CAJ | 학술논문

目的探讨高镁对高磷诱导血管钙化的影响及其可能的机制.方法体外原代培养大鼠胸主动脉血管平滑肌细胞(VSMCs),用β甘油磷酸盐(β-GP)诱导钙化.VSMCs细胞被分为4组:对照组、高磷组(10 mmol/L β-GP)、镁干预组(10 mmol/L β-GP+3 mmol/LMgSO4)、镁通道抑制剂(2-APB)干预组(10 mmol/L β-GP+3 mmol/L MgSO4+10-4 mol/L 2-APB).采用茜素红染色及邻甲酚酞络合酮比色法检测细胞钙化情况;酶联免疫吸附法(ELISA)检测细胞碱性磷酸酶(ALP)活性;RT-PCR法和Western印迹法检测细胞核心结合因子α-1(Cbfα-1)的表达.24只雄性SD大鼠被随机分为3组:对照组(甲基纤维素灌胃+高磷饮食)、血管钙化组(硫酸腺嘌呤灌胃+高磷饮食)、高镁干预组(硫酸腺嘌呤灌胃+高磷高镁饮食).造模成功后测定大鼠主动脉脉搏波速率(PWV);采用yon Kossa染色及邻甲酚酞络合酮比色法检测胸主动脉钙化情况;免疫组织化学方法检测胸主动脉Cbfα-1表达.结果细胞培养14 d后,与高磷组相比,镁干预组VSMCs钙盐沉积明显减少,ALP活性降低(P＜0.05),Cbfα-1表达下调;2-APB可抑制高镁对VSMCs的保护性作用.Cbfα-1的动态观察结果显示,镁干预组第3天Cbfα-1的表达下调(P＜0.05),其抑制效应随时间延长呈增强趋势.体内实验中,大鼠慢性肾衰竭血管钙化模型制备成功.和体外实验一致,与血管钙化组相比,高镁干预组大鼠血浆镁离子水平明显升高,胸主动脉PWV明显降低(P＜0.05),血管钙盐沉积程度亦明显减轻.免疫组织化学结果显示高镁可明显降低高磷诱导的Cbfα-1表达(P＜0.05).结论高镁可抑制血管钙化,其机制可能与其抑制VSMCs骨源性分化相关.
목적 탐토고미대고린유도혈관개화적영향급기가능적궤제.방법 체외원대배양대서흉주동맥혈관평활기세포(VSMCs),용β감유린산염(β-GP)유도개화.VSMCs세포피분위4조:대조조、고린조(10 mmol/L β-GP)、미간예조(10 mmol/L β-GP+3 mmol/LMgSO4)、미통도억제제(2-APB)간예조(10 mmol/L β-GP+3 mmol/L MgSO4+10-4 mol/L 2-APB).채용천소홍염색급린갑분태락합동비색법검측세포개화정황;매련면역흡부법(ELISA)검측세포감성린산매(ALP)활성;RT-PCR법화Western인적법검측세포핵심결합인자α-1(Cbfα-1)적표체.24지웅성SD대서피수궤분위3조:대조조(갑기섬유소관위+고린음식)、혈관개화조(류산선표령관위+고린음식)、고미간예조(류산선표령관위+고린고미음식).조모성공후측정대서주동맥맥박파속솔(PWV);채용yon Kossa염색급린갑분태락합동비색법검측흉주동맥개화정황;면역조직화학방법검측흉주동맥Cbfα-1표체.결과 세포배양14 d후,여고린조상비,미간예조VSMCs개염침적명현감소,ALP활성강저(P＜0.05),Cbfα-1표체하조;2-APB가억제고미대VSMCs적보호성작용.Cbfα-1적동태관찰결과현시,미간예조제3천Cbfα-1적표체하조(P＜0.05),기억제효응수시간연장정증강추세.체내실험중,대서만성신쇠갈혈관개화모형제비성공.화체외실험일치,여혈관개화조상비,고미간예조대서혈장미리자수평명현승고,흉주동맥PWV명현강저(P＜0.05),혈관개염침적정도역명현감경.면역조직화학결과현시고미가명현강저고린유도적Cbfα-1표체(P＜0.05).결론 고미가억제혈관개화,기궤제가능여기억제VSMCs골원성분화상관.
Objective To explore the effect and mechanism of magnesium on calcification induced by hyperphosphate.Methods Vascular smooth muscle cells (VSMCs) were primarily cultured in vitro and induced calcification by β-glycerophosphate (β-GP).VSMCs were randomly divided into control group,high phosphorus group (10 mmol/L β-GP),magnesium intervèntion group(10 mmol/L β-GP + 3 mmol/L MgSO4) and 2-aminoethoxy-diphenylborate (2-APB,an inhibitor of magnesium transporter) intervention group(10 mmol/L β-GP+3 mmol/L MgSO4+ 10-4 mol/L 2-APB).Calcium deposition and alkaline phosphatase (ALP) activity were measured by alizarin red staining,quantification of calcium and euzyme linked immunosorbent assay.RT-PCR and Western blotting were used to observe the expression of core binding factor α-1 (Cbfα-1) mRNA and protein,respectively.In vivo,male Sprague-Dawley rats (n=24) were randomly divided into control group (methylcellulose+high phosphorous diet),vascular calcification group (adenine suspension + high phosphorous diet),high magnesium intervention group(adenine suspension+high phosphorous and magnesium diet).The aortic pulse wave velocity (PWV) was measured,and vascular calcification was determined by von Kossa stain and quantification of calcium.Cbfα-1 in aortic was measured by immunohistochemistry.Results In vitro,compared with high phosphorus group,calcification,ALP activity (P ＜ 0.05) and Cbfα-1expression in VSMCs were significantly decreased in magnesium intervention group after incubation for 14 days,but the addition of 2-APB might inhibit the protective effect of magnesium on VSMCs.Dynamic observation of Cbfα-1 showed that magnesium significantly inhibited the expression of Cbfα-1 (P ＜ 0.05) on the third day and the inhibitory role was obviously increased in a time-dependent manner.Consistent with the findings in vitro,the aortic PWV,calcification were all significantly reduced (P ＜ 0.05) in high magnesium intervention group with high serum magnesium level,when compared with vascular calcification group.Immunohistochemistry showed that hypermagnesemia downregulated obviously the expression of Cbfα-1 induced by hyperphosphatemia(P ＜ 0.05).Conclusion Magnesium protects against vascular calcification by inhibiting osteogenic differentiation of VSMCs.