炎症对高磷所致大鼠血管平滑肌细胞钙化的影响及机制探讨

摘要

目的：探讨炎症对高磷所致大鼠血管平滑肌细胞钙化的影响及可能机制。方法原代培养大鼠血管平滑肌细胞，分为空白对照组、高磷组、炎症组及高磷＋炎症组。高磷组、高磷＋炎症组在加入4．8 mmol／L磷的基础培养基中培养，建立高磷诱导的大鼠血管平滑肌细胞钙化模型。高磷＋炎症组加入10μg／mL细菌脂多糖（LPS），炎症组只加入10μg／mL细菌脂多糖（LPS），空白对照组在基础培养基中培养。各组均培养48 h后，检测培养液C反应蛋白（CRP）水平、细胞碱性磷酸酶（ALP）活性及钙沉积量；Realtime-PCR法检测细胞骨形成蛋白-2（BMP-2）、肿瘤抑制基因4（Smad4）、肌肉片段同源盒2（Msx2）及成骨细胞特异性转录因子（Osterix）mRNA相对表达量。结果高磷组、炎症组及高磷＋炎症组培养液中 CRP水平、细胞 ALP 活性及钙沉积量均高于空白对照组（P均＜0．05），且高磷＋炎症组培养液中CRP水平、细胞ALP活性及钙沉积量均高于高磷组及炎症组（P均＜0．05），炎症组培养液中CRP水平和细胞钙沉积量均低于高磷组（P均＜0．05）。高磷组、炎症组及高磷＋炎症组细胞中BMP-2、Smad4、Msx2及Osterix mRNA相对表达量均高于空白对照组（P均＜0．05），且高磷＋炎症组细胞中 BMP-2、Smad4、Msx2及Osterix mRNA相对表达量均高于高磷组及炎症组（P均＜0．05），炎症组细胞中Smad4、Msx2及Os-terix mRNA相对表达量均低于高磷组（P均＜0．05）。结论炎症可显著加重高磷所致的大鼠血管平滑肌细胞钙化，其作用机制可能与上调BMP-2、Smad4、Msx2、Osterix表达及ALP活性等有关。%Objective To study the effects and possible mechanisms of inflammation on the calcification of rat vascu-lar smooth muscle cells (VSMCs)primarily caused by high phosphate.Methods Rat VSMCs were primarily cultured and then were divided into the control (Ctr)group,high phosphate (Pi)group,inflammation (LPS)group,high phosphate and inflammation (Pi+LPS)group.Rat VSMCs in the Pi group and Pi+LPS group were cultured in basal medium contai-ning 4.8 mmol/L Pi to establish the calcification models successfully.Rat VSMCs in the Pi +LPS group were cultured in basal medium containing 4.8 mmol/L Pi and 10 μg/mL LPS.Rat VSMCs in the LPS group were cultured in basal medium only containing 10 μg/mL LPS.Rat VSMCs in the Ctr group were cultured in basal medium.The C-reactive protein (CRP)level of culture medium,alkaline phosphatase (ALP)activity and calcium deposition of cells in each group were respectively measured after 48-hour culture;real-time PCR was used to detect the relative mRNA expression of bone mor-phogenetic protein-2 (BMP-2),Smad4,Msx2 and Osterix.Results The CRP level of culture medium,ALP activity and calcium deposition of cells in the Pi group,LPS group and Pi+LPS group were higher than those of the Ctr group (all P<0.05).The CRP level of culture medium,ALP activity and calcium deposition of cells in the Pi+LPS group were higher than those in the Pi group and LPS group (all P<0.05).The CRP level of culture medium and calcium deposition of cells in the LPS group were lower than those of the Pi group (all P<0.05).The relative mRNA expression of BMP-2,smad4, msx2,and Osterix in the Pi group,LPS group and Pi+LPS group was higher than that of the Ctr group (all P<0.05 ). The relative mRNA expression of BMP-2,smad4,msx2 and Osterix in the Pi+LPS group was higher than that of the Pi group amd LPS group (all P<0.05 ).The relative mRNA expression of smad4,msx2 and Osterix in the LPS group was lower than that of the Pi group (all P<0.05).Conclusions The calcification of rat VSMCs cultured in vitro could be in-duced by both high phosphate and inflammation.Inflammation combined with high phosphate could aggravate the calcifica-tion of rat VSMCs cultured in vitro significantly.The mechanisms may be ralted with the up-regulation of BMP-2,Smad4, Msx2,Osterix expression and the ALP activity.