【作者】 李晓娜；

【导师】 吴文周； 安美文；

【作者基本信息】 太原理工大学 ， 生物医学工程， 2009， 博士

【摘要】 胞质分裂是一个涉及生物学和力学的复杂过程,其分裂模式也是多种多样。其中不依赖于肌球蛋白II、与细胞周期耦合的、黏附状态下的胞质分裂模式B在真核生物和动物细胞中普遍存在,可能是比经典的收缩环模式更原始的一种分裂方式。弄清胞质分裂模式B的机制,不仅可以深入了解促进细胞进行胞质分裂的各个调节途径之间的关系,从更广泛意义上阐明胞质分裂的机制,而且能够为揭示细胞增殖和生命延续的奥秘,认识与胞质分裂相关疾病(癌症等)的发病机理、药物设计和疾病治疗提供理论依据。以往研究主要针对粘菌细胞胞质分裂模式B机制进行了一定探索,但未见针对哺乳动物细胞的研究。本研究以贴壁生长的鼠肾上皮细胞(NRK)为研究对象,采用显微图像动态采集和分析、细胞免疫荧光和细胞力学等实验技术,研究了肌球蛋白II缺失对NRK细胞分裂的影响,首次从从细胞材料力学特性和细胞运动角度对粘附状态下、肌球蛋白II缺失的哺乳动物细胞胞质分裂机制进行了探索。主要工作内容和结论如下:1.对正常NRK细胞胞质分裂过程进行了观察和描述,讨论了起泡行为的可能机制和子细胞铺展模式与间期母细胞的关系。并利用微管吸吮技术及相关理论对正常间期NRK细胞的表面张力、粘弹性进行了测定。结合形态学、细胞力学特性和肌球蛋白II马达做功,分析了NRK细胞胞质分裂过程中分裂沟收缩产生的力和应力,验证了肌球蛋白II在正常NRK细胞分裂沟收缩中的作用。2.研究了肌球蛋白II缺失对NRK细胞分裂的影响。分别于分裂中期和后期施加肌球蛋白II抑制剂,通过CCD对肌球蛋白II缺失的NRK细胞胞质分裂进行动态图像采集,并利用显微图像分析软件对细胞间桥形态学进行了定量分析,采用细胞免疫荧光技术检测了肌动蛋白的分布。研究发现:(1)肌球蛋白II缺失并不影响间期NRK细胞突出运动和肌动蛋白在分裂沟的组装和定位;(2)肌球蛋白II缺失可引起NRK细胞核分裂受阻或延迟,并使子细胞突出运动丧失极性。(3)对细胞间桥变细形态学进行量化发现:无论是正常组还是肌球蛋白II缺失组细胞,其间桥变细曲线均先表现出指数衰减,而后为线性下降。在Dx点(细胞间桥直径和长度相等)之前,肌球蛋白II缺失的NRK细胞间桥变细曲线明显不同于对照组细胞,细胞间桥直径迅速变细,曲线明显变陡;而Dx点之后各参数无明显差别。3.对细胞材料特性在肌球蛋白II缺失的NRK细胞胞质分裂中的作用进行了研究。采用已有的细胞间桥变细力学模型,结合NRK细胞间桥变细形态学参数及细胞力学特性,对正常、DMSO处理组和肌球蛋白II抑制组的细胞间桥变细曲线进行分析发现:细胞表面张力对肌球蛋白II缺失的NRK细胞间桥变细曲线影响很大。细胞间桥与子细胞之间的压力差有助于肌球蛋白II缺失的NRK细胞完成胞质分裂。在考虑变边界、质膜为超弹性以及胞浆为粘性不可压缩流体的基础上,建立了可以动态反映细胞间桥变形过程中形态学和力学参数时空分布的力学模型。4.采用细胞免疫荧光以及肌动蛋白抑制剂对细胞运动在肌球蛋白II缺失的NRK细胞胞质分裂中的作用进行了研究。结果发现:肌球蛋白II缺失的NRK细胞在胞质分裂早期两极的扇形板状突出中富集了大量肌动蛋白纤维。对肌球蛋白II缺失的NRK子细胞施加肌动蛋白抑制剂使胞质分裂明显受阻,细胞两极相对距离明显下降,子细胞运动参与了肌球蛋白II缺失的NRK细胞胞质分裂。由此得出:粘附生长的NRK细胞在肌球蛋白II缺失的情况下,整个细胞表面张力降低,胞质分裂所需的缢缩力明显减小,由肌动蛋白介导的子细胞极区突出运动产生方向相反的牵张力,不仅促进了子细胞的分离,而且使分裂沟的表面张力增大,增大的表面张力加大了细胞间桥与子细胞之间的压力差,胞浆从细胞间桥向两个子细胞流动加速。细胞间桥不断变细,使间桥与子细胞之间的压力差效应更加显著,从而促进了肌球蛋白II缺失NRK细胞的胞质分裂。更多还原

