摘要
目的探讨酸性环境对高磷诱导的大鼠血管平滑肌细胞(VSMCs)钙化的影响及可能机制。方法体外分离培养大鼠VSMCs,采用免疫细胞化学方法鉴定。将VSMCs按随机数字表法分为正常对照组、高磷+p H7.4、高磷+p H7.1和高磷+p H6.8(在高磷培养基的基础上调整p H值为6.8、7.1和7.4三个亚组)。刺激4 d后,采用RT-PCR检测L型钙通道(LTCC)α_(1C)、β_2和β_3亚基,Runt相关转录因子2(Runx2)及Smad1基因的表达;应用钙离子探针Fluo-3/AM检测VSMCs胞外钙离子内流的效应变化。刺激14 d后,对各组细胞进行钙化染色、钙含量和碱性磷酸酶(ALP)活性测定。结果与正常对照组比较,高磷+p H7.4组钙含量、ALP活性、Runx2和Smad1mRNA的表达明显增高(88.26±6.43比22.39±3.19、94.33±3.08比20.39±1.18、0.37±0.02比0.01±0.00、0.65±0.05比0.07±0.01,均为P<0.05);与高磷+p H7.4组比较,高磷+p H7.1组和高磷+p H6.8组的钙含量、ALP活性、Runx2和Smad1mRNA的表达均降低(69.95±1.72和50.74±3.29、51.11±2.05和34.62±1.13、0.25±0.02和0.09±0.01、0.44±0.04和0.23±0.01,均为P<0.05)。与正常对照组比较,高磷+p H7.4组的LTCCβ_3亚基mRNA表达水平增加(0.80±0.01比0.34±0.13,P<0.01),高磷+p H7.1组和高磷+p H6.8组的LTCCβ_3亚基mRNA表达水平均下降(0.64±0.11和0.43±0.01,均为P<0.01)。各组之间LTCCα_(1C)和β_2亚基mRNA的表达差异无统计学意义(P=0.08和0.74)。与正常对照组比较,高磷+p H7.4组的VSMCs胞内钙离子浓度增加(438.33±7.50比149.54±20.89,P<0.05);高磷+p H7.1组和高磷+p H6.8组的VSMCs胞内钙离子浓度均降低(329.66±16.64和234.00±17.43,均为P<0.01)。结论酸性环境可以抑制高磷诱导的大鼠VSMCs钙化,其可能是通过抑制LTCCβ_3亚基表达,降低VSMCs钙离子内流,阻滞VSMCs发生表型转化来实现的。
Objective To explore the effect and possible mechanisms of acidification on high phosphorus induced vascular smooth muscle cells （VSMCs） calcification in rats. Methods VSMCs were isolated from rat aorta, identified by immunocytochemistry, and randomly divided into control group and high phosphorus group according to the pH, the high phosphorus group was further settled into three subgroups, high phosphorus ＋ pH6. 8, ＋ pH7.1 and ＋ pH7.4, which were treated with β-glycerophosphate, and acidified by HCL to adjust the pH respectively. The VSMC mRNA expressions of L-type calcium channel （LTCC） subunitsα1c, β2, β3, Runt-related transcription factor 2 （Runx2） and Smadl mRNA were detected by RT-PCR after stimulated for 4 days; meanwhile, the VSMCs were loaded with calcium probe Fluo-3/AM, and the concentration of calcium was measured by Fluo-3/AM among different groups. The activity of alkaline phosphatase and calcium deposition were tested after culturing 14 days. Results Compared with control group, calcium concentration and the activity of ALP in VSMCs were significantly increased in high phosphorus ＋pH7.4 group after incubation for 14 days （88.26 ±6. 43 vs. 22. 39 ±3.19, 94. 33±3.08 vs. 20. 39± 1.18, both P 〈 0.05 ） , the expression of Smadl, Runx2 and LTCC 153 subunit mRNA in high phosphorus ＋ pH7.4 group was increased as well （ 0. 65 ±0. 05 vs.0. 07 ±0.01, 0. 37 ± 0. 02 vs. 0, 01 ±0. 00, 0. 80 ±0. 01 vs. 0. 34 ±0. 13, all P 〈0. 05）. Besides, Smadl, Runx2 and LTCC β3 subunit mRNA were decreased in high phosphorus ＋ pH7.1 group and high phosphorus ＋ pH6. 8 group （0.25 ±0.02 vs. 0.37 ±0.02, 0.09 ±0.01 vs. 0.37 ±0.02, 0.44 ±0.04 vs. 0.65 ±0.05, 0.23 ±0.01 vs. 0.65 ±0.05, 0.64±0.11 vs. 0.80±0.01, 0.43±0.01 vs. 0.80±0.01, all P〈0.01）, compared with those in high phosphorus ＋ pH7.4 group as the pH decreased. There was no significant difference in LTCC alc and 13z subunits mRNA among each groups （P =0. 08, P =0. 74）. Calcium influx in VSMCs was pa
出处
《中国心血管杂志》
2015年第6期455-460,共6页
Chinese Journal of Cardiovascular Medicine
基金
河北省自然科学基金资助项目(H2012206157)~~
关键词
酸性环境
肌细胞
平滑肌
钙通道
L型
RUNX2
Acidic environment
Muscle cells, smooth muscle
Calcium channel, L-type
Runx2
