抑制p53缺失和突变型白血病细胞PPP2R5A表达后细胞生物学变化
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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。
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.
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。
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.
引文
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14.刘冉录,徐勇,张志宏,王萌,孙建涛,张玥,李胜芝.靶向沉默核干因子对前列腺癌PC23细胞增殖能力的影响.NJA,2009,15(7)：593-598
15.李娟,黎友伦,程序,Nucleostemin基因在非小细胞肺癌组织中的表达及意义[J],中国组织化学与细胞化学杂志,2010,19(3)：229-233.
16. Yue BH, Lu J, Wang YY, Yu L; Wang QX, Liu SA, Zhang GY, Zhang QX etal. Effects of Nucleostemin gene silencing on morphology and cytochemistry of HL-60 cells. Life sci[J],2008,5(2):9-14
17. Zhang G, Zhang Q, Zhang Q, Yin L, Li S, Cheng K, Zhang Y, Xu H, Wu W.Expression of nucleostemin, epidermal growth factor and epidermal growth factor receptor in human esophageal squamous cell carcinoma tissues. [J] Cancer Res Clin Oncol.2010 Apr;136(4):587-594.
18. Nikpour P, Mowla SJ, Jafarnejad SM, Fischer U, Schulz WA. Differential effects of Nucleostemin suppression on cell cycle arrest and apoptosis in the bladder cancer cell lines 5637 and SW1710. Prolif,2009,42(6):762-769
19.张功员,尹磊,李晟磊等。食管鳞状细胞癌组织中核干细胞因子蛋白和mRNA的半定量表达[J].中华肿瘤杂志,2008,30(2)：125-128.
20.蔡子微,郑学芝,胡静.乳腺肿瘤组织nucleostem in基因表达与临床病理表现的关系[J].中国病理生理杂志,2007,23(11)：2191-2194.
21.童秀珍,邹外一,卜珊珊等.急性白血病患者骨髓细胞Nucleostemin基因的 表达与其类型、疗效的关系[J].中国病理生理杂志,2008,24(11)：2171-2174.
22. BaddooM, Hill K, Wilkinson R, et al. Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection[J]. J Cell Biochem,2003, 89(6):1235
23. Bernatchez R, Belkacemi L, Rassart E, et al. Differential expression of membrane and soluble adenylyl cyclase isoforms in cytotrophoblast cells and syncytiotrophoblasts of human placenta [J]. Placenta,2003,24(6):648-657
24. Chambers I, Colby D, Robertson M, et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells [J]. Cell,2003,113(5): 643
25. Tsai RY, McKay RD.Amultistep. GTP-driven mechanism controlling the dynamic cycling of nucleostemin. J Cell Biol.2005 Jan 17; 168(2):179-84
26. Kudron MM, Reinke VC. Elegans Nucleostemin Is Required for Larval Growth and Germline Stem Cell Division. PLoS Genet,2008,4(8):1-12.
27. Yang HX, Jin GL, Meng L et al. Screening and identification of proteins interacting with nucleostemin[J]. World J Gastroenterol,2005,11 (31):4812
28. Huang M, Whang P, Chodaparambil JV, Pollyea DA, Kusler B, Xu L, Felsher DW, Mitchell BS.Reactive oxygen species regulate nucleostemin oligomerization and protein degradation. J Biol Chem.2011 Apr 1;286(13):11035-46.
29. Nakajima TE, Yoshida H, Okamoto N, Nagashima K, Taniguchi H, Yamada Y, Shimoda T, Masutomi K.Nucleostemin and TWIST as predictive markers for recurrence after neoadjuvant chemotherapy for esophageal carcinoma. Cancer Sci. 2012 Feb;103(2):233-238.
30. Arumugam SB, Trentz OA, Arikketh D, Senthinathan V, Rosario B, Mohandas PV.Detection of embryonic stem cell markers in adult human adipose tissue-derived stem cells. Indian J Pathol Microbiol.2011 Jul-Sep;54(3):501-508.
