【作者】 罗迪贤；

【导师】 廖端芳； 何淑雅；

【作者基本信息】 南华大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 目的：探讨压应力对体外培养的血管平滑肌细胞(VSMCs)增殖的影响及其机制。 方法：原代培养大鼠主动脉VSMCs，用自行研制的压力可调细胞培养箱加压处理不同压力(0mmHg、120mmHg、180mmHg和240mmHg)，得出促增殖最明显的压力120mmHg后，用120mmHg处理不同时间(0、2、4、8、12和24小时)。用MTT法和细胞记数法观察压应力对VSMCs活性和增殖的影响；用细胞流式术观测不同压力不同时间VSMCs细胞周期的分布；用免疫印迹法检测Caveolin-1的表达与ERK1／2磷酸化的情况；用免疫荧光检测p-ERK1／2的核转位情况。用ERK磷酸化抑制剂PD98059和微丝骨架蛋白破坏剂细胞松弛素D干预压应力对VSMCs增殖和ERK1／2磷酸化的影响。 结果：压应力可诱导VSMCs的增殖，促进VSMCs从G0／G1期进入S／G2／M期，下调VSMCs中Caveolin-1的表达，上调ERK的磷酸化，其中120mmHg时VSMCs增殖与ERK1／2磷酸化最明显，而随着压应力增高，增殖速度下降，到240mmHg时则没有促增殖效应。时效上，4小时后VSMCs增殖进入平台期，Caveolin-1表达达到谷值，ERK1／2磷酸化达到峰值。免疫荧光发现压应力能明显促进p-ERK1／2核转位。PD98059可显著抑制ERK1／2磷酸化和VSMCs增殖。细胞松弛素D可明显抑制ERK／1／2的磷酸化。更多还原

【Abstract】 Backgroud: When atherosclerosis and vessel intervention occurs, vascular endothelial cells are injured or vascular smooth muscle cells(VSMCs) migrate under endoderm, so that mechanical forces can directly affect VSMCs. Mechanical forces include stress, circular stress and static pressure. Mechanical stress induces VSMCs proliferation by activating extracellular signal-regulated kinase(ERK). However, the effect of static pressure on VSMCs proliferation is unclear.Objective: To investigate the effect of static pressure on VSMCs proliferation.Methods: VSMCs from rat aorta were respectively treated with different pressures of 0mmHg, 120 mmHg, 180 mmHg, 240 mmHg in a self-manufactured pressure-adjustable cell incubator for 24hrs or were treated with 120 mmHg of static pressure for different time(0, 2, 4, 8,12 and 24 hours). The proliferation of VSMCs was evaluated by means of cell counting and MTT assay, and the distribution of VSMCs’ cell cycles was detected by Flow Cytometry(FCM). and then the protein amount of Caveolin-1 and phosphor-ERK (p-ERK) was analyzed by Weston Blot.Results: VSMCs proliferation and ERK activation were significantly increased by staticpressures in pressure-dependent manner, with the peak in 120 mmHg. When the static pressure was 120 mmHg, the peak was at 4 h. Interestingly, static pressure obviously inhibited caveolin-1 expression, which appeared a negative correlation with static pressure stimulated ERK activation. PD98059, an inhibitor of ERK kinase, and Cytochalasin D(Cyt D) that can destroy cell microfilament skeleton, both prohibited static pressure-induced VSMCs proliferation.Conclusion: Static pressure stimulates smooth muscle cell proliferation possibly via the caveolin-1/ERK pathway.更多还原