摘要
目的探讨17β-雌二醇对大鼠结肠平滑肌细胞（SMC）表达小电导钙激活钾通道3（SK3）的影响及其机制。方法酶解法分离SD雄性成年大鼠结肠SMC，进行原代培养。细胞免疫荧光双标观察SK3与α-肌动蛋白共表达。取2～3代SMC，不同浓度和不同时间17B-雌二醇刺激后、以反转录实时荧光定量（qRT）-PCR、Western印迹检测SMC表达SK3情况；观察雌激素受体阻断剂ICI182780在17β-雌二醇影响SK3表达中的作用及牛血清白蛋白结合的17β-雌二醇（BSA—E2）、仅受体激动剂PPT及β受体激动剂DPN对SK3表达的影响。结果细胞免疫荧光双标法发现大鼠结肠SMC存在SK3与α-肌动蛋白共表达。不同浓度17β-雌二醇（10、50nmol／L）刺激结肠SMC后SK3蛋白和mRNA表达量均显著高于溶剂对照组（0．217±0．030、0．321±0．077比0．103±O．063，1．872±0．606、2．967±0．659比0．815±0．202，均P〈0．05），50nmol/L为体外最大有效浓度；SK3表达的高峰时间为第12和24小时（蛋白表达量分别是0h组的2．91、3．30倍，mRNA表达量分别为3．46、3．37倍，均P〈0．05）。予ICI182780预处理后加17β-雌二醇，SK3蛋白表达低于17β-雌二醇组（0．111±0．050比0．351±0．084，P〈0．05），而与对照组差异无统计学意义；BSA—E2对SK3的表达无影响。α受体激动剂PPT组SK3蛋白表达与17β-雌二醇组均显著高于对照组（0．270±0．071、0．309±0．052比0．087±0．018，均P〈0．05）；β受体激动剂DPN对SK3蛋白表达无影响。结论大鼠结肠SMC上存在SK3，17β-雌二醇可促进SK3表达，该作用由α受体介导。
Objective To explore the effects and possible mechanism of 17β-estradiol on the expression of small conductance Ca2 ~ activated K＋ channel 3 （SK3） in rat colonic smooth muscle cells （SMC）. Methods The SMC isolated from male SD rats by enzymolysis were cultured. And double immunofluorescence staining was used to detect the co-expression of SK3 and α-actin. Colonic SMC were cultured with different concentrations of 17β-estradiol for 24 h or with 50 nmol/L 17β-estradiol at different timepoints respectively. The expressions of SK3 in colonic SMC were measured by real-time quantitative reverse transcription （qRT）-PCR and Western blotting. The effects of estrogen receptor （ER） inhibitor ICI 182780, albumin bovine serum-17β-estradiol （BSA-E2）, ERα selective agonist propyl pyrazole triol （PPT） and ER＋ selective agonist diarylpropiolnitrile （DPN） on SK3 expression were observed. Results Double immunofluorescence staining showed that SK3 and α-actin co-expressed in cultured colonic SMC. The expression of SK3 of 1713-estradiol at different concentration （10, 50 nmol/L） significantly higher than the control group（protein ： 0. 217 ± 0. 030 and 0. 321 ±0. 077 vs 0. 103±0. 063, mRNA ： 1. 872 ± 0. 606 and 2. 967 ± 0.659 vs 0.813 ±0.202, all P 〈0.05）. And 50 nmol/L was the most effective in vitro concentration. The peak expression of SK3 appeared at 12 and 24 hour （2. 91 and 3.30-fold in protein vs 3.46 and 3.37-fold in mRNA respectively, all P 〈 0. 05 ）. The protein levels of SK3 in ICI 182780 plus17 β-estradiol group was less than 1713-estradiol group （0. 111 ± 0. 050 vs 0. 351 ± 0. 084, P 〈 0. 05 ）. But it was not influenced by BSA-E2. The expressions of SK3 in PPT and E2 groups were both higher than control group（0. 270 ± 0. 071, 0. 309 ± 0. 052 vs 0. 087 ± 0. 018, both P 〈 0. 05 ）. However DPN had no effect on SK3 protein levels. Conclusions SK3 is localized in rat colonic SMC. And 17β-estradiol increases its expression in an ERa-dependant manner.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2013年第30期2367-2371,共5页
National Medical Journal of China
基金
基金项目：国家自然科学基金（30971354）
江苏省国际科技合作计划（BZ2011044）
关键词
雌二醇
小电导钙激活钾通道
肌细胞
平滑肌
结肠
受体
雌激素
Estradiol
Small-conductance calcium-activated potassium channels
Myocytes,smooth muscle
Colon
Receptors, estrogen
