大鼠结肠cajal细胞库容性Ca2+内流检测临床价值.docVIP

大鼠结肠cajal细胞库容性Ca2+内流检测临床价值 　【摘要】 目的：通过检测大鼠结肠cajal细胞中库容性Ca2+内流(capacitative Ca2+ entry，CCE)，探讨导致大鼠结肠cajal细胞内Ca2+稳态失衡的信号途径，为治疗相关疾病提供新的思路。方法：荧光探针Fura-2/AM标记细胞内游离Ca2+后，用荧光分光光度计检测乙二醇-双(2-氨基乙基醚)四乙酸(EGTA)耗竭胞内钙库后对分离的大鼠结肠cajal细胞Ca2+浓度([Ca2+])的影响。结果：在无Ca2+缓冲液中加入EGTA(10 mmol/L)，细胞内[Ca2+]由静息时的(69.37±3.02) nmol/L升高至(272.52±5.21 ) nmol/L;继之，向细胞外液中引入1.5 mmol/L和3.0 mmol/L的氯化钙溶液，导致细胞内Ca2+进一步升高为(466.43±4.63) nmol/L和(977.43±3.27) nmol/L;且此升高效应对维拉帕米(5 μmol/L)不敏感，但可被2APB(20～100 μmol/L)抑制。结论：酶解法并结合密度离心法分离的大鼠结肠cajal细胞上存在胞内钙库耗竭激活的Ca2+内流。这对于研究钙通道活性改变、预防和控制因胃肠动力紊乱所致的消化系统疾病具有重要意义。 【关键词】 库容性Ca2+内流·结肠cajal细胞·大鼠，Wistar 　　【ABSTRACT】 Objective: To investigate the changes of capacitative Ca2+ entry(CCE) in colon cajal cells and its mechanism. Methods: Intracellular Ca2+ changes induced by EGTA -activated Ca2+ influx were measured in colon cajal cells which was freshly isolated from colon of Wistar rats with acute enzymatic dissociation and ficoll density centrifugation method.with the fluorescent indicator Fura-2/AM. Results: In the absence of external Ca2+, the concentration of calcium-chelating agent EGTA elevated Ca2+ from (69.37±3.02)nmol/L to (272.52±5.21)nmol/L,a subsequent reintroduction of Ca2+ into the extracellular solution resulted in further Ca2+ elevating to 466.43±4.63)nmol/L and(977.43±3.27)nmol/L respectively, and the response was blocked by 2APB, but it was insensitive to L-type voltage calcium channels blocker verapamil. Conclusion: Ca2+ influx activated by store depletion are present in colon cajal cells, it has the value for further study on gastric motility disorder diseases. 　　【KEY WORDS】 Capacitative Ca2+ entry· Colon cajal· Wistar rats 　　钙离子(Ca2+)是细胞间连接和信号传递的重要成分，影响细胞重要的生理功能。钙离子浓度([Ca2+])变化对胃肠平滑肌的收缩与舒张起着决定性作用。因此，Ca2+与胃肠动力紊乱疾病的关系引起人们越来越多的关注，成为近年研究的靶点。本研究通过检测大鼠结肠cajal细胞Ca2+内流，探讨导致大鼠结肠cajal细胞内Ca2+稳态失衡的信号途径，为治疗因胃肠动力紊乱所致的消化系统疾病提供新的思路和实验依据。 　　1　材料与方法 　　1.1　溶液与配制　胶原酶Ⅱ、4-羟乙基哌嗪乙磺酸(HEPES)、乙二醇二乙醚二胺四乙酸(EGTA)、牛磺酸、二甲基亚砜(DMSO)、Fura-2/AM、Triton X-100、锥虫蓝、维拉帕米以及牛血清白蛋白均购于Sigma公司。 　　HEPES-Rin