氢化可的松联合rmIL-2、rmIL-15体外扩增小鼠NK细胞  

Combination use of hydrocortisone, rmIL-2 and rmIL-15 in amplification of mouse NK cells in vitro

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作  者:高祥[1] 黄婧[1] 刘钊[1] 骆晨[1] 罗诗樵[1] 

机构地区:[1]重庆医科大学附属第一医院肝胆外科,400016

出  处:《免疫学杂志》2015年第7期566-571,共6页Immunological Journal

基  金:国家自然科学基金(H1006/30972789);重庆市自然科学基金(cstc2013jcyj A10105);重庆市医学科技计划项目(2008-2-02)

摘  要:目的建立氢化可的松联合rmIL-2、rmIL-15体外扩增小鼠NK细胞的方法 ,观察该培养方法对扩增后NK细胞的纯度、扩增效率及功能的影响。方法应用免疫磁珠分选法(MACS)分离得到高纯度的小鼠脾脏CD3-CD49b+NK细胞,依据添加刺激因子的不同将纯化后的NK细胞分成A组(rmIL-2)、B组(rmIL-2+rmIL-15)和C组(氢化可的松+rmIL-2+rmIL-15)进行体外培养,每3日添加新鲜培养基及细胞刺激因子。采用台盼蓝拒染法进行活细胞计数;流式细胞术检测CD3-CD49b+NK细胞纯度;CFSE稀释法检测NK细胞的增殖效率;MTT比色法检测NK细胞杀伤活性;流式细胞术检测NK细胞膜表面CD107a的表达。结果磁珠分选后NK细胞纯度由分选前的(8.59±1.41)%提高为分选后的(91.60±1.33)%。体外扩增培养15 d后A组扩增的NK细胞纯度降低[(75.02±3.48)%,P<0.01],B组(85.87±4.79)%和C组(88.04±3.35)%与扩增前相比较无明显差异(P>0.05)。扩增15 d后C组NK细胞扩增倍数为(45.06±1.64)倍,显著高于A组(5.28±0.34,P<0.01)和B组(25.39±3.29,P<0.05)。细胞增殖实验结果表明培养第7天和第14天各组NK细胞增殖指数(PI)分别为A组(2.26±0.81)和(3.27±1.21),B组(4.62±1.45)和(15.82±3.92),C组(6.91±1.34)和(23.66±5.42)。培养第7天和第14天NK细胞增殖指数C组>B组>A组(P<0.05)。当效靶比为10∶1时,C组扩增后NK细胞肿瘤杀伤率为(81.27±2.32)%,显著高于A组[(37.20±7.32)%,P<0.01]和B组[(69.51±6.32)%,P<0.05]扩增后NK细胞。C组(49.32±4.36)%扩增后NK细胞CD107a表达水平显著高于A组[(9.37±1.82)%,P<0.001]和B组[(28.46±4.21)%,P<0.01)]NK细胞。结论氢化可的松联合rmIL-2+rmIL-15培养方案能够有效地体外扩增并活化小鼠NK细胞,为NK细胞的肿瘤过继免疫治疗应用于临床奠定了实验基础。This study was purposed to explore the effects of hydrocortisone, rmIL-2, and rmlL-15 on the purity, cytotoxicity and amplification efficiency of mouse NK cells in vitro. Three kinds of methods to expand mouse NK cells were compared, including methods A (rmIL-2), B (rmIL-2 +traiL-15) and C (rmlL-2 +traiL-15 + hydrocortisone). CD3-CD49b (DX5)+ mouse NK cells were separated from spleen and expanded in three groups as described. The purity of fresh and expanded NK cells was detected by flow cytometry. The numbers of NK cells were counted every 3 days and the proliferation index (PI) of expanded NK cells in three groups was measured by CFSE dilution assay at day 7 and day 14. Cytotoxicity of expanded NK cells was measured by MTT assay and CD107a release assay. Data showed the purity of NK cells increased to (91.60±1.33)% after MACS sorting. After expansion for 15 days, the purity of NK cells in group A was decreased from (91.60±1.33)% to (75.02±3.48)% (P〈 0.01), but there were no significance difference in the purity of fresh purified NK cells between group B ((85.87± 4.79)%, P〉 0.05) and group C ((88.04±3.35)%, P〉 0.05). The NK cells in group C were expanded for (45.06±1.64) folds, obviously higher than that in group A (5.28±0.34, P〈 0.01) and group B (25.39±3.29, P〈 0.05). CFSE dilution assay indicated that the proliferation index (PI) of NK cells in group C was (6.91±1.34) on day 7 and (23.66±5.42) on day 14, which was higher than that in group B ((4.62±1.45) on day 7, (15.82±3.92) on day 14) and group A ((2.26± 0.81) on day 7. (3.27±1.21] on day 14). The cytotoxicity ability of NK cells expanded in group C was (81.27±2.32)% when the E:T was 10:l, which was significantly higher than that in group A ((37.20±7.32)%, P 〈 0.01) and group B ((69.51 ±6.32)%, P〈 0.05). We also found the level of CD107a expression on NK cells in group C was (49.32±4.36)%, whi

关 键 词:NK细胞 分离 扩增增殖 氢化可的松 

分 类 号:R965[医药卫生—药理学]

 

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