【作者】 刘媛媛；

【导师】 徐涛；

【作者基本信息】 华中科技大学 ， 生物物理学， 2005， 硕士

【摘要】 细胞分泌活动是神经信号传导和内分泌激素释放等细胞生理功能的基础,是在时空上严格协调的胞吐与胞吞的循环过程,需要包括细胞信号整合、膜动力学改变、细胞骨架重组等一系列复杂的分子机制参与调控。过去的25 年,参与囊泡循环调控的许多蛋白质被发现和确认了,但是脂类物质在其中的作用仍不清楚,尤其是磷酸肌醇类物质在囊泡的释放以及囊泡转运过程中的作用,目前受到极大的关注。本文选用较常用的研究分泌和膜转运的模型细胞PC12 和INS-1 细胞,利用膜片钳技术、膜电容检测技术、荧光测钙技术、钙离子光解释放技术和全内反射荧光成像技术等现代生物物理方法,研究了磷脂酰肌醇-3 激酶和磷脂酰肌醇-3-磷酸对细胞分泌的作用。实验结果显示,抑制PI3K 的活性和阻断PtdIns-3-P 的功能,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变化,表明PI3K 和PtdIns-3-P对PC12 细胞的分泌无显著的直接影响。在INS-1 细胞上的实验结果也验证了这一点,表明PI3K 和PtdIns-3-P 对细胞的分泌功能没有直接的调控作用,但是不排除它们通过促进囊泡胞吞和循环过程而增强细胞对高强度重复刺激的反应和参与突触可塑性的调节。在INS-1 细胞上发现,抑制PI3K 的活性和阻断PtdIns-3-P 的功能都能抑制囊泡快相胞吞的幅度,却不影响囊泡快相胞吞以及慢相胞吞的钙依赖性和慢相胞吞的幅度,从而推断PI3K 和PtdIns-3-P 参与囊泡胞吞快相成分的调控,但是其具体分子机制仍有待进一步研究。更多还原

【Abstract】 Secretion forms the basis for signal transmission between neurons and hormone release from endocrine cells. It must undergo repeated rounds of exocytosis、endocytosis、refilling and mobilization, a phenomenon called recycling. The temporal and spatial coordination of exo–endocytic recycling requires a complex molecular machinery that integrates cell signaling, dynamic changes within membranes, and cytoskeletal rearrangements. Over the past 25 years, many proteins of this machinery have been identified and characterized. However, less is known about the function of lipids. Now the role of lipids, especially PIs, in the physiology of secretion and vesicle traffic is focused. In this study, we applied advanced biophysical techniques, such as patch clamp、membrane capacitance measurement、microfluoremetric technique、flash photolysis、TIRFM、and so on. Inspection on the role of phospoinositide-3 kinase and phospoinositide -3-phosphate in secretion was carried on PC12 and INS-1 cells which represent widely used models for secretion and membrane traffic. Wortmannin, a specific inhibitor of PI3Ks, and EGFP-2xFYVE fusion protein which bind specifically with PtdIns-3-P were used to block the activity of PI3K and PtdIns-3-P separately. The results showed that the block did not change PC12 cell response to flash including its kinetics and calcium dependence. So, it was demonstrated that PI3K and PtdIns-3-P had no effect on PC12 cell secretion itself, but did not exclude the possibility that they enhance cell to response to repetitive strong stimuli by their accelerating effect on vesicle endocytosis which speed the refilling of releasable vesicle. In INS-1 cells, it was found that the block inhibited fast endocytosis, but did not change the amplitude of slow endocytosis. At the same time, it also did not change calcium dependence of fast and slow endocytosis. From this, we draw a conclusion that PI3K and PtdIns-3-P may be involved in the regulation of endocytosis, but more experiments are required for precise elucidation.更多还原