31. Zhang J, Pan T, Im HJ, Fu FH, Wang JH. Differential properties of human ACL and MCL stem cells may be responsible for their differential healing capacity. BMC Med.2011 Jun 2;9:68.
32. Oktar PA, Yildirim S, Balci D, Can A. Continual expression throughout the cell cycle and downregulation upon adipogenic differentiation makes nucleostemin a vital human MSC proliferation marker. Stem Cell Rev.2011 Jun;7(2):413-24.
33. Bernardi R, Pandolfi PP. The nucleolus:at the stem of immortality. Nat Med,2003, 9(1):24-25.
34. Ma H, Pederson T.Depletion of the nucleolar protein nucleostemin causes G1 cell cycle arrest via the p53 pathway. Mol Biol Cell.2007 Jul;18(7):2630-2635.
35. Lingjun Meng, Tao Lin and Robert Y. L. Tsai. Nucleoplasmic mobilization of nucleostemin stabilizes MDM2 and promotes G2-M progression and cell survival. J Cell Sci,2008,121 (24):4037-4046.
36. Meng L, Hsu JK, Tsai RY. GNL3L depletion destabilizes MDM2 and induces p53-dependent G2/M arrest.Oncogene,2011 Apr 7,30(14):1716-1726.
37. Ma H, Pederson T. Nucleophosmin is a binding partner of nucleostemin in human osteosarcoma cells. Mol Biol Cell.2008 Jul;19(7):2870-5.
38. Avitabile D, Bailey B, Cottage CT, etal. Nucleolar stress is an early response to myocardial damage involving nucleolar proteins nucleostemin and nucleophosmin. Proc Natl Acad Sci U S A.2011 Apr 12;108(15):6145-50.
39. Romanova L, Grand A, Zhang L, etal. Critical role of nucleostemin in pre-rRNA processing [J]. J B iol Ch em,2009,284(8):4968-4977.
40. Paridaen JT, Janson E, Utami KH, Pereboom TC, Essers PB, van Rooijen C, Zivkovic D, MacInnes AW.The nucleolar GTP-binding proteins Gnl2 and nucleostemin are required for retinal neurogenesis in developing zebrafish. Dev Biol.2011 Jul 15;355(2):286-301.
41. Okamoto N, Yasukawa M, Nguyen C, Kasim V, Maida Y, Possemato R, Shibata T, Ligon KL, Fukami K, Hahn WC, Masutomi K. Maintenance of tumor initiating cells of defined genetic composition by nucleostemin. Proc Natl Acad Sci USA. 2011 Dec 20;108(51):20388-93.
42. C Deng, P Zhang, JW Harper, SJ Elledge, P Leder. Mice lacking p21 (CIP1/WAF1) undergo normal development, but are defective in G1 checkpoint control. Cell, 1995,82:675-684
43. Meletis K, Wirta V, Hede SM, Nister M, Lundeberg J, Frisenl J. p53 suppresses the self-renewal of adult neural stem cells. Development,2006,133(2):363-369
44. Nakano Y, Naoe T, Kiyoi H, Kitamura K, Minami S, Miyawaki S, Asou N, Kuriyama K, Kusumoto S, Shimazaki C, Akiyama H, Saito K, Nishimura M, Motoji T, Shinagawa K, Saito H, Ohno R. Prognostic value of p53 gene mutations and the product expression in de novo acute myeloid leukemia. Eur J Haematol, 2000,65:23-31
45. Melo B, Ahmad N, Lima S. Mutations in p53 gene in acute myeloid leukemia patients correlate with poor prognosis. Hematology,2002,7(1):13-19
46. Jafarnejad M, Mowla J, Matin M. Knocking down the expression of nucleostemin significantly decreases rate of prolife. ration of rat bone marrow stromal stem cells in an apparently p53-independent manner[J]. Cell Prol,2008,41(1):28-35
47. Konikova E, Kusenda J, Babusikova O. Flow cytometry of p53 protein expression in some hematological malignancies. Neoplasma,1999,46(6):368-376
48.杨琰,李小青,黄庆,黄士昂.PP2A激活剂对HL-60细胞增殖的影响及急性髓系白血病患者体内PP2A活性变化的研究.中国实验血液学杂志2011,19(3)：594-597
49. Kinney, BP; Qiao, LP; LeVaugh, JM; Shao, JH. B56 alpha/Protein Phosphatase 2A Inhibits Adipose Lipolysis in High-Fat Diet-Induced Obese Mice. Endocrinology 2010(151):3624-3632,
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1.刘冉录,Nucleostemin研究进展,天津医科大学学报,2007,13(1)：132-138.
