CAJ | 학술논문

目的：研究成骨相关转录因子（OSX）沉默对醛固酮诱导的血管平滑肌细胞（VSMC）成骨样分化和钙化的影响。方法体外原代培养小鼠的 VSMC。针对小鼠 OSX 基因设计合成小分子干扰 RNA（siRNA）序列，使用 Lipo 2000为载体，转染体外培养的 VSMC；FAM 荧光标记 siRNA 优化转染条件。实时 PCR 检测 OSX mRNA 表达。将 siRNA 序列转染 VSMC，细胞分为4组：①正常组；②醛固酮组（1.5 mmol/ L）；③siRNA 转染组：醛固酮+ OSX - siRNA；④阴性转染对照组：醛固酮+阴性对照 siRNA。实时 RT - PCR 及免疫印迹法检测 OSX、整联结合涎蛋白（Ibsp）基因和蛋白表达；茜素红染色观察细胞钙盐沉积。结果与醛固酮组相比，siRNA 转染组 OSX mRNA 表达在转染后24 h 及48 h 均明显低于醛固酮组（均 P ﹤0.01）；OSX 蛋白表达在转染后48 h 和72 h 均低于醛固酮组（均 P ﹤0.01），以48 h 最明显。siRNA有效沉默 OSX 基因表达后，OSX 转染组 Ibsp mRNA 和蛋白的表达低于醛固酮组（均 P ﹤0.01），而且细胞钙盐沉积低于高磷组。结论 OSX - siRNA 可以有效抑制 VSMC OSX 基因和蛋白的表达，从而抑制醛固酮诱导的 VSMC 成骨样分化和细胞钙化。OSX 可以成为 CKD 血管钙化治疗的靶点。
목적：연구성골상관전록인자（OSX）침묵대철고동유도적혈관평활기세포（VSMC）성골양분화화개화적영향。방법체외원대배양소서적 VSMC。침대소서 OSX 기인설계합성소분자간우 RNA（siRNA）서렬，사용 Lipo 2000위재체，전염체외배양적 VSMC；FAM 형광표기 siRNA 우화전염조건。실시 PCR 검측 OSX mRNA 표체。장 siRNA 서렬전염 VSMC，세포분위4조：①정상조；②철고동조（1.5 mmol/ L）；③siRNA 전염조：철고동+ OSX - siRNA；④음성전염대조조：철고동+음성대조 siRNA。실시 RT - PCR 급면역인적법검측 OSX、정련결합연단백（Ibsp）기인화단백표체；천소홍염색관찰세포개염침적。결과여철고동조상비，siRNA 전염조 OSX mRNA 표체재전염후24 h 급48 h 균명현저우철고동조（균 P ﹤0.01）；OSX 단백표체재전염후48 h 화72 h 균저우철고동조（균 P ﹤0.01），이48 h 최명현。siRNA유효침묵 OSX 기인표체후，OSX 전염조 Ibsp mRNA 화단백적표체저우철고동조（균 P ﹤0.01），이차세포개염침적저우고린조。결론 OSX - siRNA 가이유효억제 VSMC OSX 기인화단백적표체，종이억제철고동유도적 VSMC 성골양분화화세포개화。OSX 가이성위 CKD 혈관개화치료적파점。
Objective To explore the effect of OSX gene silenced by siRNA on osteogenic differentiation and calcification of vascular smooth muscle cells(VSMC),induced by aldosterone in vitro. Methods VSMCs were cultured in vitro and passaged 4 to 9 times. Transfection condition had been optimized by FAM fluorescent labeling - siRNA. After transfection with siRNA sequence,VSMCs were divided into 4 groups:①control,②aldosterone(1. 5 mmoL/ L),③siRNA transfection:aldosterone + OSX - siRNA,④negative transfection control:aldosterone +negative control siRNA. OSX and Ibsp mRNA or protein expression had been detected by realtime - PCR and Western blot. Calcium deposition was visualized by Alizarin staining. Results At 24 and 48 h after transfection,the expression of OSX mRNA in siRNA transfection group was sig-nificantly lower than that of aldosterone group(all P ﹤ 0. 01). At 48 and 72 h after. transfection,the expression of OSX protein in siRNA trans-fection group was significantly lower than that of aldosterone group(all P ﹤ 0. 01). While OSX gene was silenced by siRNA in siRNA transfection group,the mRNA and protein expression of Ibsp was significantly declined( all P ﹤ 0. 01)and calcium deposited. Conclusion OSX - siR-NAKiSS - 1 can suppress the expression of VSMC OSX gene and protein,therefore,osteogenetic differentiation and calcification of vascular smooth muscle cells induced by aldosterone is inhibited.