中心体复制调控相关蛋白亚细胞结构定位的研究
摘要
采用现代胚胎生物工程技术是使畜牧业向高产、高效、优质、集约化发展的有效途径。但是,体外受精胚胎和克隆胚胎发育率低是制约这两项技术走向生产实践的最大障碍。
本研究借助免疫荧光染色的方法,从中心体复制的关键调控基因CDK2和P53入手,研究CDK2和P53在经体外成熟18-26小时的牛成熟卵母细胞中的亚细胞定位,揭示它们在中心体复制过程中位置,分析CDK2和P53在牛成熟卵母细胞中的表达情况,从中心体异常复制的角度阐明体外受精胚胎和克隆胚胎染色体的不稳定性。研究结果如下:
研究哺乳动物胚胎的免疫荧光染色条件和步骤。通过对小鼠受精卵中CDK2、P53和γ-tubulin蛋白的免疫荧光染色研究,对免疫荧光染色的条件和步骤不断优化,修改、简化和发展,摸索出一套完善的针对卵母细胞的免疫荧光染色步骤。在小鼠受精卵1细胞G1期,通过固定、封闭、染色、在荧光显微镜下观察,发现CDK2、P53和γ-tubulin蛋白均出现染色阳性。CDK2和P53在细胞有丝分裂G1期定位于中心体,分布于细胞核区。
分别培养18h,22h,26h后的牛成熟卵母细胞,通过与试验一相同的固定、封闭、染色,在荧光显微镜下观察发现,(1)培养18h后的牛成熟卵母细胞,CDK2、P53和γ-tubulin蛋白均出现染色阴性。(2)培养22h后的牛成熟卵母细胞,大多数卵母细胞CDK2、P53和γ-tubulin蛋白出现染色阴性,仅有少数卵母细胞CDK2、P53和γ-tubulin蛋白出现染色阳性。(3)培养26h后的牛成熟卵母细胞,大多数卵母细胞CDK2、P53和γ-tubulin蛋白出现染色阴性,仅有少数卵母细胞CDK2、P53和γ-tubulin蛋白出现染色阳性。(4)在出现染色阳性的牛成熟卵母细胞中,CDK2和P53蛋白定位于中心体,分布于细胞核区。
CDK2和P53蛋白是与细胞中心体复制密切相关的两个蛋白,其功能和细胞的正常分裂发育有关。在用于牛体外胚胎生产的牛卵母细胞中,CDK2和P53蛋白的正常表达与否,是直接影响牛体外胚胎生产效率的原因之一。
Embryo adoption of modern bio-engineering technology is to enable livestock to the high-yield,high efficiency,high-quality,intensive development of an effective way.However,in vitro fertilization and embryo cloning and embryo development constraints is the low rate of these two techniques to produce the greatest obstacle to practice.
This study by immune staining methods,from the Centre copy of the key regulatory genes start CDK2 and P53,and P53 on CDK2 in the 18-26 hours of in vitro mature cattle mature oocytes in the subcellular localization,and reveal them in the centre Copy of the process of location,of CDK2 and P53 in cattle mature oocytes of expression,from the centre of abnormal copy clarify the perspective of in vitro fertilization and embryo cloning and embryo chromosome instability.The results are as follows:
Study of the mammalian embryo immune staining conditions and steps.Through the fertilized eggs of mice CDK2,P53 andγ-tubulin protein the immune staining on the immune staining the conditions and steps to continuously optimize,modify, simplify and development,worked out a comprehensive view of the immune oocytes Staining steps.1 cells in mouse embryos G1 phase,through a fixed,closed,dyeing,in the fluorescent microscope observation,found that CDK2,P53 andγ-tubulin protein were found positive.CDK2 and P53 in cell mitosis G1 phase of positioning at the centre,located in the nucleus area.
Training were 18 h,22h,26h after the cattle mature oocytes,with the same test a fixed,closed,dyeing,in the fluorescent microscope observed that(1) 18 h after the train cattle mature oocytes,CDK2,P53 andγ-tubulin protein were found negative.(2) 22 h after the train cattle mature oocytes,the majority of oocytes CDK2,P53 andγ-tubulin protein were negative in only a small number of oocytes CDK2,P53 andγ-tubulin protein appears positive staining.(3) 26 h after the train cattle mature oocytes,the majority of oocytes CDK2,P53 andγ-tubulin protein were negative in only a small number of oocytes CDK2,P53 andγ-tubulin protein appears positive staining.(4) were positive in the event of the cattle mature oocytes,CDK2 and P53 protein location at the centre of distribution in the nuclear area.
