【作者】 陈海蓉；

【导师】 周建伟；

【作者基本信息】 南京医科大学 ， 卫生毒理学， 2004， 硕士

【摘要】 人类疾病的发生往往由遗传因素和环境因素相互作用决定的。机体暴露于不同环境因素(应激因子)时，可在分子、细胞和整体等多个水平和层次作出相应的应答反应，启动复杂的细胞内和细胞间信号转导通路以及神经-体液-内分泌等调节机制，通过改变一系列基因的表达水平以及神经递质和激素等释放机制，最终使细胞、组织器官乃至整个机体在各种生理、生化功能和协调功能等方面获得新的平衡，从而在一定限度内维持机体正常的生命活动。探讨基因与环境的相互作用及其规律，有助于进一步理解和认识环境因素导致疾病的分子机制，从而为从分子水平上构建环境因素致机体损伤的预警体系提供科学依据。1998年，周建伟等从培养的原代人的气管和支气管上皮细胞中，利用差异显示高通量筛选技术，分离克隆了一个在生物进化上保守、参与调节细胞分化和凋亡的新基因JWA，进一步研究发现，该基因编码一种新的细胞骨架相关蛋白。本课题组随后用多种分子和细胞生物学实验手段证实JWA蛋白与微管蛋白β亚单位紧密结合，在细胞内与其分布基本平行，并在对环境物理化学因素的应答(聚合或解聚)中表现出与微管(β亚单位)同步的动力学变化，并可能参与细胞有丝分裂过程。JWA基因受多种分化诱导剂如维甲酸(RA)、13顺-维甲酸(13-cRA)和南京医科大学硕士学位论文佛波醋(T队)等调控，参与急慢性白血病细胞分化和凋亡，以及细胞对环境热应激、氧化应激应答的信号转导通路。除此以外，国外学者和本课题组的研究还发现，J认叭基因参与调节细胞内氛基酸的平衡，尤其对谷氛酸(兴奋性氨基酸)和牛磺酸(抑制性氨基酸)具有特别显著的调节作用。对调控机制的初步研究结果表明，J认A的作用可能与细胞内PKC相关的信号通路以及钙稳态的改变有关。本课题是在课题组对J认叭基因研究积累的基础上，进一步探讨J认叭基因与微管蛋白a一亚单位(或称。一微管蛋白)的结构和功能关系。研究内容分为二个部分:一，J场叭蛋白与。一微管蛋白的细胞内共定位;二，JWA蛋白参与调节细胞有丝分裂过程及微管动力学的机制。 第一部分JM战蛋白与份微管蛋白的细胞内共定位 本课题组以往的研究工作已证实J认叭蛋白和p一微管蛋白可被免疫共沉淀、两者在细胞内分布平行、对作用于微管的药物(秋水仙素和诺考达吐)的反应和及所引起的结构变化也基本一致。为进一步探讨JW人蛋白是否和微管的另一亚单位a一微管蛋白也有类似的结构和功能关系，本研究分别用抗J认叭多克隆抗体和杭。一微管蛋白单克隆抗体对经微管解聚剂(2 x 10一smol·L一’诺考达吐)处理及对照的PC12细胞裂解液进行免疫共沉淀，然后用协厄Stem blot检测各沉淀物中是否含有J从伙蛋白。结果表明在PC12细胞中，无论微管处于解聚或聚合状态，。一微管蛋白与J认叭蛋白都是结合在一起的。然而，对照的无关抗体(Caspase一3抗体)的免疫共沉淀物中则检测不出JWA蛋白。 为了解J认伙蛋白在PC12细胞内的定位，探讨不同影响因素下J认人蛋白和份微管蛋白相互关系及其变化规律，用免疫荧光显微镜对JWA蛋白在PC12细胞内的分布进行了观察。结果显示:冰上处理PC12细胞不同时间，JWA蛋白和。一微管蛋白均发生不同程度的解聚;冰上处理PC12细胞2h，37℃恢复不同时间后，解聚的J认人蛋白和a一微管蛋白又可重新聚合起来，表明在微管聚合和南京医科大学硕士学位论文解聚的过程中，J认叭蛋白和a一微管蛋白在细胞内呈现同步的动力学过程且分布基本平行。 为进一步探讨J认叭蛋白高表达或低表达后对a一微管蛋白在细胞内分布的影响及其与。一微管蛋白的相互关系的变化，选择较高转染效率的HEK293细胞转染JWA表达载体，冰上处理2h后，对J认叭蛋白和a一微管蛋白进行双重荧光标记观察，可见二者均发生解聚，在此过程中两种蛋白在细胞内分布的变化几乎是一致的。用5又10一， mol.L一，J认叭反义寡核普酸处理PC12细胞48h，并用1 xlo一smol.L一’秋水仙素处理不同时间后，发现J认伙蛋白表达被下调，与对照相比，J认人蛋白和a一微管蛋白解聚速度加快。可见无论J认叭蛋白高表达或低表达，其在细胞内的分布与a一微管蛋白基本一致。这些结果提示J场叭蛋白不仅能与p一微管蛋白紧密结合、而且也与a一微管蛋白结合;J认叭不仅与这两种微管蛋白亚单位有十分相似的功能，还对微管的动力学稳定性有一定的调节作用。第二部分J从叭蛋白参与调节细胞有丝分裂过程及微管动力学的 机制 为深入了解J认伙蛋白在微管动力学变化中的作用或调节机制，本研究进一步探讨了J认叭蛋白在各细胞周期中的变化。用0.5%，J、牛血清处理36h，Zxlo一6mol·L一’舟基脉处理16h，lxlo一，mol·L一’诺考达吐处理2h使PC12细胞分别停滞于G0一Gl期、S期和GZ一M期，免疫荧光显微镜结果发现处于细胞周期不同阶段的细胞其J认A蛋白和a一微管蛋白分布几乎完全重叠。 已知J认叭蛋白在对环境物理化学因素的应答(聚合或解聚)中表现出与微管(p亚单位)同步的动力学变化，并可能参与细胞有丝分裂过程。进一步观察了J认试蛋白的细胞内分布?更多还原

