【作者】 孙炳奇；

【导师】 于秉治；

【作者基本信息】 中国医科大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 前言 我们的前期工作已经证明了在小鼠受精卵早期发育信号传导过程中,蛋白激酶A（PKA）和蛋白激酶C（PKC）是调节有丝分裂促进因子（MPF）活性的重要物质。当小鼠受精卵进入第一次细胞周期的M期时,cAMP浓度和PKA活性都降低,而MPF活性升高。 诸多数据显示MPF在小鼠受精卵早期发育中处于中心地位。首先,MPF,Plk1和Cdc25形成一个正反馈通路,其次;有丝分裂原激活的蛋白激酶激酶激酶（Mos）活性随着MPF活性变化,且形成Mos-MEK-MAPK级联系统;再次,Myt1和Wee1激酶通过磷酸化Cdc2上Thr14和Tyr15位点而抑制MPF的活性,但是Plk1能够可通过磷酸化Myt1上的特定位点进而抑制Myt1的活性。一旦MPF活性达到最大值,APC通过对CyclinB的降解而使MPF活性下降。在M期后期,因为PP_2A的作用,Cdc25、Mos、MEK、MAPK的活性开始下降。这时,cAMP浓度和PKA活性重新开始升高,且小鼠受精卵一分为二,由此我们认为,cAMP就如同扳机一样触发了小鼠受精卵的早期发育过程。 蛋白相互作用的网络在本质上是动态的,蛋白间的一系列动态作用关系,可转化成一系列描述系统的时空进化的数学方程,方程的类型取决于所要处理的生物学问题。对于细胞周期控制系统,适合应用常微分方程,在数学上来说,应用微分方程知识捕获了我们关于蛋白合成和分解,磷酸化和去磷酸化的直觉认识,且这些方程允许我们计算每个蛋白的浓度变化。对于不同的反应类型,我们应用不同的速率法则,从而可以得到不同的微分方程: Gepasi（general pathway simulator）,通用通路模拟器。它是一个用来模拟生物化学系统的软件包。包括模型的参数估算;优化模型功能;代谢物控制分析和线性稳定性分析。本文正是用Gepasi建立数学模型,初步模拟了更多还原

