中华大蟾蜍小肠黏膜上皮斯钙素-1基因表达与质膜Ca^(2+)-ATP酶活性分析

Analysis of stanniocalcin-1 expression and plasma membrane Ca^(2+)-ATPase activity in enterocytes of toad Bufo bufo gargarizans

目的探讨小肠黏膜上皮斯钙素-1(STC1)基因表达与质膜Ca2+-ATP酶活性的关系。方法基于耐受实验结果,将72只成体中华大蟾蜍随机分为高钙环境组(加0.1 mol/L CaCl2的自来水)、低钙环境组[加0.03mol/L乙二胺四乙酸(EDTA)的自来水]及正常对照组(自来水),分别于暴露前及暴露后12h、24h、48h、72h、96h、120h、144h取血浆和小肠黏膜,利用比色法和半定量RT-PCR法分别检测血浆钙水平、质膜Ca2+-ATP酶活性和STC1 mRNA表达水平。结果 0.1mol/L氯化钙暴露致中华大蟾蜍血浆钙水平在12h和24h时显著高于对照组水平(1.9mmol/L)(P<0.05),48h时回复至正常,72h后持续升高;而12～48h内小肠黏膜上皮质膜Ca2+-ATP酶活性增强和STC1 mRNA水平上调,72h后回复至正常水平。0.03mol/L EDTA暴露则使中华大蟾蜍血浆钙水平下降,但质膜Ca2+-ATP酶活性和STC1 mRNA水平与对照组相比未出现明显变化。结论血浆钙水平升高致小肠黏膜上皮质膜Ca2+-ATP酶活性升高,进而增强STC1表达,而STC1表达上调又以负反馈方式抑制质膜Ca2+-ATP酶的活性。

Objective To investigate the relation between plasma membrane Ca^2＋-ATPase （PMCA） activity and stanniocalein-1 （STC1） gene expression in enteroeytes in toad, Bufo bufo gargarizans. Methods Based on tolerance test in this study, 72 adult toads were randomly divided into a high calcium group [ tap water supplied with 0. lmol/L CaCl2 ） , a low calcium group （tap water supplied with 0. 03mol/L ethylene diamine tetraaeetie acid （EDTA） ] and a control group （tap water）. The small intestinal mucosa was excised on the pre-exposure and post-exposure 12, 24, 48, 72, 96, 120, 144 hours, respectively. Both plasma Ca^2＋ level and PMCA activity were detected with eolorimetry, and STC1 gene expression was analyzed with semi-quantitative RT-PCR. Results The results showed that： （ 1 ） After 0. lmol/L CaCl2 exposure, the plasma calcium level increased at the 12th and 24th hour （ P 〈 0.05 ） , restored to the normal level at the 48 th hour, and went up at the 72 th hour （P 〈 0.05）. Additionally, PMCA activity was enhanced and STC1 gene expression was up-regulated during 12th-48th hour after 0. lmol/L CaC12exposure. （2） After 0.03 mol/L EDTA exposure, the plasma calcium level decreased, but both PMCA activity and STC1 gene expression did not change. Conclusion High plasma calcium level stimulates PMCA activity and STC1 expression in enterocytes, whereas up-regulated STC1 may inhibits PMCA activity in a negative feedback fashion.