RAW264.7巨噬细胞源性泡沫细胞模型的建立及鉴定

The Model Estabilishment and Identification of RAW264.7 Macrophage-Derived Foam Cell

目的以小鼠巨噬细胞RAW 264.7为对象,建立简便而又准确的泡沫细胞诱导及鉴定方法。方法将实验分为正常对照组和不同浓度氧化型低密度脂蛋白与细胞孵育组,在以孵育时间为24 h的前提下,用MTT法和流式细胞术来确定诱导泡沫细胞的氧化型低密度脂蛋白合适浓度区间范围,并用总胆固醇试剂盒和游离胆固醇试剂盒测定不同程度泡沫细胞内胆固醇酯含量。结果氧化型低密度脂蛋白浓度范围为20～30 mg/L时,细胞存活率已受到显著抑制,并随着氧化型低密度脂蛋白浓度的增高细胞凋亡率和坏死率逐渐增大,起初以细胞凋亡为主,当氧化型低密度脂蛋白浓度大于40 mg/L时凋亡的细胞不断坏死。20 mg/L和30 mg/L氧化型低密度脂蛋白与RAW 264.7细胞共孵育24 h,细胞内胆固醇酯比重分别为66.26%和71.19%,而10 mg/L的氧化型低密度脂蛋白诱导24 h的泡沫细胞不典型,40 mg/L以上的氧化型低密度脂蛋白诱导的泡沫细胞泡沫化程度严重,贴壁不牢,大部分细胞破裂或凋亡,脂滴散布于细胞外。结论 20～30 mg/L氧化型低密度脂蛋白诱导的RAW 264.7巨噬泡沫细胞模型稳定且形态较完整,符合泡沫细胞的形态学特征。

Aim To establish the model of murine macrophage RAW264.7 derived foam cell and identify them with simple and accurate method. Methods The experiment was devided into normal control group and six experimental groups of different concentration of oxidized low density lipoprotein（ox-LDL） incubated with cell.On the incubation time for 24 hours that MTT method and flow cytometry（FCM） were used to determine the suitable range of ox-LDL concentration,then measure intra-cellular cholesterol ester in different level of macrophage foam cell with total cholesterol and free cholesterol kit. Results Cell viability had been significantly inhibited when ox-LDL concentration was at the range of 20～30 mg/L,when ox-LDL concentration was greater than 40 mg/L,apoptosis cells continuously necrosis.Ox-LDL at the concentration of 20 and 30 mg/L,incubated with cell for 24 hours,the propotion of intra-cellular cholesterol ester were 66.26% and 71.19%.Foam cell induced by 10 mg/L of ox-LDL was not typical,and by 40 mg/L of ox-LDL or more,severe degree of foam cell led to cells floating,most cells were rupture or apoptosis,lipid droplet dispersed in extra-cellular. Conclusion Ox-LDL at the concentration range of 20～30 mg/L,incubated with RAW264.7 for 24 h,induced foam cell has good morphology,and model stability,meet the morphology feature of foam cell.