CDK2 and P53 protein is the central body and cell reproduction of the two closely related proteins,their function and a normal cell development of the split.In vitro embryos for cattle production in cattle oocytes,CDK2 and P53 protein expression of the normal or not,is a direct impact on the efficiency of cattle production in vitro embryonic one of the reasons.
本研究借助免疫荧光染色的方法,从中心体复制的关键调控基因CDK2和P53入手,研究CDK2和P53在经体外成熟18-26小时的牛成熟卵母细胞中的亚细胞定位,揭示它们在中心体复制过程中位置,分析CDK2和P53在牛成熟卵母细胞中的表达情况,从中心体异常复制的角度阐明体外受精胚胎和克隆胚胎染色体的不稳定性。研究结果如下:
研究哺乳动物胚胎的免疫荧光染色条件和步骤。通过对小鼠受精卵中CDK2、P53和γ-tubulin蛋白的免疫荧光染色研究,对免疫荧光染色的条件和步骤不断优化,修改、简化和发展,摸索出一套完善的针对卵母细胞的免疫荧光染色步骤。在小鼠受精卵1细胞G1期,通过固定、封闭、染色、在荧光显微镜下观察,发现CDK2、P53和γ-tubulin蛋白均出现染色阳性。CDK2和P53在细胞有丝分裂G1期定位于中心体,分布于细胞核区。
分别培养18h,22h,26h后的牛成熟卵母细胞,通过与试验一相同的固定、封闭、染色,在荧光显微镜下观察发现,(1)培养18h后的牛成熟卵母细胞,CDK2、P53和γ-tubulin蛋白均出现染色阴性。(2)培养22h后的牛成熟卵母细胞,大多数卵母细胞CDK2、P53和γ-tubulin蛋白出现染色阴性,仅有少数卵母细胞CDK2、P53和γ-tubulin蛋白出现染色阳性。(3)培养26h后的牛成熟卵母细胞,大多数卵母细胞CDK2、P53和γ-tubulin蛋白出现染色阴性,仅有少数卵母细胞CDK2、P53和γ-tubulin蛋白出现染色阳性。(4)在出现染色阳性的牛成熟卵母细胞中,CDK2和P53蛋白定位于中心体,分布于细胞核区。
CDK2和P53蛋白是与细胞中心体复制密切相关的两个蛋白,其功能和细胞的正常分裂发育有关。在用于牛体外胚胎生产的牛卵母细胞中,CDK2和P53蛋白的正常表达与否,是直接影响牛体外胚胎生产效率的原因之一。
Embryo adoption of modern bio-engineering technology is to enable livestock to the high-yield,high efficiency,high-quality,intensive development of an effective way.However,in vitro fertilization and embryo cloning and embryo development constraints is the low rate of these two techniques to produce the greatest obstacle to practice.
This study by immune staining methods,from the Centre copy of the key regulatory genes start CDK2 and P53,and P53 on CDK2 in the 18-26 hours of in vitro mature cattle mature oocytes in the subcellular localization,and reveal them in the centre Copy of the process of location,of CDK2 and P53 in cattle mature oocytes of expression,from the centre of abnormal copy clarify the perspective of in vitro fertilization and embryo cloning and embryo chromosome instability.The results are as follows:
Study of the mammalian embryo immune staining conditions and steps.Through the fertilized eggs of mice CDK2,P53 andγ-tubulin protein the immune staining on the immune staining the conditions and steps to continuously optimize,modify, simplify and development,worked out a comprehensive view of the immune oocytes Staining steps.1 cells in mouse embryos G1 phase,through a fixed,closed,dyeing,in the fluorescent microscope observation,found that CDK2,P53 andγ-tubulin protein were found positive.CDK2 and P53 in cell mitosis G1 phase of positioning at the centre,located in the nucleus area.
