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目的建立不同阶段的泡沫细胞,并对其生物特性进行鉴定。方法体外培养人THP-1来源的巨噬细胞(THP-M),由ox-LDL诱导其转化为不同阶段泡沫细胞,ELISA方法检测不同阶段泡沫细胞内总胆固醇和胆固醇酯含量;透射电镜和油红O染色光镜观察细胞内脂质,噻唑蓝染色法测定不同阶段泡沫细胞的活性变化规律。Western blot方法测定II型微管相关蛋白1轻链3(LC3II)蛋白表达水平。结果 24h开始,THP-M细胞质内出现大量的红染颗粒或脂质空泡,总胆固醇和胆固醇酯均较对照组增加,细胞胆固醇酯含量大于总胆固醇的50%,泡沫细胞已经形成。噻唑蓝染色结果显示,自48h开始少量泡沫细胞出现死亡现象(存活率93.21±3.66%),60h和72h细胞存活率率分别为72.15±2.23%和63.06±1.83%。电镜扫描结果发现,24h细胞内脂滴聚集,24h内细胞浆自噬小体逐渐增多。36h、48h细胞内脂滴大小和形态各异,可见吞噬脂滴后尚未消化完全的自噬溶酶体,部分细胞膜破裂;60h和72h,细胞内自噬小体数量明显减少,细胞大量崩解。Western blot检测结果表明自噬标志性蛋白LC3II表达水平变化规律与电镜观察自噬小体结果一致。结论利用oxLDL诱导THP-M24h、36h-48h、60h-72h后,细胞形态和细胞内脂含量分别符合早期、中期和晚期泡沫细胞的生物特征,成功建立了早、中、晚期泡沫细胞模型,其中细胞活力变化可能与II型凋亡(自噬性死亡)相关。为不同病程AS研究提供支持。
Objective To establish foam cells in different stages and identify their biological characteristics. Methods Human THP-1-derived macrophages (THP-M) were cultured in vitro and transformed into foam cells by ox-LDL. The levels of total cholesterol and cholesteryl ester in foam cells at different stages were measured by ELISA. Transmission electron microscopy and oil Red O staining was used to observe intracellular lipid. The activity of foam cells at different stages was determined by thiazolyl blue staining. The expression of type II microtubule-associated protein 1 light chain 3 (LC3II) protein was determined by Western blot. Results At 24 h, a large amount of red staining granules or lipid vacuoles appeared in the cytoplasm of THP-M cells. The total cholesterol and cholesterol esters increased compared with the control group. The content of cholesterol ester in cells was more than 50% of total cholesterol, and foam cells had formed. The results of thiazolyl blue staining showed that a small amount of foam cells died after 48h (survival rate 93.21 ± 3.66%). The survival rates at 60h and 72h were 72.15 ± 2.23% and 63.06 ± 1.83%, respectively. Electron microscopy results showed that lipid droplets accumulate within 24h cells within 24 hours of autophagosome gradually increased. 36h, 48h lipid droplets within the cell size and shape, showing phagocytic lipid droplets have not yet been digested completely autophagic lysosomes, some of the cell membrane rupture; 60h and 72h, significantly reduced the number of intracellular autophagosomes, a large number of cell disintegration . Western blot results showed that the expression pattern of LC3II, an autophagy marker protein, was consistent with the result of electron microscopy. Conclusions The cell morphology and intracellular lipid content of THP-M24h, 36h-48h and 60h-72h induced by oxLDL were consistent with the biological characteristics of early, middle and late foam cells, respectively. The early, middle and late foam cell models were successfully established, in which Changes in cell viability may be associated with type II apoptosis (autophagic death). Support for different duration of AS research.