【Abstract】 The most human diseases are the result of interactions between environmental factors and individial genetic susceptibility, but the mechanisms of the interactions are far to be understood. An increasing number of environmental responsive genes are cloned and found to be associated with human diseases. Some of environmental responsive genes are found involved in multi-signal transduction pathways and with a cross-talk manner to against the environmental stresses and keep the cell and body in homeostasis. JWA, a novel retinoic acid inducible and cytoskeleton associated gene, was primary cloned by Zhou et al (AF070523, 1998). The further evidences have shown in this research group that JWA seems a multi-functional gene. In one hand, it regulates cellular differentiations and apoptosis in multi tumor cell lines andprimary leukemia cells and associated with PKC signal pathway. In structurally, JWA protein binds with beta-tubulin and the both showed parallel distributions and could changed by treatment of physical and chemical microtubule targeting agents. In another hand, we found that JWA effectively involves in the regulation of intracellular amino acids transportation in PC 12 cells and this role might be associated with intracellular calcium signals. In this study, we focused on the relationships between the structure and function of JWA binds to alpha-tubulin. Firstly, we examined the dynamic changes in morphology of JWA and microtubule under disassembling agents treatment in PC 12 cells. Secondary, we designed series of models to investigate if JWA is also binds with alpha-tubulin and involves in cell cycle, especially in cell mitosis; and probe to observe the cellular localization of JWA in the PC 12 cells treated with environmental chemical podophyllotoxin.1. The relationships between JWA and a-tubulin in PC12 cellsWe previously confirmed that JWA protein and B-tubulin showed simultaneously and co-existed distribution patterns with treatment of antimitotic chemical colchicines. To understand the intracellular binding of JWA protein and a-tubulin by immunoprecipitation, the results showed that JWA protein was identified by either anti-JWA antibody or anti-a-tubulin antibody, with or without antimitotic chemical Nocodazole. JWA protein was unable detected by a non-specific IgG (Caspase-3 ip). These results suggested that JWA protein and a-tubulin are naturally intracellular binding each other and with a parallel distribution.Pretreatment of PC 12 cells on ice 2 hours (0C) to deploymerize the both proteins, and then put the treated cells back into 37C culture condition to recover them to original distribution patterns. The both proteins are still showed simultaneously and co-existed distributionpatterns during the processes. These results suggested that JWA protein and a-tubulin are polymerized and depolymerized simultaneously and with almost completely co-localization patterns. To clarify the influence of increased and decreased expression of JWA protein on the relationships between a-tubulin and JWA protein, we firstly successfully established JWA expression vector and was transfected HEK293 cells and then exposed on ice treatment for 2h. The both proteins showed depolymerized and co-existed distribution patterns. Secendly, after antisense-JWA oligonucleotide (5 X 10-5 mol.L-1) treatment for 48h and with colchicines (1 X 10"5 mol.L-1) treatment at different time periods, the expression of JWA protein was down-regulated; the depolymerization rate of both JWA protein and a-tubulin was increased when compared with the control . These results suggested that JWA protein stably bound to a-tubulin no matter it was at a higher or lower expressing level; however, the lower expression of JWA protein might speed up the depolymerizing rate of a-tubulin. Therefore, JWA might be an effective regulator of the a-tubulin polymerization.2. JWA involves in cell mitosis and regulates microtubularstabilityIt is likely that JWA protein is also associated with cell cycle. We treated PC12 cells with 0.5% bovine serum for更多还原