【作者】 刘莹；

【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 蛋白激酶C（PKC）是一组丝／苏氨酸蛋白激酶，参与多种信号传导系统。包括细胞分化、生长调控、肿瘤发生和细胞凋亡。PKC家族可分为三组：cPKC，又称传统PKC，包括α、β、γ等，是一组Ca²⁺依赖，二脂酰甘油激活的同工酶；nPKC，也叫新PKC，包括δ、ε、η及θ，是非依赖Ca²⁺、但二脂酰甘油依赖的一组同工酶；aPKC，即非典型PKC，包括ζ和λ两种亚型，体外实验证明是即不依赖Ca²⁺，也非二脂酰甘油激活。PKC的多种亚型在不同细胞中的定位、表达和功能的不同使得PKC可以参与多种信号传递，其参与生长调控的分子机制，也因细胞本身可以合成几种PKC同工酶而变得复杂起来。 真核细胞的分裂是一系列受调节过程，这一过程可被一组高度保守的蛋白激酶家族-cyclin依赖激酶（cdks）所调节。Cdks的活化需要结合一种特异的调节亚基-cyclin。cyclin／cdk复合物作为广泛的影响细胞分裂调节物而发挥作用。每一种复合物参与不同的细胞周期各时段的调控。现在，已有大量的不同种类的cyclin／cdk复合物被认识并纯化出来。 G2／M的过渡可受到cdks的调控。在这一过程中一个关键、重要的物质就是MPF（M-phase promoting factor, MPF），即M-期促进因子，又称成熟促进因子（maturation-promoting factor，MPF）。它最先由Masui和Markert在1971年发现，并于1988年从Xenopus laevis卵中纯化。MPF是调节细胞进、出M期所必需的蛋白激酶，具有广泛的生物功能，通过促进靶蛋白的磷酸化而改变其生理活性。MPF自身的活性随着细胞周期的运转而发生周期性变化。MPF是一包含催化亚基p34^cdc2和调节亚基cyclin B的二聚体，p34^cdc2的活性严格依赖cyclin B。在Xenopus卵中，缺乏cyclin B的合成MPF将不能活化。Cyclin B随着细胞周期进程而周期性改变。G1期开始合成，并逐渐增多，在G2期末达到最高量。而在只有cyclin B的量达到一定程度时，p34^cdc2-cyclin B复合物才能从前-MPF（非活化状态）而活化，从而促进细胞周期从G2进入到M期，引起细胞分裂。p34^cdc2有三个重要调节位点Thr14、Tyr15和Thr161分别被其他各种因素调节，当Thr161处于磷酸化，而Thrl4、Tyrls处于脱磷酸化时，p34抛才能被激活。那么，所有能使这些位点的磷酸化水平改变的因素都可能调节 p34“拟的活性，进而调节细胞周期进程。CdC25作为一种磷酸酶在其中可能发挥了作用。在血管内皮细胞，Cdc25磷酸酶表达下降引起 p34”M的 Tyr残基脱磷酸化的抑制而使P34测活性下降。Cyclin B作为调节亚基在整个 MPF的调节过程中也受到多种因素的影响，Cyclin B的表达含量的改变也同样影响着细胞周期。 另外，细胞内 ca人在细胞周期进程中也是一重要调节因素。主要的调节方式是通过对P34““的活性调节起作用的。有研究表明，Ca卜和地”螫合剂分别在可通透性细胞和无细胞系统中激活和灭活 p34抛。而 Ca卜在细胞内暂时性升高对 cyhin B的合成并无影响。Ca卜活化 P34”拟的机制第一步是结合cycin B，并且处于磷酸化状态，即 Thr14、Tyrls和 Tlirl61都处于磷酸化状态；第二步是 CdC25磷酸酶通过是 Thr14、Tyr15脱磷酸化而激活 p34”拟。而 Ca从浓度升高降低 p34”怕是通过使 Thr161脱磷酸化及CylcinB 7K解，同时使 P34”帕一cyclin B复合物分解。为了避免 PKC的激动剂OAG（l，Oleoyl－2＊。Cetyl＊Sfl－gy。efol）的加人而影响到细胞内C。‘”浓度，我们在选择OAG的浓度时，则应注意OAG的加人不要影响到细胞内Ca＊浓度的改变。 我们的研究主要集中在PKC如何调控MPF，进而调节细胞周期进程的。实验中，应用了PKC的激活剂 OAG和特异性PKC抑制剂CalPhOStin C作用于NIH3Th细胞，来检测在GZ／M期进程中 P34”“和cyclinB转录水平和蛋白表达水平及其活性改变，以及作用后对细胞周期进程，特别是对GZ／M期过渡的影响。 方 法 ＊细胞培养与细胞周期同步化 小鼠NIH3Th细胞系（中国医科大学细胞生物学教研室提供人用DMEM（d讪ecco’s mo腼ed E8ge’S meditun）培养基加 10％（VOI／VOL）的56℃灭活的胎牛血清，滤膜除菌。细胞周期同步化在m期末用phidicolin（DMSO溶成浓度为 Zllg／ul人在培养瓶中指数生长期的细胞稀释至 2．5 XJO～rnl加人 aPhidicolin，终浓度为 lug／ml培养液，37T CO。培养箱中培养24小时后，用灭菌的PBS洗2次，后加人新鲜培养液。此时细胞停止在 ·二·GI＊期。 细胞用胰蛋白酶N．2％）消化后，在 PBS中洗2次，1200 x g离心 10分钟去上清，加人 70％（VOI／VOL）的冷乙醇，于一20t 30分钟。去除乙醇后，细胞中加人含有 RNase AN．lin牙IIl）的 PBS中放在 37 t培养箱 30分钟。然后，细胞被含有50u才IIl ProPidium iodide（si）PBS中染色30分钟，用（BECTON DICKINSON FACScan）型流式细胞仪腻 DNA含量。· 2．RNA提取及 RT—PCR NIH3D细胞（IX 10‘／瓶），DEPC处理的四S洗2次，力人 ’vim＊＊l试剂更多还原

【Abstract】 The term protein kinase C ( PKC ) stands for a group of at least twelve serine /threconine kinases that are characterized by a high degree of similarity in their catalytic kinase domains and cysteine - rich regions. The PKC family is subdivided into three groups; the classical PKC members (abr) are Ca2 + and diacylg-lycerol - dependent, the novel PKCs are Ca2+ -independent but diacyl-glycerol -dependent and the atypical PKCs are not activated by Ca2+and diacylglycerol in vitro . This may account for the multiplicity and diversity of the cellular activities implicted to be mediated by PKC, since individual PKC enzymes are thought to execute distince cellular functions, at different cellular locations. Harald Mischak ,et al found that in NIH3T3 cells express high amounts of PKC -m RNA ,low but detectable amounts of