2. Tsai RY, McKay RD. A nucleolar mechanism controlling cell proliferation in stem cells and cancer cells [J]. Genes Dev,2002,16(23):2991-3003
3. Lin S, Yu HS. Clinical significance of nucleostemin expression and its correlation with cyclin D1 expression in malignant ovarian tumors. Int J Gynecol Cancer. 2011 Oct;21(7):1166-1171.
4. Paridaen JT, Janson E, Utami KH, Pereboom TC, Essers PB, van Rooijen C, Zivkovic D, Maclnnes AW, The nucleolar GTP-binding proteins Gnl2 and nucleostemin are required for retinal neurogenesis in developing zebrafish. Dev Biol.2011 Jμl 15;355(2):286-301.
5.岳保红,朱晓燕,王清霞,刘帅,张功员,张秋堂,张慧,张钦宪.核干细胞因子基因沉默对HL-60细胞分化特征的影响.郑州大学学报,2007,42(4)：657-659.
6. Gil-Ranedo J, Mendiburu-Elicabe M, Garcia-Villanueva M, Medina D, del Alamo M, Izquierdo M. An off-target nucleostemin RNAi inhibits growth in human glioblastoma-derived cancer stem cells. PLoS One.2011;6(12):e28753.
7. Avitabile D, Bailey B, Cottage CT, Sundararaman B etal. Nucleolar stress is an early response to myocardial damage involving nucleolar proteins nucleostemin and nucleophosmin. Proc Natl Acad Sci USA.2011 Apr 12;108(15):6145-6150
8. Yoshida R, Fujimoto T, Kudoh S, Nagata M, Nakayama H, Shinohara M, Ito T. Nucleostemin affects the proliferation but not differentiation of oral squamous cell carcinoma cells. Cancer Sci.2011Jul;102(7):1418-23.
9. Meng L, Yasumoto H, Tsai RY. Multiple controls regulate nucleostemin partitioning between nucleolus and nucleoplasm Cell Science,2006,119: 5124-5136.
10. Matsuo E, Nagamine T, Matsumoto S, Tsuneizumi K. Drosophila GTPase nucleostemin 2 changes cellular distribution during larval development and the GTP-binding motif is essential to nucleoplasmic localization. Biosci Biotechnol Biochem.2011,75(8):1511-1515.
11. Bardutzky J, Shen Q, Henninger N, etal. Differences in ischemiclesion evolution in different rat strains using diffusion and perfusion imaging [J]. Stroke,2005, 36(9):2000
12. Duncan PW, Jorgensen HS, Wade DT. Outcome measures in acute stroke trials:a systematic review and some recommendations to improve practice [J]. Stroke, 2000,31(6):1429
13.郭俣,夏俊,杨清玲,陈昌杰,刘长青。核干细胞因子在肿瘤细胞中的差异表达及意义。蚌埠医学院学报2011,36(7)：669-672
14.刘冉录,徐勇,张志宏,王萌,孙建涛,张玥,李胜芝.靶向沉默核干因子对前列腺癌PC23细胞增殖能力的影响.NJA,2009,15(7)：593-598
15.李娟,黎友伦,程序,Nucleostemin基因在非小细胞肺癌组织中的表达及意义[J],中国组织化学与细胞化学杂志,2010,19(3)：229-233.