Training were 18 h,22h,26h after the cattle mature oocytes,with the same test a fixed,closed,dyeing,in the fluorescent microscope observed that(1) 18 h after the train cattle mature oocytes,CDK2,P53 andγ-tubulin protein were found negative.(2) 22 h after the train cattle mature oocytes,the majority of oocytes CDK2,P53 andγ-tubulin protein were negative in only a small number of oocytes CDK2,P53 andγ-tubulin protein appears positive staining.(3) 26 h after the train cattle mature oocytes,the majority of oocytes CDK2,P53 andγ-tubulin protein were negative in only a small number of oocytes CDK2,P53 andγ-tubulin protein appears positive staining.(4) were positive in the event of the cattle mature oocytes,CDK2 and P53 protein location at the centre of distribution in the nuclear area.
CDK2 and P53 protein is the central body and cell reproduction of the two closely related proteins,their function and a normal cell development of the split.In vitro embryos for cattle production in cattle oocytes,CDK2 and P53 protein expression of the normal or not,is a direct impact on the efficiency of cattle production in vitro embryonic one of the reasons.
引文
[1]程金华,朱化彬,戴蕴平,等.我国体细胞克隆牛的研究进展.中国生物工程杂志[J],2006,26(10):77-82.
[2]侯志富,王维忠,印璞,等.大肠肿瘤分子调控模式初探.中国肿瘤临床[J].1999.26(1):856-857.
[3]和占星,文际坤,赵开典.日本牛胚胎工程技术的现状与趋势.黄牛杂志[J].1999,25(3):72-73.
[4]刘建民.牛卵母细胞体外受精及体外发育的研究.生物技术[J],1993,3(3):18-21.
[5]李荣凤,薛晓先,刘哲,等.牛卵母细胞皮层颗粒荧光染色法质成熟鉴定.畜牧兽医学报[J],2000,31(3):203-210.
[6]马云.牛卵泡卵母细胞体外成熟、体外受精及受精卵体外培养的研究.陕西杨凌,西北农林科技大学硕士学位论文[D].2001.
[7]马平,马云.牛的体外受精技术研究进展.家畜生态学报[J],2006,27(6):251-255
[8]蒙少宁.从试管牛到“克隆牛”.光明日报[Z],1998年2月23日.
[9]杨叔培.世界第一头第二代克隆牛在日诞生.生物技术通报[J],2000,2:41-42.
[10]旭日干,贾士荣.农业生物技术进展与展望[M].台肥,中国科学技术大学出版社,1993.
[11]花田章.山羊的体外受精.家畜繁殖学杂志[J].1985,31(5):21-26.
[12]王建英,兰邹然,杨桂文,等.纺锤体检验点的信号通路及其作用.山东医药[J].2006.(46)16:95-96.
[13]周佳勃 吴韩东,刘丽清,等.精子和卵母细胞质量控制对山羊卵胞质内精子注射(ICSI)受精的影响.实验生物学报[J].2004;37:367-374.
[14]Abeieu A,Magnaghi-Jaulin L,Kahana JA,et al.X-Mps-1,an essential component of the mitotic checkpoint,is necessary for Mad1,Mad2 and CENP-E association with kinetochores,in Xenopus egg extracts.Cell[J].2001.106:83-93.
[15]Bailly E,Doree M,Nurse P.et al.P34cde2 is located in both nucleus and cytoplasm:part is centrosomally associated at G2/M and enters vesicles at anaphase.EMBO J[J],1989,8(13):3985-3995.
[16]Bomens M.Centmsome composition and microtubule anchoring mechanisms.Curr Opin Cell Biol[J],2002,14(1):25-34.
[17]Chen D Y,Li J S,Han Z M,et al.Somatic cel bovine cloning:Efect of donor cel an d recipients.Chinese Science Buletin[J],2003,48:549-554
[18]Chen SS.EMBO J,2002,21(17):4491-4499.
[19]Dai YP,Wang LL,Wang HP,et al.Fate of centrosomes following somatic cell nuclear transfer (SCNT) in bovine oocytes. Reproduction[J],2006,131:1051～1061.
[20] D'Assoro AB, Banett SL, Folk C,et al. Amplified centrosomes in breast cancer: a potential indicator of tumor aggressiveness.Breast Cancer Res Treat[J], 2002, 75(1):25～34.
