核心结合因子α-1基因沉默对高磷诱导的血管平滑肌细胞钙化的影响  

Effects of core-binding factor α1 gene silenced by siRNA on calcification of vascular smooth muscle cells induced by high phosphate

作　　者：余毅[1] 魏培丹[1] 

机构地区：[1]南京军区福州总医院血液净化科,350025

出　　处：《中华医学杂志》2014年第4期251-255,共5页National Medical Journal of China

基　　金：福建省科技计划重点项目（2013Y0069）

摘　　要：目的研究核心结合因子α-1（Cbfα-1）基因沉默对高磷诱导的血管平滑肌细胞（VSMC）成骨样分化和钙化的影响。方法体外原代培养大鼠VSMC。针对大鼠Cbfα-1基因设计合成4对候选小分子干扰RNA（siRNA）序列，以Lipo2000为载体，转染体外培养的VSMC；FAM荧光标记siRNA优化转染条件。反转录（RT）．PCR检测Cbfα-1 mRNA表达，筛选有效siRNA序列。将有效siRNA序列转染VSMC，细胞分为4组：（1）正常磷组（1．3mmol／L）；（2）高磷组（2．6mmol／L）；（3）siRNA转染组：高磷＋Cbfα-1-siRNA；（4）阴性转染对照组：高磷＋阴性对照siRNA。RT-PCR和Western印迹法检测Cbfα-1、骨桥蛋白（OPN）基因和蛋白表达；茜素红染色观察细胞钙盐沉积。结果在Cbfα-1-siRNA浓度为100nmol／L、Lipo为8μL／孔转染条件下，转染效率约55％；筛选最佳干扰序列Cbfa．1siRNAl952，转染24h沉默效率可达81．8％。与高磷组相比，siRNA转染组Cbfα-1mRNA表达在转染后24和48h均明显低于高磷组（24h：0．335±0．059比0．714±0．106，48h：O．574±0．036比0．726±0．086，均P〈0．01）；Cbfα-1蛋白表达在转染后48和72h均显著低于高磷组（均P〈0．01），以48h最明显。siRNA有效沉默cbfα-1基因表达后，siRNA转染组OPNmRNA和蛋白的表达明显低于高磷组（均P〈0．05），且细胞钙盐沉积亦明显低于高磷组。结论化学合成的Cbfα-1 siRNA可有效抑制VSMC Cbfα-1基因和蛋白的表达，从而抑制高磷诱导的VSMC成骨样转分化和细胞钙化。Cbfα-1可望成为CKD血管钙化治疗的靶点。Objective To explore the effects of core-binding factor αl （Cbfα-1） gene silenced by siRNA on osteogenic differentiation and calcification of vascular smooth muscle cells （VSMC） induced by high phosphate in vitro. Methods VSMC were cultured in vitro and passaged 3 to 8 times. Four pairs of Cbfα-1 siRNA were designed and synthesized. Transfection was performed with cationic lipid vectors （Lipofectamine 2000）. Transfection conditions were optimized by the FAM fluorescent labeling-siRNA to screen effective siRNA sequences by reverse transcription-polymerase chain reaction （RT-PCR）. After transfection with effective siRNA sequences, VSMCs were divided into 4 groups ： （ 1 ） normal phosphate （ Pi 1.3 mmol/L） ; （2） high phosphate （ Pi 2.6 mmol/L） ; （3） siRNA transfection ： high phosphate ＋ Cbfα-1- siRNA; （4） negative transfection control： high phosphate ＋ negative control siRNA. Cbfα-1 and osteopontin （OPN） mRNA and protein expression were detected by RT-PCR and Western blotting. Calcium deposition was visualized by Alizarin stain method. Results The transfeetion efficiency was around 55% with a concentration of Cbfα-1 siRNA 100 nmol/L and Lipo 8 μl/ well. Cbfα-1 siRNA 1952 was chosen as the effective sequence with a suppression ratio up to 81.8%. At 24 and 48 h post-transfection, the expression of Cbfα-1 mRNA was significantly lower in siRNA transfection group than that in high phosphate group （0. 335± 0. 059 vs 0.714 ±0. 106, 0.574 ± 0.036 vs 0. 726 ± 0. 086, all P 〈 0.01 ）. At 48 and 72 h post- transfection, the expression of Cbfα-1 protein in siRNA transfection group was significantly lower than that in high phosphate group （ both P 〈 0.01 ）. While Cbfα-1 gene was silenced by siRNA in siRNA transfection group, the mRNA and protein expression of OPN significantly declined（ all P 〈 0.05 ） and calcium depositionin cell layers decreased. Conclusions Cbfα-1 siRNA can effectively inhibit the expression of Cbfα-1 mRNA and protein in VS

关 键 词：血管中膜 肾机能不全 慢性 动脉硬化 RNA 小分子干扰 核心结合因子 d1亚基 

分 类 号：R363[医药卫生—病理学]