碱性环境和高磷条件下大鼠胸主动脉平滑肌细胞成骨样表型转化中钙激活钾通道mRNA的表达

Expression of Calcium-activated Potassium Channel mRNA in Rat Thoracic Aorta Smooth Muscle Cells Bone-like Phenotypic Transformation in the Condition of Alkaline Environment and High Phosphorus

目的观察碱性环境中高磷诱导大鼠胸主动脉平滑肌细胞中电导钙激活钾通道(KCa3.1)与大电导钙激活钾通道(KCa1.1)表达的变化,以及探究钙激活钾通道与大鼠胸主动脉平滑肌细胞表型转化之间的关系。方法采用组织块贴壁法培养原代大鼠主动脉平滑肌细胞,利用10 mmol/Lβ-甘油磷酸钠制备血管平滑肌细胞钙化模型。使用HCl和Na HCO3调节培养基p H值。细胞随机分为5组:正常p H 7.4组、高磷p H 7.4组、高磷p H 7.7组、高磷p H 8.0组、TRAM-34干预组,共培养4天。用逆转录-聚合酶链反应检测各组细胞中KCa3.1、KCa1.1α、KCa1.1β、Runt相关转录因子2(Runx2)和平滑肌22α(SM22α)表达。结果与正常p H 7.4组相比,高磷组Runx2水平明显升高,且随着p H升高而表达量增加(P<0.05);高磷组SM22α水平明显下降,且随着p H升高而表达量减少(P<0.05)。与正常p H 7.4组相比,高磷p H 7.4组KCa3.1表达升高(P<0.05),KCa1.1α表达下降(P<0.05)。在高磷组中,随着p H升高KCa3.1、KCa1.1α表达量增加(P<0.05)。在同一组中KCa3.1表达高于KCa1.1α(P<0.05)。KCa1.1β表达在3个高磷组间未见统计学差异(P>0.05)。与高磷p H 8.0组相比,TRAM-34干预组Runx2mRNA水平明显下降(P<0.05),SM22αmRNA水平明显上升(P<0.05)。相关分析显示,KCa3.1表达与Runx2表达呈正相关(r=0.945,P<0.01),与SM22α表达呈负相关(r=-0.926,P<0.01);在正常p H 7.4组、高磷p H 7.4组中KCa1.1α表达与Runx2表达呈负相关(r=-0.746,P=0.029),与SM22α表达呈正相关(r=0.971,P=0.002);在高磷p H 7.7组、高磷p H 8.0组中KCa1.1α表达与Runx2表达呈正相关(r=0.805,P=0.002),与SM22α表达呈负相关(r=-0.806,P=0.005);KCa1.1β表达与Runx2、SM22α表达不相关(r=0.414,P=0.356;r=-0.155,P=0.714)。结论碱性环境中平滑肌细胞钙激活钾通道表达参与高磷诱导的大鼠胸主动脉平滑肌细胞的表型转化。

Aim To observe the expression changes of intermediate conductance calcium-activated potassium channel（ KCa3. 1） and large conductance calcium-activated potassium channel（ KCa1. 1） in rat thoracic aorta smooth muscle cells induced by high phosphorus in alkaline environment; To explore the relationship between the KCa and the phenotype transformation of rat thoracic aorta smooth muscle cells. Methods Tissue block adherence method was used to culture primary rat aortic smooth muscle cells. Vascular smooth muscle cell（ VSMC） calcification model was prepared with 10 mmol / L β-glycerol sodium phosphate. PH value of culture medium was regulated by using HCl and Na HCO3.Then the cells were divided into 5 groups： normal p H 7. 4 group,high phosphorus p H 7. 4 group,high phosphorus p H 7. 7group,high phosphorus p H 8. 0 group,TRAM-34 intervention group,and cultured for 4 days. The expressions of KCa3. 1,KCa1. 1α,KCa1. 1β,runt-related transcription factor 2（ Runx2） and smooth muscle 22 α（ SM22α） were detected by reverse transcription polymerase chain reaction（ RT-PCR） in each group cells. Results Compared with normal p H 7. 4 group,the expression of Runx2 was increased in high phosphorus groups,and increased with the increase of p H（ P〈 0. 05）; the expression of SM22α was reduced in high phosphorus groups,and reduced with the increase of p H（ P〈 0. 05）. Compared with normal p H 7. 4 group,the expression of KCa3. 1 was increased and the expression of KCa1. 1αwas reduced in high phosphorus p H 7. 4 group（ P〈 0. 05）. In the high phosphorus groups,the expressions of KCa3. 1 and KCa1. 1α were increased with the increase of p H（ P〈 0. 05）. In the same group,the expression of KCa3. 1 was more than KCa1. 1α（ P〈 0. 05）. There was no significant difference in KCa1. 1β expression among 3 high phosphorus groups（ P〈 0. 05）. Compared with high phosphorus p H 8. 0 group,the expression of Runx2 was decreased and the expression of SM22α added in TRAM-34 intervention group（ P〈 0. 05）. Correlation analysis showed that KCa3. 1 expression was positively correlated with Runx2 expression（ r = 0. 945,P〈 0. 01） and was negatively correlated with SM22α expression（ r =-0. 926,P〈 0. 01）. KCa1. 1α expression was negatively correlated with Runx2 expression（ r =- 0. 746,P = 0. 029） and was positively correlated with SM22α expression（ r = 0. 971,P = 0. 002） in normal p H 7. 4 group and high phosphorus p H7. 4 group. KCa1. 1α expression was positively correlated with Runx2 expression（ r = 0. 805,P = 0. 002） and was negatively correlated with SM22α expression（ r =-0. 806,P = 0. 005） in high phosphorus p H 7. 7 group and high phosphorus p H 8. 0 group. The expression of KCa1. 1β was not correlated with the expression of Runx2 and SM22α（ r = 0. 414,P =0. 356; r =- 0. 155,P = 0. 714）. Conclusion The expression of calcium-activated potassium channel in smooth muscle cells is involved in the phenotypic transformation of rat thoracic aorta smooth muscle cells induced by high phosphorus in alkaline environment.