PKC - 8and PCKe ,and barely detectable amounts of PKC-and-m RNA. PKC-rand - ?m RNA could not be detected at all. Using Western blot analysis , they found that PKC -a was the only member of the PKC family that was present in abundant a-mounts. Low amounts of PKC - s and sometimes PKC - 8 and - could be detected , while antibodies against all other isozymes did not detect PKC - specific bands.According to the current model of cell cycle control, transition between different cell cycle phases is regulated at checkpoints. There are two key checkpoints ;G1/S and G2/M. Progression through the cell cycle is mediated by the activation of a highly conserved family of protein kinases, the cyclin - dependent kinases ( cdks). Activation of a cdk requires binding to a specific regulatory subunit , termed a cyclin. Together, these cyclin/cdk complexes are the universal cell cycle regulators ,with each complex controlling a specific transition between the subsequent phases in the cell cycle . Now a number of different cyclin/cdk complexes have been identified and purified. CHIYA KOSAKA et al reported that phorbol 12 - myristate 13-acetate ( PMA) and 1,2- dioctanoyl - sn - glycerol( DiC8) ,a membrane - permeable DAG,block the G1/S transition by inhibiting the expression of cyclin A and the phosphorlation of the retinoblastoma gene product (pRb) in human vascular endothelial cells and suggested that the inhibition of the activity of cdk2,a cyclin -dependent kinase (cdk) asssociated with cyclin A, is the cause of the PMA - induced Gl/S arrest. In addition , cdk4 and cdk6 bind to cyclin D also regulate Gl phase.The transition from G2/M phase is also mediated by cdk(s). A critical and central component is likely to be MPF ( M - phase promoting factor, another name is maturation - promoting factor, it was first discovered by Masui and Marikert in 1971 ,and then purified from Xenopus laevis eggs in 1988. MPF is a complex that consists of the protein kinase p34cdc2 and the regulatory protein cycli B; the protein kinase activity of p34cdc2 is strictly dependent on its association with cyclinB. In Xenopus laevis eggs the absent of cyclin B, MPF will not be activited. The amounts of cyclin B changes due to the phase of cell cycle. In Gl phase ,cyclin B start to be synthesized, getting more and more and the highest of amounts in the late of G2 phase . the amounts of cyclin B is the necessery for the activity of MPF. In addition to cyclin B, p34cdc2 can be activity or in activity in three site Thrl4,Tyrl5 and Thr 161. In all eukaryotic cells that have been studied , protein phosphorylation serves as a key regulator of cell cycle events . Entry and exit from the M phase of the cell cycle are triggered universally by the activation and inactivation of p34cdc2, a cyclin - dependent kinase that binds B -type mitotic cyclins. Only when Thrl4 and Tyrl5 are in dephosphorylation and Thrl61 is in the state of phosphorylation ,p34cdc2 is activity.In addition, activation and inactivation of p34cdc2are induced by Ca2+and prevented by Ca2+ chelators in permeabilized cells and cell - free systems. This suggests that intracellular Ca2+ transients may play an important physiological role in the control of p34cdc2 kinase activity .更多还原