16. Yue BH, Lu J, Wang YY, Yu L; Wang QX, Liu SA, Zhang GY, Zhang QX etal. Effects of Nucleostemin gene silencing on morphology and cytochemistry of HL-60 cells. Life sci[J],2008,5(2):9-14
17. Zhang G, Zhang Q, Zhang Q, Yin L, Li S, Cheng K, Zhang Y, Xu H, Wu W.Expression of nucleostemin, epidermal growth factor and epidermal growth factor receptor in human esophageal squamous cell carcinoma tissues. [J] Cancer Res Clin Oncol.2010 Apr;136(4):587-594.
18. Nikpour P, Mowla SJ, Jafarnejad SM, Fischer U, Schulz WA. Differential effects of Nucleostemin suppression on cell cycle arrest and apoptosis in the bladder cancer cell lines 5637 and SW1710. Prolif,2009,42(6):762-769
19.张功员,尹磊,李晟磊等。食管鳞状细胞癌组织中核干细胞因子蛋白和mRNA的半定量表达[J].中华肿瘤杂志,2008,30(2)：125-128.
20.蔡子微,郑学芝,胡静.乳腺肿瘤组织nucleostem in基因表达与临床病理表现的关系[J].中国病理生理杂志,2007,23(11)：2191-2194.
21.童秀珍,邹外一,卜珊珊等.急性白血病患者骨髓细胞Nucleostemin基因的 表达与其类型、疗效的关系[J].中国病理生理杂志,2008,24(11)：2171-2174.
22. BaddooM, Hill K, Wilkinson R, et al. Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection[J]. J Cell Biochem,2003, 89(6):1235
23. Bernatchez R, Belkacemi L, Rassart E, et al. Differential expression of membrane and soluble adenylyl cyclase isoforms in cytotrophoblast cells and syncytiotrophoblasts of human placenta [J]. Placenta,2003,24(6):648-657
24. Chambers I, Colby D, Robertson M, et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells [J]. Cell,2003,113(5): 643
25. Tsai RY, McKay RD.Amultistep. GTP-driven mechanism controlling the dynamic cycling of nucleostemin. J Cell Biol.2005 Jan 17; 168(2):179-84
26. Kudron MM, Reinke VC. Elegans Nucleostemin Is Required for Larval Growth and Germline Stem Cell Division. PLoS Genet,2008,4(8):1-12.
27. Yang HX, Jin GL, Meng L et al. Screening and identification of proteins interacting with nucleostemin[J]. World J Gastroenterol,2005,11 (31):4812
28. Huang M, Whang P, Chodaparambil JV, Pollyea DA, Kusler B, Xu L, Felsher DW, Mitchell BS.Reactive oxygen species regulate nucleostemin oligomerization and protein degradation. J Biol Chem.2011 Apr 1;286(13):11035-46.
29. Nakajima TE, Yoshida H, Okamoto N, Nagashima K, Taniguchi H, Yamada Y, Shimoda T, Masutomi K.Nucleostemin and TWIST as predictive markers for recurrence after neoadjuvant chemotherapy for esophageal carcinoma. Cancer Sci. 2012 Feb;103(2):233-238.
30. Arumugam SB, Trentz OA, Arikketh D, Senthinathan V, Rosario B, Mohandas PV.Detection of embryonic stem cell markers in adult human adipose tissue-derived stem cells. Indian J Pathol Microbiol.2011 Jul-Sep;54(3):501-508.
31. Zhang J, Pan T, Im HJ, Fu FH, Wang JH. Differential properties of human ACL and MCL stem cells may be responsible for their differential healing capacity. BMC Med.2011 Jun 2;9:68.
32. Oktar PA, Yildirim S, Balci D, Can A. Continual expression throughout the cell cycle and downregulation upon adipogenic differentiation makes nucleostemin a vital human MSC proliferation marker. Stem Cell Rev.2011 Jun;7(2):413-24.
