【作者】 谢丽；

【导师】 徐涛；

【作者基本信息】 华中科技大学 ， 生物物理， 2005， 博士

【摘要】 q细胞的分泌活动是神经元释放神经递质、内分泌细胞分泌激素和细胞膜上功能蛋白的镶入和内吞等生命活动的基础,对于生物体正常机能的维持是必需的。如在胰腺β细胞中,胰岛素分泌量的绝对和相对不足会导致糖尿病的发生。细胞内钙离子(Ca²⁺)作为一种重要的第二信使,在囊泡分泌过程中起着非常重要的作用。因此研究胞内Ca²⁺信号调控细胞分泌的分子机制具有非常重要的意义。本课题运用膜电容检测、荧光测钙、Ca²⁺光解释放、FM1-43 光学检测以及碳纤电极电化学检测等技术,以大鼠胰腺?细胞为研究对象,对胞外ATP 引起的胰岛素分泌机制和胞内钙信号来源进行了系统全面的研究,证实了胞外ATP 通过引起囊泡钙库而不是传统认为的内质网钙库释放而触发分泌。另外,还发现了一种从猪肠道中纯化的葡萄糖依赖的促胰岛素肽（GIP）的异构体形式-GIP1-39 也能较强的促进胰岛素分泌和胞内Ca²⁺浓度的升高。本课题的主要研究结果和结论如下: （1）在大鼠胰腺β细胞上,胞外ATP 通过引起胞内钙库释放而触发较强的分泌活动。通过胞内透析钙离子螯合剂EGTA 和BAPTA,发现ATP 引起胞内钙库释放形成的局部Ca²⁺浓度靠近囊泡释放位点,与分泌位点相偶联。ATP 通过引起胞内钙库释放（主要是内质网钙库）而使胞内Ca²⁺浓度瞬时很大的升高,但用Thapsigargin排空内质网钙库后,并没有完全阻断ATP 引起的胞内Ca²⁺升高,并且对ATP 引起的胞吐作用几乎没有影响。说明ATP 同时引起β细胞内TG 不敏感的钙库释放,并且这类钙库对ATP 引起的分泌起主要调控作用。为了验证是否为囊泡钙库的作用,我们用能够驱散酸性钙库中钙离子的质子载体——Ionmycin 和Monensin,发现Ionmycin 和Monensin 同时处理细胞后,抑制了ATP 引起的分泌和胞内Ca²⁺浓度的增加。用IP3受体的阻断剂2-APB 和Heparin 分别处理细胞发现,抑制了ATP 引起的分泌。用Ryanodine 受体的抑制剂Dantrolene 处理细胞后,却对ATP 引起的分泌没有影响。说明是IP3 敏感的钙库在起作用。另外,利用钙离子光解释放技术实验发现ATP 并没有直接作用于分泌机制。所有结果揭示了,在大鼠胰腺β细胞中囊泡钙库对ATP 引起更多还原

【Abstract】 The activity of secretion is the basis for transmitters release from neurons, hormones secretion from endocrine cells and setting or endocytosis of functional membrane proteins, which are absolutely necessary in maintaining normal physiological function of organisms. For example, the absolute or relative deficiency of insulin secretion from pancreatic ? cells will result in diabetes. Intracellular Ca²⁺ acts as a second messenger that palys important role in regulating vesicles secretion. So, it is very meaningful for study the molecular mechanism on which intracellular Ca²⁺ signal regulates vesicles secretion. In this study, using membrane capacitance, microfluorometric measurement of intracellular free Ca²⁺ concentration, flash photolysis of caged Ca²⁺, FM1-43 staining and carbon fiber technique, we studied the exact mechanism of insulin secretion and the source of intracellular Ca²⁺ induced by extracellular ATP in rat pancreatic ? cells. Our results suggest extracellular ATP promptly triggers vigorous exocytosis under resting condition via localized Ca²⁺ release from secretory granules rather than ER. In addition, GIP1-39, a novel chain length form of GIP （gastric inhibitory polypeptide or glucose-dependent insulinotropic polypeptide）, has been purified from porcine intestine and demonstrated that is capable of stimulating insulin secretion and elevating of intracellular Ca²⁺ concentration from rat pancreatic βcells. Main results of the study are as follows: （1） Extracelluar ATP could trigger vigorous exocytosis under resting condition via intracellular Ca²⁺ store release in rat pancreatic ? cells. We performed intracellular dialysis experiments with pipette solutions containing EGTA or BAPTA to chelate intracellular Ca²⁺ and concluded that the majority of ATP-induced exocytosis was due to localized Ca²⁺ release near the vesicle fusion sites. To address the question of whether endoplasmic reticulum （ER） Ca²⁺ pool plays a role in the ATP stimulated exocytosis, single ? cells were pre-incubated with thapsigargin and found SERCA inhibition hardly reduced ATP-induced capacitance increase in Ca2+-free bath solution. Then we further studied whether the granule Ca2+ stores contribute to the TG-insensitive exocytosis and [Ca2+]i elevation induced by ATP using ionomycin and monensin which have been shown to dissipate Ca2+ gradient from all acidic organelles and found pretreatment with ionomycin and monensin significantly reduced ATP-induced Cm increment and [Ca2+]i elevation. Furthermore, we pre-treated cells with IP3 receptor blockers, 2-APB and heparin, as well as ryanodine receptor blocker, dantrolene, respectively, and found IP3 coupled extracellular ATP stimulation to exocytosis. In addition, using flash photolysis of caged Ca2+, and found ATP application didn’t act directly on the secretory machinery. All these results suggest that ATP-induced exocytosis is mainly stimulated by localized Ca2+ release from secretory granules in rat pancreatic ? cells. （2） GIP1-39, a novel chain length form of GIP, has been purified recently from porcine intestine. We have characterized that GIP1-39 is an insulinotropic peptide, and demonstrated that GIP1-39 is more potent in stimulating insulin secretion from rat pancreatic islets than GIP1-42. Therefore, we have further investigated some aspects on the mechanism behind the insulinotropic effect of GIP1-39 in single rat pancreatic ? cells. GIP1-39 was able to significantly increase intracellular Ca2+ concentration （[Ca2+]i ）, and capable of enhancing exocytosis assessed by membrane capacitance measurement. The novel GIP1-39 might be a more optimal molecular pattern in stimulating insulin secretion and deserves to be further investigated biologically and clinically.更多还原