间歇性碱刺激对高磷诱导的大鼠血管平滑肌细胞钙化的影响及机制

Effect and mechanism of intermittent alkaline stimulation on high phosphorus induced calcification in vascular smooth muscle cells of rats

目的探讨间歇性碱刺激对高磷诱导的大鼠血管平滑肌细胞（VSMC）钙化的影响及其机制。方法体外分离培养大鼠VSMC，将传代培养至第4代的细胞按随机数字表法分为正常对照组、高磷＋pH7．4组、高磷＋pH7．5组、高磷＋pit7．6组和高磷＋pH7．7组。正常对照组采用含10％胎牛血清的培养基培养，其他各组采用含10mmo]／L13-甘油磷酸的高磷培养基培养，然后分别加入7．4％NaHCO，调整pH值为7．4、7．5、7．6和7．7。干预4h后，正常对照组更换正常培养基，其余4组的培养基均更换为高磷基础上pH值为7．4的培养基，然后隔1d更换培养基1次。干预4d后，分别应用RT-PCR和Westernblot检测VSMC的L型钙通道（LTCC）B，亚基和Runt相关转录因子2（Runx2）mRAN和蛋白的表达水平；干预4d后，应用钙离子探针Fluo-3／AM检测VSMC内钙离子水平；干预14d后，应用酶联免疫吸附法（ELISA）测定碱性磷酸酶（ALP）活性；干预14d后，通过测量钙含量观察细胞钙化程度。结果（1）与正常对照组比较，高磷＋pH7．4组LTCCB，亚基mRNA和蛋白表达水平均较高（分别为0．49±0．03比0．23±0．02和0．45±0．03比0．26±0．02，P均〈0．05）；与高磷＋pH7．4组比较，高磷＋pH7．5组LTCC13，亚基mRNA（0．86±0．05）和蛋白（0．62±0．04）表达水平均较高（P均〈0．05）；与高磷＋pH7．5组比较，高磷＋pH7．6组LTCC13，亚基mRNA（0．99±0．05）和蛋白（0．80±0．03）表达水平均较高（P均〈0．05）；与高磷＋pH7．6组比较，高磷＋pH7．7组LTCC13，亚基mRNA（1．16±0．05）和蛋白（0．93-I-0．03）表达水平均较高（P均〈0．05）。（2）与正常对照组比较，高磷＋pH7．4组细胞内钙离子水平较高（124．61±6．06比75．68±7．82，P〈0．05）；与高磷＋pH7．4组比较，高磷＋pH7．5组细胞内钙离子水平较高（210．85±9．75，P〈0．05）；与高磷＋pH7．5组比较，高磷＋pH7．6组细胞内钙离子水平较高（298．44±11．42，P〈0．05）；与高磷＋pH7．6组比较，高磷＋pH7．7组细胞内钙离子水平较高（401．13±11．41，P〈0．05）。（3）与正常对照组比较，高磷＋pH7．4组Runx2mRNA、蛋白表达水平和ALP活性均较高（分别为0．60±0．04比0．34±0．03、0．42±0．04比0．21±0．02、67．2±4．3比23．2±2．3，P均〈0．05）；与高磷＋pH7．4组比较，高磷＋pH7．5组Runx2mRNA（0．76±0．05）、蛋白（0．68±0．03）表达水平和ALP活性（102．1±5．4）均较高（P均〈0．05）；与高磷＋pH7．5组比较，高磷＋pH7．6组Runx2mRNA（0．90±0．05）、蛋白（0．90±0．05）表达水平和ALP活性（139．3±4．9）均较高（P均〈0．05）；与高磷＋pH7．6组比较，高磷＋pH7．7组Runx2mRNA（1．11±0．05）、蛋白（1．08±0．06）表达水平和ALP活性（197．0±6．7）均较高（P均〈0．05）。（4）与正常对照组比较，高磷＋pH7．4组钙含量较高[（75．4±4．3）mg／g蛋白比（25．2±2．1）mg／g蛋白，P〈0．05]；与高磷＋pH7．4组比较，高磷＋pH7．5组钙含量较高[（100．8±5．7）mg／g蛋白，P〈0．05]；与高磷＋pH7．5组比较，高磷＋pH7．6组钙含量较高[（143．5±6．1）mg／g蛋白，P〈0．05]；与高磷＋pH7．6组比较，高磷＋pH7．7组钙含量较高[（205．1±8．2）mg／g蛋白，P〈0．05]。结论间歇性碱刺激可促进高磷诱导的大鼠VSMC钙化，可能是通过促进LTCC13，亚基表达，增加钙离子内流，从而促使VSMC发生表型转化来实现。

