钙化血管平滑肌细胞肾上腺髓质素及其受体系统的基因表达上调

Upregulation of Gene Expression of Adrenomedullin and its Receptor System in Calcified Vascular Smooth Muscle Cells

探讨钙化的血管平滑肌细胞肾上腺髓质素生成和肾上腺髓质素受体系统一降钙素受体样受体和受体活性修饰蛋白基因表达的改变及其病理意义。采用β-甘油磷酸盐诱导培养的大鼠血管平滑肌细胞钙化;放射免疫法测定血管平滑肌细胞分泌的肾上腺髓质素含量;半定量逆转录聚合酶链反应测定细胞肾上腺髓质素、降钙素受体样受体和受体活性修饰蛋白的mRNA水平;原子吸收分光光度计测定细胞钙含量;碱性磷酸酶试剂盒测定血管平滑肌细胞碱性磷酸酶活性;β液体闪烁计数仪测定45Ca2+放射活性。结果发现,与非钙化血管平滑肌细胞比较,钙化血管平滑肌细胞内钙含量、45Ca2+摄入及碱性磷酸酶活性分别增加118％、174％和7倍(P<0.01);钙化细胞肾上腺髓质素分泌量增高99％(P<0.01),肾上腺髓质素、降钙素受体样受体、受体活性修饰蛋白2和3的mRNA水平分别增加78％、93.7％、91.8％和109.5％(P均<0.01)。肾上腺髓质素与降钙素受体样受体、受体活性修饰蛋白2和3的mRNA水平呈正相关,其相关系数分别为0.83、0.92和0.93(P均<0.01)。结果提示,血管平滑肌细胞肾上腺髓质素 旁/自分泌功能改变可能参与血管钙化的调节过程。

Aim To explore the production of adrenomedullin (ADM), changes and significance of ADM, ADM receptor system-calcitonin receptor like receptor (CRLR) and receptor activity modifying proteins (RAMP) mRNA in calcified vascular smooth muscle cells (VSMC). Methods Calcification of cultured rat VSMC was produced by incubation with β-glycerophos phate. Content of ADM released by VSMC was measured by radioimmunoassay (RIA). The amount of ADM, CRLR and RMAP mRNA was determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The intracellular calcium content, alkaline phosphatase activity and cellular 45 Ca2+ uptake were determined. Results The content of calcium, 45 Ca2+ uptake and alkaline phosphatase activity in calcified VSMC increased by 118% , 174% and 7-fold (all P < 0.01), respectively, compared with control VSMC. Content of ADM in medium increased by 99% (P < 0.01). Furthermore, it was found that the levels of ADM, CRLR, RAMP 2 and RAMP 3 mRNA in calcified cells were elevated by 78% , 93.7% , 91.8% and 109.5 % (all P < 0.01), respectively, compared with control. The elevated levels of CRLR, RAMP 2 and RAMP 3 mRNA were positively correlated with ADM mRNA ( r = 0.83, 0.92 and 0.93, respectively, P < 0.01) in calcified VSMC. Conclusion Calcified VSMC generated an increased amount of ADM, up-regulated gene expressions of ADM, CRLR, RAMP 2 and RAMP 3, which suggested that changes of autocrine/paracrine function of vessel might be involved in the regulation of vascular calcification .