钠钙交换体亚型2在大鼠逼尿肌细胞中的表达与功能

目的 检测大鼠逼尿肌细胞是否表达钠钙交换体亚型2(NCX2),并测试其功能.方法 10只大鼠(7只雌性,3只雄性)为实验对象,取膀胱组织为实验组,大脑组织为对照组.首先利用急性酶分离技术,制备和培养大鼠逼尿肌细胞,再采用反转录-聚合酶链式反应和免疫印迹技术,检测大鼠膀胱组织中是否表达NCX2基因及蛋白;免疫荧光技术进一步明确NCX2与逼尿肌细胞的关系;膜片钳技术测试逼尿肌细胞是否存在NCX2电流.结果 首先制备了光镜下以梭形为主,免疫组化染色α-actin呈阳性的逼尿肌细胞;利用逆转录-聚合酶链式反应,在大鼠大脑组织、膀胱组织中分别发现NCX2 mRNA表达;免疫印迹技术在上述组织中检测NCX2的蛋白相对表达量,差异均未见统计学意义;免疫荧光双抗进一步证实NCX2蛋白与逼尿肌细胞共表达;膜片钳检测到逼尿肌细胞上的NCX2电流,电流密度为(0.79±0.45) pA/pF.结论 发现NCX2表达于大鼠逼尿肌细胞,电生理证实NCX2具有维持逼尿肌细胞内钙离子平衡的能力,其生理作用需要进一步的探索.

Expression of sodium/calcium exchanger subtype 2 and its physiological role in detrusor cells of rats

Objective To investigate the expression of sodium/calcium exchanger subtype 2 (NCX2) and explore its physiological role in detrusor cells of rats.Methods The bladder tissue was taken from 7 female and 3 male Wistar rats with the brain tissue as the control,and the detrusor cells were isolated from the bladder tissue using acute enzyme digestion technique.RT-PCR and Western blotting were used to detect the expression of NCX2 at mRNA and protein respectively in the bladder and brain tissues of the rats.Double-label immunofluorescence assay was used to verify the localization of NCX2 expression in the detrusor cells.NCX2 current was recorded using the whole-cell patch-clamp technique on the detrusor cells in the presence of the antagonist KB-R7943 (5 μmol/L).Results The acutely isolated detrusor cells showed predominantly a spindle-shaped morphology under microscope and were immunocytochemically positive for α-actin.NCX2 expression was detected in both the bladder and brain tissues of the rats with similar expression levels between the 2 tissues.Double-label immunofluorescence confirmed that NCX2 expression was localized on the detrusor cells,and NCX2 currents were detected in the detrusor cells with a current density of 0.79 ± 0.45 pA/pF.Conclusion NCX2 is expressed on the detrusor cells of rats and the electrophysiological evidence obtained suggests that NCX2 contributes to the maintenance of intracellular calcium homeostasis in the detrusor cells,but its exact physiological role still needs further investigation.