【Abstract】 PrefaceOur lab group previous work has demonstrated that both protein kinase A （PKA） and prtein kinase C （PKC） are important elements to regulate the activity of M - phase promoting factor （MPF） in signal transduction pathway of mouse zygote early development. When the mouse zygote entrys into M phase of the first cell cycle, both the adenosine 35’- monophosphate （cAMP） concentration and the PKA activity decrease, whereas the MPF activity increases.All the reported data have pointed out that MPF is playing a central role in signal transduction pathway of mouse zygote first cell cycle. First, MPF, Plk1 （polo like kinase 1） and Cdc25 form a positive feedback loop. Secondly, the Mitogen - activated protein kinase kinase kinase （Mos） activity follows the MPF activity pattern, and Mitogen - activated protein kinase （MAPK） is activated through the Mos - MEK - MAPK cascade （MEK: Mitogen - activated protein kinase） Thirdly, Myt1 and Wee1 can inhibit the MPF activity by phosphoryla-tion Cdc2 on Thr14 and Tyr15 site, but the Mytl activity can be inhibited through a phosphorylation by Plk1. Once the MPF activity reaches the maximum , APC begins to degrade CyclinB, thus reducing the MPF activity. In the later period of the M phase, all the active Cdc25, Mos, MEK, MAPK become inactive due to the dephosphorylation by protein phosphatase 2A (PP_2A). At this point, the cAMP concentration and the PKA activity increase again, and the mouse zytoge divides into two cells. Overall, cAMP acts as a trigger to induce mouse zygote to development in this whole process.The protein interactive network is dynamic in essentially. The dynamic relationship between a serial of protein effection can be converted into a serial ofmath equation to describe system space - time evolution. The equation type is decided by biology question to be treated. To cell cycle control system, it is suitable to use ordinary differential equatins （ ODEs）. In terms of mathematical, the ordinary differential equatins used can assist us capture the intuitive gnosia about protein synthesis and degradation, phosphorylation and dephosphorylation. And, these equations can permit us to calculate the concentration change of each protein. We used diffferent rate laws to different reaction types, and we get different ordinary differential equatins: Gepasi jxgeneral pathway simulator) , Gepasi is a software package for modeling biochemical systems. It simulates the kinetics of systems of biochemical reactions and provides a number of tools to fit models to data, optimize any function of the model, perform metabolic control a-nalysis and linear stability analysis. We just constructed a math model by using gepasi and simulated the signal transduction in the first cell cycle about mouse zytoge early development.Materials and MethodsMaterials:1. experimental animal: Kunming mouse supply by CMU experimental animal department2. instrumenttation: a Personal computer （Intel Pentium 2. 4G CPU, HY 512M Memory, MicroStar 845E MainBoard, MicroSoft?XP System）.3. Software for math model. Gepasijxgeneral pathway simulator), free download from www. gepasi. org for Windows,version is 3. 30,ZIP format.Methods:1. The ovaries were removed from Kunming pregnant female mice （5 - 6w） and transferred to prewarmed （37T! ） M2 medium. Cumulus cells were removed by 300|xg/ml hyaluronidase. Zygote were cultured in M16. Zygotes were collected at appointed time.2. Collect experimental data about mouse zytoge early development.3. Construct quantitative model by using ordinary differential equations. We use Gepasi to simulate this math model on PC. That is, We write outreaction formula, then Gepasi can automatic convert reaction formula to ordinary differential equations automaticly and can plot simulation graph.Results1. The cAMP concentration and the PKA activity lower at mitosis, higher after M/Gl transition point until the next mitosis. When the cAMP concentration and the PKA activity decrease, the MPF activity rises. Our simulated results u-sing Gepai approach exhibit the similarities of The cAMP concentration , the active PKA and MPF to that of the experimental results.2. The quantity of Plkl protein fluctuates slightly during mitotic after fertilization. However, the activity of Plkl after phosphorylation has a remarkable change during the first cell cycle in mouse zygotes, which reaches the maximum level at the M phase. Gepai simulation also shows a similar change of Plkl activity from Gl to M phase.3. When mouse oocytes are arrested at Mil, both MAPK activity and MPF activity stayed at a higher level. After fertilization, both activities begin to decrease and reach the minimal level after 7 hours. The simulation results show MAPK activity are higher at Mil phase and decreases to a minimal level after fertilization. At mitsis of first cell cycle, MAPK activity rise, but it’s activity could not get to Mil level.4. CyclinB continues to be accumulated before metaphase, and start to degrade near the metaphase in the case that no activators and inhibitors are added. In PKI - microinjected embryo, CyclinB maintains a higher quantity level even after exiting the M phase. CyclinB simulation result;CyclinB accumulate to top at M phase and degradate when exit M phase. This is in coincidence with experimental data.DiscussinEarly zygote division is characterized by a rapid succession of interphase and mitotic state in many species including mouse. In our study, all the experi-mental evidence has shown that cAMP is an upstream signal substance whose concentration influences the activity of downstream kinases such as PKA, MPF, Plkl, Cdc25 , Weel et al. PKA as a negative regulator whose activity changes is opposite to MPF activity, mediates cAMP signal pathway to downstream. It only in xenopus ocytes that PKA can phosphorylate Cdc25 protein, and cause meio-genic Gil phase block. In mouse zygotes, it unclear about the relation between PKA and Cdc25. Cdc25a, Cdc25b and Cdc25c are all activators of cyclin - dependent kinases,but maybe Cdc25b plays chief effect in mouse zygotes early development.In the signal transduction pathway of first mouse embryo mitosis, cAMP acts as a trigger. Namely, cAMP concentration drops first, then a serial of ki-nase change its activity,mouse zygotes entry intocell cycle and complete mitosis. We just construct a math model byusing Gepasi algorithm has been used to explore this process, and the simultion results demonstrated a greemnet with the experimental data.The model may not answer all the question we posed, but it can integrate our knowledge to a system. It is a start point to ask questious regarding the system as a whole, with the help of more experimental results, the mode may be verified for reliability. And the simulation process may help us to have a good comprehension about experimental phenomenon.Based on the complexity in signal transduction pathway of mouse zygotes early development, our model is far from complete and accurate in details and need to be expanded and corrected in future. But, such a model can be extended in continuely till we have a complete mode to reveal the cell cycle regualte mechanism overall in mouse zygotes early development.Conclusion1. The math model can rudiment reflect the basic state about the first cell cycles signal transduction pathway in mouse zygotes early development.2. We think cAMP is the trigger that induce mouse zygote to develop in the first cell cycle.更多还原