不同阶段泡沫细胞模型的建立与鉴定

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。

Establishment and Identification for Different Stages of Foam Cells

Objective To establish and identify different stages of foam cell models. Methods THP-1 cell derived macrophage（THP-M） was induced with ox-LDL（80μg/ml） into different stages of foam cells models. Total cholesterol and cholesterol ester content in foam cells were detected by ELISA; Transmission electron microscopy（SEM） and oil red O staining were used to observe accumulated lipid in cells, MTT staining was used to determine foam cells viability at different stages. Results A lot of oil red O positive cells was observed in ox-LDL group since 24 h; Total cholesterol and cholesterol ester significantly increased in 24h-THP-M than control group. Cholesterol ester content is more than 50% of the total cholesterol, indicating that foam cells have been formed. MTT staining results showed that the survival rate of foam cells were respectively 93.21±3.66%, 72.15±2.23% and 63.06±1.83% after THP-M cells have cocultured with ox-LDL for 48 h, 60 h and 72 h; SEM results showed that intracellular lipid drops gathered apparently, cytoplasmic autophagosome increased gradually within 24 h. when incubated with ox-LDL for 36 h or 48 h, intracellular lipid droplets are different from each other both in size and shape. Autophagolysosome with half-digested lipid drops were visible. Some foam cells burst; when cocultured for 60 h and 72 h, the number of autophagosomes decreased significantly in the cell and more cells disrupted. Western blot test results showed that LC3II protein expression level was consistent with the results from SEM at different stages. Conclusion When cocultured with ox-LDL for 24h, or 36 h-48 h, or 60h-72h, THP-M cell morphology and lipid content were in line with the characteristics of foam cells at early stage, middle stage or late stage, respectively. So, we obtained different stages of foam cell models successfully. And cell vitality changes during those courses may be linked to type II apoptosis（autophagy death）.