【摘要】 磷脂酰肌醇-3激酶(PI3K)是磷脂酰肌醇代谢过程中一种重要的酶,通过其代谢产物参与了对多种细胞生理活动的调节,如囊泡运输、细胞骨架重组、细胞存活、吞噬作用、细胞凋亡等.为研究其对细胞分泌功能的作用,使用磷脂酰肌醇-3激酶家族的特异性抑制剂渥曼青霉素(wortmannin)阻断磷脂酰肌醇-3激酶的活性,以EGFP-2xFYVE融合蛋白与磷脂酰肌醇-3-磷酸(PtdIns-3-P)的结合为指征,使用荧光显微成像技术检测渥曼青霉素对磷脂酰肌醇-3激酶的抑制作用,采用膜片钳膜电容测量方法及光解钙离子释放技术检测渥曼青霉素对PC12细胞分泌功能的影响.实验结果表明,wortmannin阻断了磷脂酰肌醇-3激酶的活性,抑制了磷脂酰肌醇-3-磷酸(PtdIns-3-P)的产生,并使FYVE与PtdIns-3-P解离,但渥曼青霉素处理之前和处理30min后的PC12细胞分泌反应的幅度、动力学特性和分泌的钙依赖性均无显著差异,表明磷脂酰肌醇-3激酶对PC12细胞的分泌无显著的直接影响.更多还原

【Abstract】 Phosphoinositide3-kinase (PI3K) is involved in regulation of many kinds of physiological processes in cells, such as vesicle transportation, cytoskeleton reorganization, cell survival, phagocytosis, apoptosis, and so on. It is an important regulator of vesicle edocytosis. According to preview reports, PI3K seems to be one of regulators of cell secretion. To inspect this, the effect of PI3K on PC12 cell secretion was checked by using its specific inhibitor wortmannin. Wortmannin inhibited PI3K activity indicated by loss of EGFP-2xFYVE fusion protein binding to the PtdIns-3-P which is localized in early endosome, and did not change PC12 cell response to flash including its kinetics and calcium dependence. The result demonstrated that PI3K has no effect on PC12 cell secretion itself, but does not exclude the possibility that it enhances cell to response to repetitive strong stimuli by its accelerating effect on vesicle endocytosis which speeds up the refilling of releasable vesicle.更多还原