【作者】 宋鸣子；

【导师】 顾洛；

【作者基本信息】 南京医科大学 ， 生理学， 2007， 硕士

【摘要】 染料木黄酮(genistein)是一种从大豆中提取的天然异黄酮类物质，它作为蛋白酪氨酸激酶的抑制剂，能够促进肿瘤细胞的凋亡，抑制其增殖、浸润和转移，但其确切的作用机制尚不清楚。众所周知，肿瘤细胞与细胞外基质的黏附是其发生浸润和转移的始动因素，肿瘤细胞黏附能力的改变势必会影响其浸润和转移。具有维持细胞形态，调节细胞运动等功能的肌动蛋白细胞骨架，在肿瘤的浸润和转移过程中发挥着重要的作用。细胞内存在数十种肌动蛋白结合蛋白(actin binding protein)通过与肌动蛋白的结合与解离，调节其聚合／解聚过程，改变F-肌动蛋白在细胞内的含量和分布，影响肌动蛋白细胞骨架的结构和功能，最终实现对众多细胞功能的调控。热休克蛋白27(heat shock protein 27，Hsp27)作为一种重要的肌动蛋白结合蛋白，在细胞内可被p38激酶信号途径磷酸化，进而参与调节肌动蛋白聚合／解聚过程，使肌动蛋白细胞骨架发生重组。已有研究表明，在体外染料木黄酮具有抑制p38激酶活性的作用，因此，我们推测，染料木黄酮可能通过抑制细胞内p38激酶的活性，降低Hsp27磷酸化水平，从而诱导肿瘤细胞肌动蛋白细胞骨架重组，影响肿瘤细胞的黏附能力。为证实以上推测，本研究选用具有高转移潜能的人乳腺癌细胞株(MDA-MB-435细胞)，设计并进行了以下两部分实验：(1)观察染料木黄酮对肌动蛋白细胞骨架重组和细胞黏附能力的影响，进而阐明两者之间的关系；(2)通过研究p38激酶和Hsp27是否介导染料木黄酮诱导的肌动蛋白细胞骨架的重组，从而初步探讨染料木黄酮所致肌动蛋白细胞骨架重组的作用机制。第一部分不同浓度染料木黄酮(12.5、25、50和100μmol／L)体外孵育MDA-MB-435细胞24h后，分别用荧光染色法、流式细胞术和免疫印迹法观察肌动蛋白细胞骨架的重组情况；噻唑蓝比色法(MTT)检测细胞对人工重组基底膜(Matrigel)黏附能力的改变。结果发现，染料木黄酮使MDA-MB-435细胞内F-肌动蛋白排列和分布明显改变，并且其含量明显降低；同时细胞与Matrigel的黏附率与对照组相比明显降低，其效应具有剂量依赖性。本研究结果表明，染料木黄酮可促进MDA-MB-435细胞内F-肌动蛋白解聚，诱导肌动蛋白细胞骨架重组是其体外降低细胞黏附能力的关键环节。第二部分不同浓度染料木黄酮(12.5、25、50和100μmol／L)体外孵育MDA-MB-435细胞24h后，分别用流式细胞术观察细胞的凋亡情况；用免疫印迹法研究细胞内ERK1／2和p38激酶的表达及活性的改变，同时分析Hsp27的表达、磷酸化水平及其在细胞内定位的改变。结果发现，染料木黄酮处理24h未能诱导细胞发生明显凋亡，细胞内ERK1／2活性在对照组和各实验组之间无显著性差异。p38激酶和Hsp27的蛋白总量无明显改变，但二者磷酸化水平随着染料木黄酮处理浓度的增加逐渐降低；与此相应的是Hsp27在胞浆中含量明显减少，在细胞骨架中含量明显增多。本研究结果表明，染料木黄酮引起F-肌动蛋白解聚，进而降低细胞的黏附能力并非由于其改变了细胞内ERK1／2的活性，诱导细胞发生凋亡所致；p38激酶途径以及Hsp27磷酸化／脱磷酸化过程参与介导染料木黄酮所致MDA-MB-435细胞肌动蛋白细胞骨架的重组。通过以上研究，我们得出以下结论：1．染料木黄酮所致MDA-MB-435细胞内F-肌动蛋白解聚，肌动蛋白细胞骨架重组是其体外降低细胞黏附能力的关键环节之一。2．p38激酶途径以及Hsp27磷酸化／脱磷酸化过程参与介导染料木黄酮所致MDA-MB-435肌动蛋白细胞骨架的重组。更多还原

【Abstract】 Genistein is an isoflavone present in soybeans, and is a verypotent inhibitor of the protein-tyrosine kinase (PTK) activity of theEGF receptor in vitro. The biological effects of genistein are relatedto cell growth, apoptosis, invasion and metastasis of cancer cells.But the mechanism involved in the inhibition of invasion andmetastasis in cancer cells was unclear. It is well known that complexcommunications through the cell-ECM (extracellular matrix)adhesion initiate the invasion and migration of cancer cell. Actincytoskeleton plays a key role in cell shape and movement. And thereorganization of actin cytoskeleton is involved in the invasion andmigration of cancer cells. There are a number of actin bindingproteins regulating the state and distribution of polymerization offilaments. Hsp27 (heat shock protein 27) is an actin binding protein,in which regulate actin dynamics via p38 mitogen-activated proteinkinase signaling pathway. Recently it is reported that genisteincould inhibit p38 MAPK activation. We hypothesized that genisteinhave a direct effect on the inhibition of the p38 MAPK activation and subsequently inhibit the phosphorylation of Hsp27, leading toloss of cell adhesive ability in human breast cancer cells through thereorganization of actin cytoskeleton. To address this issue, thefollowing studies were designed and performed.In the first study, the MDA-MB-435 cells were treated withdifferent concentrations of genistein (12.5, 25, 50 and 100μmol/L)for 24h. The reorganization of actin cytoskeleton was detected byFITC-phallodin staining and the F-actin contents measured by flowcytometer and western blotting. The adhesive ability of cells toartificial basement membrane (Matrigel) was measured by MTTassay. Compared to the control cells, genistein significantly changedthe redistribution of the F-actin, reduced F-actin contents andreduced the adhesive rate to Matrigel. There results suggest that theless adhesive ability of MDA-MB-435 cells to Matrigel induced bygenistein was related to the reorganization of actin cytoskeleton.In the second study, the MDA-MB-435 cells were treated withdifferent concentrations of genistein (12.5, 25, 50 and 100μmol/L)for 24h. The cell apoptosis was analyzed by flow cytometer. Theexpression of ERK1/2, p38 and Hsp27, phosphorylation of ERK1/2,p38 and Hsp27 was evaluated by western blot analysis. Theexpression of Hsp27 in cytosolic fraction and cytoskeletal fractionwas also detected by by western blotting. After treated by genisteinfor 24h, the cell apoptosis and ERK1/2 activation had no changes inall groups. However, the phosphorylation of p38 and Hsp27 wasattenuated compared to the control cells. Simultaneously Hsp27translocated from the cytosolic fraction to the cytoskeletal fraction. These findings suggest that the reorganization of actin cytoskeletoninduced by genistein was mediated by translocation of Hsp27 incells via p38 signaling pathway.Together with our studies, we conclude that:1. The depolymerization of F-actin induced by genistein maylead to the less adhesive ability of MDA-MB-435 cells to Matrigel.2. The reorganization of actin cytoskeleton induced bygenistein was mediated by translocation of Hsp27 in cells via p38signaling pathway.更多还原