[21] Derenzini M, Sirri V, Trere D, et al. The quantity of nuclelar proteins nucleolin and protein B23 is related to cell doubling time in human cancer cells. Lab Invest[J]. 1995. 73: 497—502.
[22] Doxsey S. The centrosome—a tiny organelle with big potential. Nat Genet[J], 1998,20(2):104～106.
[23] El.Deiry WS. To kino T, Velculescu VE, et al. WAF1, a potential mediator of p53 tumor suppression. Cell[J], 1993, 75(4): 817-825.
[24] Evan GI, Vouaden KH. Proliferation,cell cycle and apoptosis in cancer. Nature[J], 2001,411:342-348.
[25] Feuerstein N, Mond JJ. "Numatrin," a nuclear matrix protein associated with induction of proliferation in B lymphocytes. Biochem Biophys Res Commun[J], 1987,147 :335—391
[26] Fukasawa K, Choi T. Kuriyama R Abnormal centrosome amplification in the absence of p53. Science[J],1996, 271(5256): 1744-1747
[27] Gong G C, Dai Y P, Zhu H B, et al. Production of transgenicblastoc yst by nuclear transfer from diferent types of somatic cells in cattle. Sci China C Life Sci[J], 2004, 47: 183—189
[28] Goats S, Flanagan WM, Nourse J. et al. Requirement of p27Kipl for restriction point control of the fibroblast cell cycle. Scicence[J],1996,272(5263):877
[29] GrisendiS. Nature[J]. 2005. 437: 147-153
[30] Harper JW, Adami GR, Wei N, et al. The p21 Cdk-interacting protein Cipl is a potent inhibitior of G1 cyclin-dependent kinases. Cell[J], 1993, 75(4): 805—806.
[31] Hassold T, Hunt P. To en (meiotically) is human:the genesis of human aneuploidy. Natire Reviews Genetics[J],2001,2:280—291.
[32] Hinchcliffe EH, Sluder G."It takes two to tango":understanding how centrosome duplication is regulated throughout the cell.Genes Development[J],2001,15:1167—1181
[33] Hollander MC, Alamo F, Jackman J, et al. Analysis of the mammalian GADD45 gene and its response to DNA damage. J Biol Chem[J]. 1993,268(32):24385～24393
[34] Hollander M C, Sheikh M S, Bulavin D. Genomic instability in Gadd45a-deficient mice. Nat Genet[J],1999,23(2):176～ 184
[35] Itahana K, Bhat KP, Jin A, et al. Tumor suppressor ARF degrades B23. a nucleolar protein involved in ribosome biogenesis and cell proliferation. MolCell[J], 2003, 12: 1151—1164.
[36] Jin S, Tong T, Fan W, et al. GADD45-induced cell cycle G2-M arrest associates with altered subcellular distribution of Cyclin B1 and is independent of p38 kinase activity. Oncogene[J], 2002, 21(57): 8696-8704
[37] Kawamura K, Izumi H, Ma Z,et al.Induction of centrosome amplification and chromosome instability in human bladder cancer cells by p53 mutation and cyclin E overexpression. Cancer Res[J], 2004, 64(14):4800～4809
[38] Kishigami S, Bui HT, Wakayama S, et al. Successful mouse cloning of an outbred strain by trichostatin A treatment after somatic nuclear transfer. Reprod Dev[J]. 2007,53:165～170.
[39] Kramer A, Lukas J, Bartek J. Checking out the centrosome. Cell Cycle[J], 2004,3(11): 1390～1393.
[40] Lange BM. Integration of the centrosome in cell cycle control,stress response and signaltransduction pathways. Curr Opin Cell Biol[J],2002,14(1):35～43.
[41] Lanza RP, Cibelli JB, Faber D, Sweeney RW, Henderson B, Nevala W, West MD, Wettstein PJ. Cloned cattle can be healthy and normal. Science[J],2001,294:1893～1894.
[42] Lischwe MA, Smetana K, Olson MO, et al. Proteins C23 and B23 are the major nucleolar silver staining proteins. Life Sci[J], 1979,25 : 701～708.
[43] Mamts Gupta, Shivkumar Gupta, Barbara Hoffman, et al.Gadd45aand Gadd458protect the hematopoietie cells from UV induced apoptosis via distinct signaling pathway including P38 activation an JNK inhibition. JBC[J], 2006,281(26):17552～ 17558.
