CAJ | 학술논문

目的观察高磷对大鼠血管平滑肌细胞(RVSMC)钙化的影响,及探讨阿托伐他汀在RVSMC钙化中的保护作用及相关机制.方法用不同的试剂培养RVSMC,分别为正常磷组(Pi 1.4 mmol/L)、高磷组(Pi 2.0 mmol/L、2.6 mmol/L)、凋亡抑制组(Pi 2.6 mmol/L+ZVAD-FMK 0.1 μmol/L、Pi 2.6 mmol/L+ZVAD-FMK 1.0 μmol/L、Pi 2.6 mmol/L+ZVAD-FMK2.0 μmol/L)和阿托伐他汀组(H 2.6 mmol/L+Statin 1 nmol/L、Pi 2.6 mmol/L+Stain 10 nmol/L、Pi 2.6 mmol/L+Statin 100 nmol/L).甲O-酚酞络合酮方法测定平滑肌细胞钙含量,BCA法测定蛋白含量,用蛋白含量标化钙含量,同时以von Kossa染色观察细胞钙化情况.ELISA方法测定不同时间各组细胞的相对凋亡量.结果 (1)培养第3、6、9天时,Pi 2.0 mmol/L、Pi 2.6mmol/L组与Pi 1.4 mmol/L组相比,RVSMC钙沉积较多(P<0.05).(2)培养第6天时,Pi 2.6mmol/L+ZVAD-FMK 1.0 μmol/L、Pi 2.6 mmol/L+ZVAD-FMK 2.0 μmol/L组与Pi 2.6 mmol/L组相比,RVSMC钙沉积较少(P<0.05);Pi 2.6 mmol/L+Statin 10 nmol/L、Pi 2.6 mmol/L+Statin100 nmol/L组与Pi 2.6 mmol/L组相比,细胞钙沉积较少(P<0.05).von Kossa染色显示Pi2.6 mmol/L组细胞有大量黑色颗粒沉积,而Pi 2.6 mmol/L+Statin 100 nmol/L组较少.(3)培养第3、6、9天时,Pi 2.6 mmol/L与Pi 1.4 mmol/L相比,细胞相对凋亡量较多(P<0.05);培养第12、24小时,Pi 2.0 mmol/L、Pi 2.6 mmol/L组与Pi 1.4 mmol/L组相比,细胞相对凋亡量较多(P<0.05);培养第6天、第24小时时,Pi 2.6 mmol/L+Statin 10 nmol/L、Pi 2.6 mmol/L+Statin100 nmol/L与Pi 2.6 mmol/L组相比,细胞相对凋亡量较少(P<0.05).结论高磷培养可诱导RVSMC体外钙化.阿托伐他汀对高磷诱导的RVSMC钙化有保护作用,此作用可能与其降低RVSMC凋亡有关.
목적 관찰고린대대서혈관평활기세포(RVSMC)개화적영향,급탐토아탁벌타정재RVSMC개화중적보호작용급상관궤제.방법 용불동적시제배양RVSMC,분별위정상린조(Pi 1.4 mmol/L)、고린조(Pi 2.0 mmol/L、2.6 mmol/L)、조망억제조(Pi 2.6 mmol/L+ZVAD-FMK 0.1 μmol/L、Pi 2.6 mmol/L+ZVAD-FMK 1.0 μmol/L、Pi 2.6 mmol/L+ZVAD-FMK2.0 μmol/L)화아탁벌타정조(H 2.6 mmol/L+Statin 1 nmol/L、Pi 2.6 mmol/L+Stain 10 nmol/L、Pi 2.6 mmol/L+Statin 100 nmol/L).갑O-분태락합동방법측정평활기세포개함량,BCA법측정단백함량,용단백함량표화개함량,동시이von Kossa염색관찰세포개화정황.ELISA방법측정불동시간각조세포적상대조망량.결과 (1)배양제3、6、9천시,Pi 2.0 mmol/L、Pi 2.6mmol/L조여Pi 1.4 mmol/L조상비,RVSMC개침적교다(P<0.05).(2)배양제6천시,Pi 2.6mmol/L+ZVAD-FMK 1.0 μmol/L、Pi 2.6 mmol/L+ZVAD-FMK 2.0 μmol/L조여Pi 2.6 mmol/L조상비,RVSMC개침적교소(P<0.05);Pi 2.6 mmol/L+Statin 10 nmol/L、Pi 2.6 mmol/L+Statin100 nmol/L조여Pi 2.6 mmol/L조상비,세포개침적교소(P<0.05).von Kossa염색현시Pi2.6 mmol/L조세포유대량흑색과립침적,이Pi 2.6 mmol/L+Statin 100 nmol/L조교소.(3)배양제3、6、9천시,Pi 2.6 mmol/L여Pi 1.4 mmol/L상비,세포상대조망량교다(P<0.05);배양제12、24소시,Pi 2.0 mmol/L、Pi 2.6 mmol/L조여Pi 1.4 mmol/L조상비,세포상대조망량교다(P<0.05);배양제6천、제24소시시,Pi 2.6 mmol/L+Statin 10 nmol/L、Pi 2.6 mmol/L+Statin100 nmol/L여Pi 2.6 mmol/L조상비,세포상대조망량교소(P<0.05).결론 고린배양가유도RVSMC체외개화.아탁벌타정대고린유도적RVSMC개화유보호작용,차작용가능여기강저RVSMC조망유관.
Objective To investigate the protective effects of atorvastatin on hyperphosphate-induced rat vascular smooth muscle ceils (RVSMCs) calcification and to discuss the mechanism. Methods RVSMCs were placed in various culture media, including normal phosphate medium, high phosphate medium, ZVAD-FMK medium and atorvastatin medium.Calcium content and cell protein content were quantified by the o-cresolphthalein complexone method and BCA protein assay respectively. Calcification was visualized by yon Kossa staining. And cell apoptosis was quantified by ELISA. Results (1)At day 3, 6, 9, RVSMCs calcification occurred more frequently in high phosphate medium than that in normal phosphate medium (P<0.05). (2)At day 6, RVSMCs calcification was significantly inhibited in 1.0 μmol/L and 2.0 μmol/LZVAD-FMK medium (P<0.05). And in 10 nmol/L and I00 nmol/L statin medium, RVSMCscalcium deposition significantly decreased (P<0.05). (3)RVSMCs apoptosis and calcification occurredfrequently in high phosphate medium. And atorvastatin significantly inhibited RVSMCs apoptosisboth in long-term and short-term (P<0.05). Conclusions Hyperphosphate can induce the calcium deposition of RVSMCs in vitro. Atorvastatin protects RVSMCs from phosphate-induced calcification by inhibiting apoptosis.