Ficoll和Percoll法分离结直肠癌相关巨噬细胞的比较  

A comparative study of tumor-associated macrophages isolation in colorectal carcinoma by Ficoll-Hypaque density gradient centrifugation and Percoll density gradient centrifugation

作　　者：杨芳[1] 伍菲凡 王晓燕[1] 胡志燕[1] 李祖国[1] 

机构地区：[1]南方医科大学基础医学院病理科,广州市510515

出　　处：《实用医学杂志》2015年第9期1394-1398,共5页The Journal of Practical Medicine

摘　　要：目的：比较Percoll非连续密度梯度离心法、Ficoll-Hypaque 密度梯度离心法分离结直肠癌中的巨噬细胞的效果，并进一步探讨贴壁分离巨噬细胞的最适宜贴壁时间。方法：取结直肠癌患者的肿瘤组织，制成单个细胞的悬液，分别使用100％ Ficoll、35％ Percoll 以及25％与65％双梯度 Percoll 进行巨噬细胞分离，37℃培养2 h后，去除未贴壁的细胞，使用免疫荧光实验来检测巨噬细胞的纯度。之后，将Ficoll分离得到的单个核细胞，分别培养1、2、4和9 h后比较巨噬细胞的纯度。结果：Ficoll-Hypaque 密度梯度离心法分离的巨噬细胞的纯度为60．18％，Percoll 非连续密度梯度离心法分离的巨噬细胞的纯度分别为54．33％和10．93％，均低于Ficoll法分离所得到的巨噬细胞的纯度（P ＜0．05）。 Ficoll分离后贴壁1、2、4 h 分离得到的巨噬细胞纯度均达到60％以上，高于贴壁培养9 h得到的巨噬细胞的纯度（34．67％）。结论： Ficoll-Hypaque密度梯度离心法分离巨噬细胞，纯化程度好，是一种简单、高效的巨噬细胞分离方法，适于临床和科研中广泛应用。贴壁培养2～4h是贴壁分离巨噬细胞最适宜的时间。Objective Percoll density gradient centrifugation and Ficoll-Hypaque density gradient cen-trifugation, which are frequently-used methods for separation of tumor-associated macrophages （TAMs） from solid carcinoma were compared, in order to find an effective way to separate TAMs from colorectal carcinoma （CRC）. Furthermore, we studied the best adherence time of separating macrophage among mononuclear cells. Methods specimens were collected from CRC patients , after digesting into single cells , TAMs were separated from the same specimen by 100% Ficoll, 35% percoll and 25% combined with 65% percoll respectively. After these pre-liminary separation, the collected cells were purified a second time by adherence separation. The purity of TAMs were detected by immunofluorescence. Results TAMs purity from Ficoll-Hypaque density gradient centrifugation was 80.18%, statistically higher than that from Percoll density gradient centrifugations （54.33% and 10.93% re-spectively）. Conclusion Compared to Percoll density gradient centrifugation, Ficoll-Hypaque density gradient centrifugation is a more effective and simple way to isolate TAMs from colorectal carcinoma , suggesting it can be wildly used in clinical and basic medical research. 2-4 hours is the best adherence time for isolating macrophage.

关 键 词：结直肠肿瘤 肿瘤相关巨噬细胞 Ficoll-Hypaque密度梯度离心法 Percoll非连续密度梯度离心法 贴壁分离 

分 类 号：R735.34[医药卫生—肿瘤]