【作者】 王亚杰；

【导师】 于秉治；

【作者基本信息】 中国医科大学 ， 细胞生物学， 2002， 博士

【摘要】 前言 细胞增殖是细胞生命的一个基本特征，细胞增殖与机体的生长、生理性再生、创伤修复、细胞凋亡及肿瘤发生都有密切关系，它也是细胞分化形成组织、器官、系统的基础。细胞生长到一定阶段开始分裂，细胞分裂后产生的新细胞生长增大，此后又平均地分裂成两个遗传学上相同的子细胞。细胞这种周而复始的生长分裂周期就称为细胞周期(cell cycle)。细胞周期从形态学上分为4个阶段：G1期(DNA合成前期)、S期(DNA合成期)、G2期(DNA合成后期)及M期(有丝分裂期)；其中G1，S及G2期又统称为分裂间期。细胞周期调控的研究对揭示个体生长、发育、衰老、癌变的机制有重要意义。 在许多物种的早期胚胎细胞分裂过程中，由间期到M期的连续分裂是一个重要特点。MPF(M期促进因子，M-phase promoting factor，MPF，又可称为成熟促进因子maturation promoting factor，MPF)由Cdc2和周期素B两个亚基组成。其中周期素B是MPF的调节亚基，与Cdc2结合而使其活化，是Cdc2具有蛋白激酶活性的前提。催化亚基Cdc2具有丝／苏氨酸蛋白激酶活性，活化后可作用于多种底物，如组蛋白H1，核层蛋白，核仁素，P53等，引起染色体凝集，核膜破裂，核仁消失，细胞骨架重建等分裂期征象。MPF的活性变化调节着细胞周期进程。 蛋白激酶A(protein kinase A，PKA)是依赖于cAMP的丝／苏氨酸蛋白激酶，是重要的信号传导途径之一，广泛参与许多生命过程，包括生长、分化、肿瘤发生、细胞凋亡、细胞周期调控等。以小鼠卵母细胞为实验对象，给予PKA激动剂cAMP、8-Br-cAMP或磷酸二酯酶抑制剂IMBX(isomethyl butyl xanthine)均可抑制小鼠卵母细胞的胚泡破裂及减数分裂成熟。通过对非洲爪赡胚胎细胞周期的研究发现JKA活性也随细胞周期变化而波动，在M期出现时PKA活性下降，而M／间期转换时PKA活性达最大值，一直维持高值直到下一个M期出现，其对MPF活性调节与CdC25磷酸化状态及周期素B表达有关。 小鼠受精卵是脊椎动物中与人类种属较为接近的简单而天然的细胞周期模型，但有关小鼠受精卵中PKA对其发育的影响及与MPF活性关系以及调节机制至今国内外尚未见报道。本实验应用PKA激动剂CAMP及抑制剂PKI显微注射入小鼠二一细胞期受精卵并观察卵细胞M期形态学变化及 PKA对 MPF活性的影响以及 CdC25 C，CdCZ电泳迁移率，CdcZ的磷酸酪氨酸 PTyrl及周期素 B含量，为揭示 PKA在哺乳动物细胞周期调控机制，对生长、发育、癌变、死亡的研究提供理论依据。 实验材料 昆明系小白鼠，中国医科大学实验动物部提供。 测活用试剂除nacl山innI 为国产分析纯对rr为amesa产品，［y／乍j ATP为北京亚辉生物医学工程公司产品外，余者均为 Sipoa产品。 免疫印迹分析用抗体： Cdc25 C免多克隆抗体购于 Santa Cruz公司。 CdcZ，周期素m鼠单克隆抗体购于Neomarkers公司。 CdcZ磷酸酪氨酸 PTyrls抗体购于 New England Biolabs。 Hgy标记羊抗鼠及羊抗兔二抗 IgG为北京中山生物技术有限公司产 口口口口 化学发光 ECL试剂盒购自于 Santa Cruz生物技术公司。 实验方法 1．小鼠超排卵及取卵： 2．CAMP及 PKI显微注射：NARISHIGE显微操作系统（日本产X O－lylnPus IX－70倒置显微镜，Hoffman调制相差观察。注射量为细胞总体积 ·二·5呢（10 PI）。cAMP浓度分别为 10 nunol／L，40 mrno＊L，100 nun＊L水溶液，P助抑制剂P0（Protein inase inhibitor，type）溶于5。of／L的2－（N一吗啡琳）乙磺酸（2－［N－morpholino］ethannes讪nic眈id，MES），PH6．5，浓度分别为200呷oVL，800pnunl／L，2 IIUndL。对照组分非注射组及注射 MES以及 MZ培养液组。 3．相差显微镜下观察卵裂情况并照相： 毗MPJ 处理组及对照组细胞于M16培养液中培养，相差显微镜下观察并照相，记录三组细胞卵裂情况，于HCG后30小时计数各实验组了一细胞期受精卵分裂率。35小时计数三组细胞死亡率。 4．酶活性测定的鼠卵收集： 根据实验需要每隔30 min从培养基中取出3个受精卵，收集液中清洗，移人0．5 ml的Eppendorf管中，放人一70t冰箱中速冻保存直至测活Bu。 5．MPF及PKA活性测定： 将收集的鼠卵冻融3次，使细胞裂解，分别加人＊＊F反应液25山，30 T水浴反应7 ruin。取25 PI点于Whatlnan pei强阳离子交换滤纸上，将滤纸置于液问瓶内，用Beckman液问计数仪测定cPm值。 PKA活性测定与MPF活性测定过程相同，只除PKA反应液中PKA的特异性底物肯普肽（kemPhde）替代了MPF的特异底物组蛋白HI，反应液中不含PKA抑制剂。 以注射HCG后时间为横坐标，以cPm值为纵坐标绘制MPF／PKA活性变化时间曲线。 6．CAMP浓度测定：未经注射的卵细胞，按时间要求从m 培养液中取出100只卵细胞，离心去上清，卵细胞沉淀于一70℃冻存。浓度测定前将收集的卵细胞冻（－70更多还原