【Abstract】 Cytokinesis is a complicated life process involved biology and mechanics. Literature findings demonstrated a diversity of mechanisms by which animal cells carried out cytokinesis. Cytokinesis mode B was a cell cycle-coupled, adhesion-dependent cytokinesis that did not depend on myosin II. This cytokinesis mode existed universally in adherent eukaryotes and higher animal cells and might be a more primary cytokinesis mode than classic contractile ring-depended model. However, the mechanism of cytokinesis mode B was still not clear. Since not only adherent eukaryotes but also higher animal cells employed cytokinesis mode B, it must play an important role in cell division. Understanding the mechanism of cytokinesis mode B will not only provide insight into how the multiple modes of division worked in cooperative manner from a comprehensive view, but also provide theoretical evidence for revealing the mystery of cell proliferation and disease related cytokinesis failure (such as cancer).The previous studies of the mechanism of cytokinesis B were mainly focus on Dictyostelium cells. However, such investigation has not been done on the mammalian cells. In the present study, Micro-image dynamic collecting and analysis system, immunofluorescence method and cell mechanics techniques were used to explore the effects of myosin II absence on cytokinesis using NRK cells. Our findings provided the first direct evidences that cell material mechanical properties and cell motility orchestrated to dictate a well-controlled cytokinesis mode B of adherent mammalian cells. The main work and conclusions are as follows: 1. First, the cytokinesis process of normal NRK cells was observed and described in detail. The possible mechanism of blebbing behavior and the relation between the daughter cells adhesion pattern and the mother cell at interphase were discussed. Second, cell surface tension and vicroelastic properties of NRK cells at interphase were determined using micropipette aspiration technique and its relative theoretical model. The change of force and strrss in dividing furrow regions during cytokinesis were analyzed according to the morphology parameters,cell mechanical properties and myosin II motor work.2. The behavior of NRK cells absence of myosin II undergoing division was explored. The Micro-images of NRK cells under division were collected and analyzed after myosin II inhibitor was released at metaphase and anaphase respectively. The distribution of actin at anaphase and early cytokinesis were examined using immunofluorescence method. Our findings showed that:(1) Myosin II absence neither affected the lamellipodia motion of NRK cells at interphase nor assembly and recruitment of actin to the dividing furrow.(2) Inhibition of myosin II activity at metaphase resulted in failure of karyokinesis or delay into cytokinesis. The lamellipodia motion of daughter cells lost their polarity.(3) The results of intercellular bridge thinning morphology analysis showed that whether in the control or NRK cells absence of myosin II, the trajectories of intercellular bridge thinning behaved a combination of a linear decrease and an exponential decay, with the exponential decay preceding the linear decrease. The intercellular bridge trajectories of cells absence of myosin II was obvious differently from the controls with relative diameter decrease more quickly before Dx point, while the trajectories were identical after Dx point.3. The role of cell material properties on the cytokinesis absence of myosin II was in investigated using the existed intercellular bridge thinning mechanics model. The intercellular bridge thinning trajectories of the control and NRK cells absence of myosin II were analyzed respectively. Intercellular bridge thinning morphology parameters and cell mechanical properties from experiments were employed during analysis. The results showed that cell surface tension contributed greatly to the intercellular bridge thinning trajectories. It indicated that internal pressure difference between the intercellular and the two daughters could help NRK cells absence of myosin II to complete cytokinesis.A new intercellular bridge thinning dynamics model was established on the base of fluid mechanics principle. This model can allow the parameters of morphology or mechanics to vary temporally or spatially as intercellular bridge-thinning proceeded.4. The role of cell motility in myosin II absent cytokinesis was investigated using cyto-immunofluorescence method and actin flament polymerization inhibitor (cytohalasin D). The results showed that a mass of actin filament recruited to the leading lamellipodia of daughter cells from the cytoplasm in cells treated with myosin II inhibitor. The process of cytokinesis absence of myosin II was obviously held back by releasing cytohalasin D. The relative pole to pole diatance decreased in contrast with other groups. Our results provided the first evidence that cell motility was involved the cytokinesis absence of myosin II.Our results indicated that the force required in cytokinesis might obviously reduce because the whole cell surface tension decreased during cytokinesis of adherent NRK cells absence of myosin II. Traction force generated from daughter cells motility not only facilitated two daughter cells separate, but also led to the surface tension increase in intercellular bridge, which resulted in the internal pressure difference increase between the intercellular bridge and two daughter cells, the latter accelerated the cytoplasm flow from the intercellular bridge into two daughter cells. While the intercellular bridge thinning further facilitated internal pressure difference effect to help the cytokinesis of adherent NRK cells absence of myosin II.更多还原