best concentration of siRNA-PPP2R5A and transfect time.(3) Observe the cells growth by inverted microscope.(4) Observe the cells form by Wright-Giemsa dye.(5) Test the change of cell apoptosis and cell cycle by FCM. (6) SPSS17.0statistical software was applied to analyse the data statistics. And (x±s) and one-way AN OVA were used to compare the quntitative data, Chi-Square Test was used for qualitative data. a=0.05was considered as the significance level.Result:(1) The result of RT-PCR and immunocytochemistry show that when the cell concentration is1×105/ml and the transfect time is48h, the expression of PPP2R5A changes is the most obvious.(2) Under the inverted microscope, the cells become vary size, scattered and can’t bunch up. Some cells are not full and the cell debris become more than control group.(3) Wright-Giemsa dye show that the cells become broken, and have much cell debris. Many cells’nuclear become gather and form to apoptotic body.(4) When we analysis the result of cell’s apoptosis and cell cycle, we find the apoptosis cells in treatment group are more than control groups, and the cells at G1phase and G2/M phase in treatment group are more than control groups, while the cells at S phase are less than control groups, which indicates that the proliferation become weak.Conclusion:(1) This study successfully transfect the siRNA into the leukemia cells and silent the expression, and the cells present a series of biologische merkmale on apoptosis and cell cycles. (2) We test and verify the relation between PPP2R5A and cell apoptosis and differentiation, laying a foundation of finding the signal transduction pathway independently on P53.更多还原