摘要
目的对比胰酶消化法和混合酶(Ⅱ型胶原酶+Ⅰ型DNA酶)消化法分离小鼠胶质瘤浸润免疫细胞的效果。方法没有分组情况提取接种GL261胶质瘤细胞系21天后C57BL/6小鼠的脑组织,剪碎后A组用胰酶消化法,B组采用混合酶(Ⅱ型胶原酶+Ⅰ型DNA酶)消化法分别消化组织,通过Percoll密度梯度离心法获取肿瘤浸润免疫细胞,使用流式细胞术比较提取免疫细胞的表型特点。结果混合酶(Ⅱ型胶原酶+Ⅰ型DNA酶)消化法(B组)后,提取的小胶质细胞上CD11b的荧光强度比胰酶消化的A组强(P<0.05);肿瘤相关巨噬细胞(tumor-associated macrophages,TAM)、浸润的淋巴细胞、CD8^+T细胞的比例和数量明显高于A组胰酶消化后得到的细胞(P<0.05);CD4^+T细胞的数量明显高于A组胰酶消化后得到的细胞(P<0.05)。结论混合酶(Ⅱ型胶原酶+Ⅰ型DNA酶)消化法得到的肿瘤浸润免疫细胞增多,细胞表面膜蛋白不受破坏,适用于对胶质瘤浸润免疫细胞的分析。
Objective Objective To compare the effects of trypsin and multi-enzyme(TypeⅡcollagenase+DNaseⅠ)digestion on the isolation of murine glioma-infiltrating immune cells.Methods Brain tissues were collected from day 21 after the injection of GL261 glioma cells.After homogenization,the tissues were digested by trypsin(A group)and multi-enzyme(TypeⅡcollagenase+DNaseⅠ)(B group),respectively.Immune cells were collected by Percoll density gradient centrifugation and analyzed by flow cytometry.Results After the digestion of multi-enzyme(TypeⅡcollagenase+DNaseⅠ)(B group),the mean fluorescence intensity(MFI)of CD11b in microglia was significantly higher than that obtained with the digestion of trypsin(P<0.05).The frequency and absolute number of TAMs,infiltrating lymphocytes and CD8^+T cells were increased using the digestion of multi-enzyme(TypeⅡcollagenase+DNaseⅠ)compared to that using the digestion of trypsin(P<0.05).The number of CD4^+T cells was higher when tissues digested with multi-enzyme(TypeⅡcollagenase+DNaseⅠ)than that digested with trypsin(P<0.05).Conclusion After multi-enzyme(TypeⅡcollagenase+DNaseⅠ)digestion,the integrity of cell surface proteins of the immune cells was maintained.This method could be applied to the study of glioma-infiltrating immune cells.
作者
段瑾
杜铭
王笑
孙逊
Duan Jin;Du Ming;Wang Xiao;Sun Xun(Department of Immunology,Basic Medical college,China Medical University,Shenyang 110013,China)
出处
《国际免疫学杂志》
CAS
2019年第4期337-342,共6页
International Journal of Immunology
基金
国家自然科学基金(31370921,81771753).