[44] Murphy KL. FASEB J,2000,14(14):2291～2302.
[45] Okuda M, Horn HF, Tarapore P. Nucleophos min/B23 is a target of CDK2/cyclin E in centrosome duplication. Cell[J],2000,103(1):127～140.
[46] Okuda M. Oncogene[J],2002,21:6170～6174.
[47] Okuda M. The role of nuclephosmin in centrosome duplication. Oncogene[J]. 2002. 21: 6170～ 6174.
[48] Ouyang X.Biochim Biophys Acta[J],2001,1541(3): 212-220.
[49] Ouyang X, Wang X, Xu K. et al. Effect of P53 on centrosome amplification in prostate cancer cells. Biochim biophys Acta[J],2001,1541(3):212～220.
[50] Patrick Allard. Stem-loop binding protein accumulates during oocyte maturation and is not cell-cycle-regulated in the early mouse embryo. Journal of Cell Science[J],2002,115(23):4577～ 4586.
[51] Racowsky C. High rates of embryonic loss, yet high incidence of multiple brirths in human ART: is this paradoxical?. Theriogenology[J],2002,57:87～96.
[52] Salisbury J L, D'Assoro A B, Lingle W L. Centrosome amplification and the origin of chromosomal instability in breast cancer. J Mammary Gland Biol Neoplasia[J], 2004,9(3):275～283.
[53] Schuyler SC, Pellman D. Search, capture and signal: games microtubules and centrosomes play. Cell Sci[J],2001,114:247～255.
[54] Stearns T. Centrosome duplication. A centnolar pas de deux. Cell[J],2001,105(4):417～420.
[55] Tarapore P, Fukasawa K. Loss of p53 and centrosome hyperamplification. Oncogene[M], 2002,21(40):6234～6240.
[56] Tarapore P, Horn HF, Tokuyama Y, et al. Direct regulation of the centrosome duplication cycle by the p53-p21Wafl/Cipl pathway. Oncogene[M], 2001, 20(25): 317-384.
[57] Tarapore P, Okuda M, Fukasawa K. A mammalian in vitro centriole duplication System: evidence for involvement of CDK2/cyclin E and nucleophosmin/B23 in centrosome duplication. Cell Cycle[J],2002,l(1):75～81.
[58] Tarapore P. Oncogene[M],2002,21:6234～5240.
[59] Wilmut A, Schnieke AE, McWhir J, et al. Viable offspring derived from fetal and adult mammalian cells. Nature[J],1997,385:810～813.
[60] Wilmut I, Beaujean N, de Sousa PA, et al. Somatic cell nuclear transfer. Nature[J],2002,419:583～ 586.
[2]侯志富,王维忠,印璞,等.大肠肿瘤分子调控模式初探.中国肿瘤临床[J].1999.26(1):856-857.
[3]和占星,文际坤,赵开典.日本牛胚胎工程技术的现状与趋势.黄牛杂志[J].1999,25(3):72-73.
[4]刘建民.牛卵母细胞体外受精及体外发育的研究.生物技术[J],1993,3(3):18-21.
[5]李荣凤,薛晓先,刘哲,等.牛卵母细胞皮层颗粒荧光染色法质成熟鉴定.畜牧兽医学报[J],2000,31(3):203-210.
[6]马云.牛卵泡卵母细胞体外成熟、体外受精及受精卵体外培养的研究.陕西杨凌,西北农林科技大学硕士学位论文[D].2001.
[7]马平,马云.牛的体外受精技术研究进展.家畜生态学报[J],2006,27(6):251-255
[8]蒙少宁.从试管牛到“克隆牛”.光明日报[Z],1998年2月23日.
[9]杨叔培.世界第一头第二代克隆牛在日诞生.生物技术通报[J],2000,2:41-42.
[10]旭日干,贾士荣.农业生物技术进展与展望[M].台肥,中国科学技术大学出版社,1993.
[11]花田章.山羊的体外受精.家畜繁殖学杂志[J].1985,31(5):21-26.
[12]王建英,兰邹然,杨桂文,等.纺锤体检验点的信号通路及其作用.山东医药[J].2006.(46)16:95-96.