【Abstract】 INTRODUCTIONA rapid succession of interphase and mitotic states characterizes the early embryonic divisions of many species. The onset of mitosis is induced by activation of MPF, a highly conserved complex consisting of a kinase, Cdc2, and an activating subunit, cyclin B. During the cell cycle the concentration of the catalytic subunit Cdc2 remains constant while cyclin accumulates from interphase to mitosis and is degraded at each metaphase - anaphase transition. In interphase, association with cyclin induces phosphorylation of Cdc2 at key tyrosine and thre-onine residues. In this state the complex is enzymatically inactive. At mitosis, the complex is dephosphorylated and activated by Cdc25.Protein kinase A is cAMP - dependent kinase, one of the most important signal transduction pathways, plays a pivotal role in growth, differentiation, tumor occur, cell cycle control, etc. PKA activators such as cAMP, 8 - Br -cAMP or phosphodiesterase(PDE) inhibitor isomethyl butyl xanthine(IMBX) or purified PKA catalytic subunit all can inhibit germinal vesicle breakdown (GVBD) and meiotic maturation in mouse oocytes, also in Xenopus oocytes. In Xenopus embryonic extracts cAMP concentration and PKA activity also oscillations accompany with the cell cycle, low in mitosis and increased during M/G1 transition, keep high until next mitosis, and the mechanism concern with the Cdc25 phosphorylation state and cyclin B concentration. Mouse fertilized egg is the most simple and natural model for cell cycle that near to human, but little is know about PKA on MPF also PKA on mitosis. In this article, we microinjected cAMP (as activator of PKA) and protein kinase inhibitor (PKI) (as inhibitor ofPKA) into mouse 1-cell stage fertilized eggs, the cAMP concentration, PKA and MPF activaty were detected, also the Cdc25C, Cdc2 phosphorylated state and the concentration of pTyr15 for Cdc2, cyclin B1.MATERIALSFemales of 4-5 week-old KUMING mice and males of 8 week-old KUMING mice were supplied from the Department of Laboratory Animals, China Medical University. cAMP, PKA inhibitor peptide (PKI) , Kemptide, histone H1 (type III-S), polyvinyl alcohol, Na3VO4, NaF, B-glycerophosphate, p- nitrophenylphosphate, 3-[N-morpholino]-propanesulfonic acid (MOPS), genistein, ML-9, leupeptin, aprotonin, pepstatin, chymostatin, trypsin-chymotrypsin inhibitor were purchased from Sigma Chemical Co. [r-32 P] ATP was obtained from Yahui biomedical Enginnering co. Anti -Cdc25C rabbit polyclonal antibody was purchased from Santa Cruz Biotechnology, Anti-Cdc2 and anti - cyclinBl mouse monoclonal antibody were from Neo-markers, Anti - Cdc2 pTyrlS monoclonal antibody was obtained from New England biolabs. HRP - conjugated anti - mouse or anti - rabbit IgG secondary antibody, purchased from Beijing Zhongshan Biotechnology.METHODS1. Superovulation, natural mating, collection of fertilized eggs;2. Microinjection: Microinjection was performed using NARISHIGE micro-injection system ( JAPAN) , Olypus Model EX - 70 inverted microscope with Hoffmann optics. Typical injection volume was 5% of total cell volume, or 10 pl, one - cell stage embryos in G2 were microinjected with cAMP, 10 mmol/L, 40 mmol/L, 100 mmol/L solution in water, or PKI, 200nmol/L, 800mol/ L, 2 mmol/L soluted in 5 mmol/L 2 - [ N - morpholino ] ethane - sulfonic acid (MES), pH6.5; no microinjection or microinjected with M2 medium or 5 mmol/L MES as control.3. Observed under a dissecting microscope and registered the percentage of division and death at 30 or 35 hours after HCG, respectively.4. Histone HI kinase assay; Three eggs were removed from M16 medium, washed in collection buffer and transferred to a 0. 5 ml Eppendorf tube in 5 l of collection buffer. The eppendorf tube was immediately stored at - 80 C until the kinase assay was performed. The frozen eggs were frozen and thawed for three times, then added into 25l MPF buffer. The reaction was allowed to proceed for 7 min at 30C , then 25 L aliquots was removed and spotted on whatman p81 paper, counted t更多还原