33. Bernardi R, Pandolfi PP. The nucleolus:at the stem of immortality. Nat Med,2003, 9(1):24-25.
34. Ma H, Pederson T.Depletion of the nucleolar protein nucleostemin causes G1 cell cycle arrest via the p53 pathway. Mol Biol Cell.2007 Jul;18(7):2630-2635.
35. Lingjun Meng, Tao Lin and Robert Y. L. Tsai. Nucleoplasmic mobilization of nucleostemin stabilizes MDM2 and promotes G2-M progression and cell survival. J Cell Sci,2008,121 (24):4037-4046.
36. Meng L, Hsu JK, Tsai RY. GNL3L depletion destabilizes MDM2 and induces p53-dependent G2/M arrest.Oncogene,2011 Apr 7,30(14):1716-1726.
37. Ma H, Pederson T. Nucleophosmin is a binding partner of nucleostemin in human osteosarcoma cells. Mol Biol Cell.2008 Jul;19(7):2870-5.
38. Avitabile D, Bailey B, Cottage CT, etal. Nucleolar stress is an early response to myocardial damage involving nucleolar proteins nucleostemin and nucleophosmin. Proc Natl Acad Sci U S A.2011 Apr 12;108(15):6145-50.
39. Romanova L, Grand A, Zhang L, etal. Critical role of nucleostemin in pre-rRNA processing [J]. J B iol Ch em,2009,284(8):4968-4977.
40. Paridaen JT, Janson E, Utami KH, Pereboom TC, Essers PB, van Rooijen C, Zivkovic D, MacInnes AW.The nucleolar GTP-binding proteins Gnl2 and nucleostemin are required for retinal neurogenesis in developing zebrafish. Dev Biol.2011 Jul 15;355(2):286-301.
41. Okamoto N, Yasukawa M, Nguyen C, Kasim V, Maida Y, Possemato R, Shibata T, Ligon KL, Fukami K, Hahn WC, Masutomi K. Maintenance of tumor initiating cells of defined genetic composition by nucleostemin. Proc Natl Acad Sci USA. 2011 Dec 20;108(51):20388-93.
42. C Deng, P Zhang, JW Harper, SJ Elledge, P Leder. Mice lacking p21 (CIP1/WAF1) undergo normal development, but are defective in G1 checkpoint control. Cell, 1995,82:675-684
43. Meletis K, Wirta V, Hede SM, Nister M, Lundeberg J, Frisenl J. p53 suppresses the self-renewal of adult neural stem cells. Development,2006,133(2):363-369
44. Nakano Y, Naoe T, Kiyoi H, Kitamura K, Minami S, Miyawaki S, Asou N, Kuriyama K, Kusumoto S, Shimazaki C, Akiyama H, Saito K, Nishimura M, Motoji T, Shinagawa K, Saito H, Ohno R. Prognostic value of p53 gene mutations and the product expression in de novo acute myeloid leukemia. Eur J Haematol, 2000,65:23-31
45. Melo B, Ahmad N, Lima S. Mutations in p53 gene in acute myeloid leukemia patients correlate with poor prognosis. Hematology,2002,7(1):13-19
46. Jafarnejad M, Mowla J, Matin M. Knocking down the expression of nucleostemin significantly decreases rate of prolife. ration of rat bone marrow stromal stem cells in an apparently p53-independent manner[J]. Cell Prol,2008,41(1):28-35
47. Konikova E, Kusenda J, Babusikova O. Flow cytometry of p53 protein expression in some hematological malignancies. Neoplasma,1999,46(6):368-376
48.杨琰,李小青,黄庆,黄士昂.PP2A激活剂对HL-60细胞增殖的影响及急性髓系白血病患者体内PP2A活性变化的研究.中国实验血液学杂志2011,19(3)：594-597
49. Kinney, BP; Qiao, LP; LeVaugh, JM; Shao, JH. B56 alpha/Protein Phosphatase 2A Inhibits Adipose Lipolysis in High-Fat Diet-Induced Obese Mice. Endocrinology 2010(151):3624-3632,