[13]周佳勃 吴韩东,刘丽清,等.精子和卵母细胞质量控制对山羊卵胞质内精子注射(ICSI)受精的影响.实验生物学报[J].2004;37:367-374.
[14]Abeieu A,Magnaghi-Jaulin L,Kahana JA,et al.X-Mps-1,an essential component of the mitotic checkpoint,is necessary for Mad1,Mad2 and CENP-E association with kinetochores,in Xenopus egg extracts.Cell[J].2001.106:83-93.
[15]Bailly E,Doree M,Nurse P.et al.P34cde2 is located in both nucleus and cytoplasm:part is centrosomally associated at G2/M and enters vesicles at anaphase.EMBO J[J],1989,8(13):3985-3995.
[16]Bomens M.Centmsome composition and microtubule anchoring mechanisms.Curr Opin Cell Biol[J],2002,14(1):25-34.
[17]Chen D Y,Li J S,Han Z M,et al.Somatic cel bovine cloning:Efect of donor cel an d recipients.Chinese Science Buletin[J],2003,48:549-554
[18]Chen SS.EMBO J,2002,21(17):4491-4499.
[19]Dai YP,Wang LL,Wang HP,et al.Fate of centrosomes following somatic cell nuclear transfer (SCNT) in bovine oocytes. Reproduction[J],2006,131:1051～1061.
[20] D'Assoro AB, Banett SL, Folk C,et al. Amplified centrosomes in breast cancer: a potential indicator of tumor aggressiveness.Breast Cancer Res Treat[J], 2002, 75(1):25～34.
[21] Derenzini M, Sirri V, Trere D, et al. The quantity of nuclelar proteins nucleolin and protein B23 is related to cell doubling time in human cancer cells. Lab Invest[J]. 1995. 73: 497—502.
[22] Doxsey S. The centrosome—a tiny organelle with big potential. Nat Genet[J], 1998,20(2):104～106.
[23] El.Deiry WS. To kino T, Velculescu VE, et al. WAF1, a potential mediator of p53 tumor suppression. Cell[J], 1993, 75(4): 817-825.
[24] Evan GI, Vouaden KH. Proliferation,cell cycle and apoptosis in cancer. Nature[J], 2001,411:342-348.
[25] Feuerstein N, Mond JJ. "Numatrin," a nuclear matrix protein associated with induction of proliferation in B lymphocytes. Biochem Biophys Res Commun[J], 1987,147 :335—391
[26] Fukasawa K, Choi T. Kuriyama R Abnormal centrosome amplification in the absence of p53. Science[J],1996, 271(5256): 1744-1747
[27] Gong G C, Dai Y P, Zhu H B, et al. Production of transgenicblastoc yst by nuclear transfer from diferent types of somatic cells in cattle. Sci China C Life Sci[J], 2004, 47: 183—189
[28] Goats S, Flanagan WM, Nourse J. et al. Requirement of p27Kipl for restriction point control of the fibroblast cell cycle. Scicence[J],1996,272(5263):877
[29] GrisendiS. Nature[J]. 2005. 437: 147-153
[30] Harper JW, Adami GR, Wei N, et al. The p21 Cdk-interacting protein Cipl is a potent inhibitior of G1 cyclin-dependent kinases. Cell[J], 1993, 75(4): 805—806.
[31] Hassold T, Hunt P. To en (meiotically) is human:the genesis of human aneuploidy. Natire Reviews Genetics[J],2001,2:280—291.
[32] Hinchcliffe EH, Sluder G."It takes two to tango":understanding how centrosome duplication is regulated throughout the cell.Genes Development[J],2001,15:1167—1181
[33] Hollander MC, Alamo F, Jackman J, et al. Analysis of the mammalian GADD45 gene and its response to DNA damage. J Biol Chem[J]. 1993,268(32):24385～24393
[34] Hollander M C, Sheikh M S, Bulavin D. Genomic instability in Gadd45a-deficient mice. Nat Genet[J],1999,23(2):176～ 184
[35] Itahana K, Bhat KP, Jin A, et al. Tumor suppressor ARF degrades B23. a nucleolar protein involved in ribosome biogenesis and cell proliferation. MolCell[J], 2003, 12: 1151—1164.
