炎症致巨噬细胞和血管平滑肌细胞低密度脂蛋白受体反馈调控差异的研究

Investigation of differences in LDLr feedback regulation in macrophages and vascular smooth muscle cells under inflammatory stress

目的：探讨在生理及炎症应激条件下，人单核细胞系（T HP-1）巨噬细胞和血管平滑肌细胞（VSMCs）、低密度脂蛋白受体（LDLr）的负反馈调控的细胞特异性差别及其可能的机制。方法脂多糖（LPS）加入T HP-1巨噬细胞和VSMCs培养基中诱导炎症应激，酶学法检测细胞内胆固醇水平，反转录聚合酶链反应（RT-PCR）法检测LDLr、SCAP、固醇调控元件结合蛋白2（SREBP2）mRNA水平。结果生理条件下，LDL负荷增加细胞内胆固醇水平，进而减少 THP-1巨噬细胞和 VSMCs LDLr mRNA 水平。VSMCs IC50为11．25μg/mL ，低于THP-1巨噬细胞的18．125μg/mL。0～400 ng/mL LPS 呈剂量依赖性上调两种细胞 LDLr mRNA 水平，但VSMCs LDLr曲线比THP-1巨噬细胞LDLr曲线平坦，200 ng/mL LPS处理下，THP-1巨噬细胞LDLr mRNA上调倍数远高于VSMCs（0．33和0．04）。LDLr阻断剂肝素钠减少两种细胞内由LPS诱导的胆固醇沉积。生理条件下，LDL负荷减少SREBP2和SCAP mRNA水平，而LPS增加SREBP2和SCAP mRNA水平。结论炎症应激在两种细胞中均扰乱细胞内胆固醇水平介导的LDLr负反馈调控，THP-1巨噬细胞LDLr上调程度更大，这可能是炎症应激下T HP-1巨噬细胞更易泡沫化的一个原因。

Objective To investigate cell-specific regulation of LDLr in THP-1 macrophages and human VSMCs under physiological and inflammatory conditions and its potential mechanisms .Methods Inflammatory stress was induced by adding LPS to human THP-1 macrophages and human VSMCs .Intracellular total cholesterol （TC） , free cholesterol （FC） and cholesterol ester （CE） were examined by an enzymic assay .Total cellular RNA was isola-ted from cells for detecting LDLr ,SREBP-2 and SCAP mRNA levels using real-time PCR .Results LDL loading in-creased intracellular cholesterol level ,thereby reduced LDLr mRNA level in both T HP-1 macrophages and VSMCs under physiological conditions .The IC50 in VSMCs was 11 .25 μg/mL ,which is much lower than 18 .125 μg/mL in THP-1 macrophages .With the increase in concentration of LPS （0-400 ng/mL） ,the LDLr mRNA levels were up-regulated in both cells ,but the curve of LDLr mRNA in VSMCs showed more flat than that of THP-1 macrophages . Under the treatment of 200 ng/mL of LPS ,the upregulation fold （URF） of the LDLr mRNA in THP-1 macrophages was much higher than that of VSMCs （0 .33 VS 0 .04） .LDLr blocking agent heparin decreased lipid droplets induced by LPS significantly in THP-1 macrophages and VSMCs .LDL loading reduced the SREBP2 and SCAP mRNA level under physiological conditions .Exposure to LPS caused over-expression of SREBP2 and SCAP despite a high concen-tration of LDL in the culture medium .Conclusion Inflammatory stress disrupts LDLr negative feedback regulation induced by intracellular cholesterol in both cell types ,to a greater degree in THP-1 macrophages ,which could be one reason why THP-1 macrophages are more prone to become foam cells under inflammatory stress .