摘要
目的:建立小鼠NK细胞的分离及培养扩增的方法。方法:用两步黏附分离法和磁珠活化的细胞分选(MACS)法,从小鼠脾脏的单个核细胞(MNC)中分离NK细胞,计数所得细胞的总数,并用FITC抗小鼠CD3和PE抗小鼠NK1.1单克隆荧光抗体染色后,用流式细胞仪(FACS)检测NK细胞的纯度。以YAC -1为靶细胞,用MTT比色法检测NK细胞的杀伤活性。结果:将两步黏附分离法分离的2×107个脾MNC培养5、10、15、20d,计数细胞的总数并检测CD3-NK1.1+细胞百分率,分别为0.5×107、1.4×107、2.6×107、3.0×107和18.36%、43.44%、55.68%、60.03%。效靶细胞25∶1时,NK细胞的杀伤率分别为54.38%、66.54%、79.38%和83.86%,明显高于脾MNC(41.93%)(P<0.01)。MACS法纯化前后细胞的总数和CD3-NK1.1+细胞的百分率,分别为1.0×108、1.5×106和2.54%、93.60%;但纯化后的细胞在体外培养20d时,只扩增到1.9×106个。结论:用两步黏附分离法分离的小鼠NK细胞,培养2～3wk可获得大量的NK细胞,纯度可达55%～60%;在效靶细胞为25∶1时,NK细胞对YAC1细胞的杀伤率约为80%。
AIM: To establish a method to isolate, purify, and culture mouse nature killer (NK)-cells in vitro. METHODS: NK-cells were isolated from splenic mononuclear cells (MNC) by a two-step adherence system and magnetic microbeads actived cell sorting (MACS), then these cells were cultivated with feeder cells and IL-2 in RPMI 1640 medium for 5, 10, 15, and 20 days. The enriched cells were counted and stained with anti-mouse CD3-FITC and Anti-mouse NK1.1-PE. The purity of the NK-cells was determined by flow cytometrx and the cytotoxicity to YAC-1 targets was detected by MTT assay. RESULTS: In the two-step adherence system, the enriched cells cultivated for 5, 10, 15, and 20 days were 0.5×10~7, 1.4×10~7, 2.6×10~7, and 3.0×10~7 respectively, and the percent of CD3^- NK1.1^+ cells was 18.36%, 43.44%, 55.68%, and 60.03% respectively. The cytotoxicity to YAC-1 targets was significantly higher than that of splenic MNC(P<0.01), and increased from 41.93% up to 54.38%, 66.54%, 79.38%, and 83.86% respectively at 25∶1 of effector∶target ratio. After NK-cells were purified by MACS, the purity reached (93.60%), the enriched cells was 1.5×10~6, but proliferated to only 1.9×10~6 20 days later. CONCLUSION: NK-cells isolated by using the two step adherence system proliferate abundantly, and the purity reaches 55%~60%. The cytotoxicity to YAC-1 targets is about 80% at 25∶1 of effector: target ratio.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2005年第3期379-381,389,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家重点基础研究发展规划(973)项目(No.G2000057006)
家自然科学基金重点项目(No.30230350)
关键词
NK细胞
分离
培养
扩增增殖
IL-2
NK cell
isolation
culture
amplified proliferation
rhIL-2