[36] Jin S, Tong T, Fan W, et al. GADD45-induced cell cycle G2-M arrest associates with altered subcellular distribution of Cyclin B1 and is independent of p38 kinase activity. Oncogene[J], 2002, 21(57): 8696-8704
[37] Kawamura K, Izumi H, Ma Z,et al.Induction of centrosome amplification and chromosome instability in human bladder cancer cells by p53 mutation and cyclin E overexpression. Cancer Res[J], 2004, 64(14):4800～4809
[38] Kishigami S, Bui HT, Wakayama S, et al. Successful mouse cloning of an outbred strain by trichostatin A treatment after somatic nuclear transfer. Reprod Dev[J]. 2007,53:165～170.
[39] Kramer A, Lukas J, Bartek J. Checking out the centrosome. Cell Cycle[J], 2004,3(11): 1390～1393.
[40] Lange BM. Integration of the centrosome in cell cycle control,stress response and signaltransduction pathways. Curr Opin Cell Biol[J],2002,14(1):35～43.
[41] Lanza RP, Cibelli JB, Faber D, Sweeney RW, Henderson B, Nevala W, West MD, Wettstein PJ. Cloned cattle can be healthy and normal. Science[J],2001,294:1893～1894.
[42] Lischwe MA, Smetana K, Olson MO, et al. Proteins C23 and B23 are the major nucleolar silver staining proteins. Life Sci[J], 1979,25 : 701～708.
[43] Mamts Gupta, Shivkumar Gupta, Barbara Hoffman, et al.Gadd45aand Gadd458protect the hematopoietie cells from UV induced apoptosis via distinct signaling pathway including P38 activation an JNK inhibition. JBC[J], 2006,281(26):17552～ 17558.
[44] Murphy KL. FASEB J,2000,14(14):2291～2302.
[45] Okuda M, Horn HF, Tarapore P. Nucleophos min/B23 is a target of CDK2/cyclin E in centrosome duplication. Cell[J],2000,103(1):127～140.
[46] Okuda M. Oncogene[J],2002,21:6170～6174.
[47] Okuda M. The role of nuclephosmin in centrosome duplication. Oncogene[J]. 2002. 21: 6170～ 6174.
[48] Ouyang X.Biochim Biophys Acta[J],2001,1541(3): 212-220.
[49] Ouyang X, Wang X, Xu K. et al. Effect of P53 on centrosome amplification in prostate cancer cells. Biochim biophys Acta[J],2001,1541(3):212～220.
[50] Patrick Allard. Stem-loop binding protein accumulates during oocyte maturation and is not cell-cycle-regulated in the early mouse embryo. Journal of Cell Science[J],2002,115(23):4577～ 4586.
[51] Racowsky C. High rates of embryonic loss, yet high incidence of multiple brirths in human ART: is this paradoxical?. Theriogenology[J],2002,57:87～96.
[52] Salisbury J L, D'Assoro A B, Lingle W L. Centrosome amplification and the origin of chromosomal instability in breast cancer. J Mammary Gland Biol Neoplasia[J], 2004,9(3):275～283.
[53] Schuyler SC, Pellman D. Search, capture and signal: games microtubules and centrosomes play. Cell Sci[J],2001,114:247～255.
[54] Stearns T. Centrosome duplication. A centnolar pas de deux. Cell[J],2001,105(4):417～420.
[55] Tarapore P, Fukasawa K. Loss of p53 and centrosome hyperamplification. Oncogene[M], 2002,21(40):6234～6240.
[56] Tarapore P, Horn HF, Tokuyama Y, et al. Direct regulation of the centrosome duplication cycle by the p53-p21Wafl/Cipl pathway. Oncogene[M], 2001, 20(25): 317-384.
[57] Tarapore P, Okuda M, Fukasawa K. A mammalian in vitro centriole duplication System: evidence for involvement of CDK2/cyclin E and nucleophosmin/B23 in centrosome duplication. Cell Cycle[J],2002,l(1):75～81.
[58] Tarapore P. Oncogene[M],2002,21:6234～5240.
[59] Wilmut A, Schnieke AE, McWhir J, et al. Viable offspring derived from fetal and adult mammalian cells. Nature[J],1997,385:810～813.
[60] Wilmut I, Beaujean N, de Sousa PA, et al. Somatic cell nuclear transfer. Nature[J],2002,419:583～ 586.