Objective To explore the effect and possible mechanisms of intermittent alkaline on rat vascular smooth muscle cells （VSMCs） calcification induced by high phosphorus. Methods VSMCs were isolated from rat thoracic aorta and cultured in vitro. The fourth generation VSMCs were randomly divided into control group, high phosphorus ＋ pH7.4, high phosphorus ＋ pHT. 5, high phosphorus ＋ pH7.6 and high phosphorus ＋ pH7.7 group with random number table. The control group was cultured in DMEM with 10% fetal bovine serum. Other groups were cultured in DMEM with 10 mmol/L [3-glycerophosphate and alkalized by 7.4% NaHCO3 to adjust the pH respectively. After the intervention of 4 hours, the control group was replaced with the normal medium containing 10% fetal bovine serum, the other 4 groups were replaced with high phosphorus based on the pH value of the culture medium, and then replaced the culture medium every other day. After 4 days intervention, the mRNA and protein expression of L type calcium channel β3 subunit （ LTCC β3 ） and Runt related transcription factor 2 （ Runx2 ） were detected by RT-PCR and Western blot. After 4 days intervention, the level of VSMC calcium ion was detected by Fluo-3/AM. After 14 days intervention, alkaline phosphatase （ALP） activity was measured by enzyme linked immunosorbent assay （ELISA） and the calcification was observed by measuring calcium content. Results （ 1 ） Compared with control group, the gene and protein expressions of LTCC β3 were higher in high phosphorus ＋ pH7.4 group （0. 49 ± 0. 03 vs. 0. 23 ± 0. 02 and 0. 45 ± 0. 03 vs. 0. 26 ± 0. 02 respectively, all P 〈 0. 05 ）. Compared with high phosphorus ＋ pH7.4 group, the mRNA （0. 86 ± 0. 05 ） and protein （0. 62 ± 0. 04） expressions of LTCC [33 were higher in high phosphorus ＋ pH7.5 group （ P 〈 0. 05 ）. Compared with high phosphorus ＋ pH7.5 group, the mRNA（0. 99 ±0. 05） and protein（0. 80 ±0. 03） expressions of LTCC 133 were higher in high phosphorus ＋ pH7.5 group （ all P 〈 0. 05 ）. Compared with high phosphorus ＋ pH7.6 group, the mRNA （ 1.16 ± 0. 05 ） and protein （0. 93± 0. 03 ） expressions of LTCC β3 were higher in high phosphorus ＋ pH7.7 group （all P 〈 0. 05 ）. （2） Compared with control group, calcium ion influx were higher in high phosphorus ＋ pH7.4 group （ 124. 61 ±6. 06 vs. 75.68 ±7. 82, P 〈 0. 05 ）. Compared with high phosphorus ＋ pH7.4 group, calcium ion influx was higher in high phosphorus ＋ pH7.5 group（210. 85 ± 9. 75 ,P 〈 0. 05 ）. Compared with high phosphorus ＋ pH7.5 group, calcium ion influx was higher in high phosphorus ＋ pH7.6 group（298.44 ± 11.42,P 〈 0. 05 ）. Compared with high phosphorus ＋ pH7.6 group, calcium ion influx was higher in high phosphorus ＋ pH7.7 group（401.13 ± 11.41, P 〈 0. 05 ）. （3） Compared with control group, the mRNA and protein expressions of Runx2 and ALP were higher in high phosphorus ＋ pH7.4 group （0.60±0.04 vs. 0.34±0.03,0.42 ±0.04 vs. 0.21 ±0.02,67.2 ±4.3 vs. 23.2±2.3 respectively,all P 〈0. 05）. Compared with high phosphorus ＋ pH7.4 group, the mRNA（0. 76 ±0. 05） and protein（0.68 ± 0. 03） expressions of Runx2 and ALP（ 102. 1 ± 5.4） were higher in high phosphorus ＋ pH7.5 group （ all P 〈0. 05）. Compared with high phosphorus ＋ pH7.5 group, the mRNA（0. 90 ±0. 05） and protein（0. 90 ± 0.05） expressions of Runx2 and ALP（ 139.3 ± 4. 9） were higher in high phosphorus ＋ pH7.6 group （ all P 〈 0. 05 ）. Compared with high phosphorus ＋ pH7.6 group, the mRNA（ 1.11 ± 0. 05 ） and protein（ 1.08 ± 0. 06） expressions of Runx2 and ALP（ 197. 0 ± 6. 7） were higher in high phosphorus ＋ pH7. 7 group （ all P 〈 0. 05 ）. （4） Compared with control group, the calcium content were higher in high phosphorus ＋ pH7.4 group （ （75.4 ± 4. 3 ） mg/g pro vs. （ 25.2 ± 2. 1 ） mg/g pro, P 〈 0. 05 ）. Compared with high phosphorus ＋ pH7.4 group, the calcium content were higher in high phosphorus ＋ pH7.5 group （ （ 100. 8 ± 5.7 ） mg/g pro,P 〈 0. 05）. Compared with high phosphorus ＋ pH7.5 group, the calcium content were higher in high phosphorus ＋ pH7.6 group （ （ 143.5 ± 6. 1 ） mg/g pro,P 〈 0. 05 ）. Compared with high phosphorus ＋ pH7.6 group, the calcium content were higher in high phosphorus ＋ pH7.7 group （ （205.1 ± 8.2） mg/g pro,P 〈 0. 05）. Conclusion Intermittent alkaline stimulation can promote high phosphorus induced rat VSMCs calcification possibly through upregulating LTCC β3 subunit gene and protein expression, increasing calcium ion influx and enhancing VSMCs phenotypic transformation.