小鼠肝脏自然杀伤细胞分离纯化及受体表达分析  

Isolation and purification of murine hepatic NK cells and analyse of the expression of NK cell receptors

作　　者：邹勇[1] 陈韬[1] 严伟明[1] 王洪武[1] 宋戈[1] 肖非[1] 罗小平[2] 宁琴[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院感染科,感染性疾病研究所,湖北武汉430030 [2]华中科技大学同济医学院附属同济医院儿科

出　　处：《中西医结合肝病杂志》2008年第5期277-279,共3页Chinese Journal of Integrated Traditional and Western Medicine on Liver Diseases

基　　金：973计划重要传染病基础研究专项(2007CB512900;2005CB522901);国家自然科学基金面上项目(NSFC30672380;30571643)

摘　　要：目的：建立一种有效分离纯化小鼠肝脏自然杀伤细胞（NK细胞）的方法,并对其表面几种NK细胞受体（NKP46、NKG2D、NKG2A/C/E和Ly49A/C/I）的表达进行分析。方法：首先利用Percoll不连续密度梯度离心分离小鼠肝脏淋巴细胞,进一步经CD5（Ly-1）磁珠阳性分选清除CD3^＋DX5^＋T细胞,最后利用CD49b（DX5）磁珠阳性分选肝脏NK（CD3^-DX5^＋）细胞。分离后的细胞经台盼蓝染色检测细胞存活率,流式细胞术检测肝脏NK细胞纯度及几种NK细胞受体的表达百分率。结果：不连续密度梯度离心能有效分离出肝脏淋巴细胞,CD5（Ly-1）磁珠清除CD3^＋DX5^＋T细胞后再用CD49b（DX5）磁珠阳性分选出肝脏NK（CD3^-DX5^＋）细胞,纯度可达到（94.8±1.0）%,存活率为92%～95%。NK细胞受体NKP46、NKG2D、NKG2A/C/E和Ly49A/C/I在小鼠肝脏NK细胞上的表达百分率分别为（97.9±0.7）%、（93.2±1.7）%、（57.2±0.9）%和（63.3±1.9）%。结论：成功建立了一种有效分离纯化小鼠肝脏NK细胞的方法,掌握了正常小鼠肝脏NK细胞上几种重要受体的表达情况,为进一步在病毒性肝炎小鼠模型中进行肝脏NK细胞功能学研究及受体基因芯片分析奠定了基础。Objective： To estabish a efficient method for isolating and purifying the murine hepatic： natural killer cells （NK） and to analyze the expression of several NK receptors including NKP46, NKG2D, NKG2A/C/E and Ly49A/C/ I. Methods ： Murine hepatic lymphocytes were first separated by percoll discontinue density gradient centrifugation. CD3^ ＋ DX5 ^＋ T cells were then deleted by a positive sorting with CD5 magnetic microbeads. At last, hepatic NK cells （ CD3^ - DX5 ^＋ ） were positively sorted by DX5 （anti-CD49b） magnetic microbeads. The purity and the cell survival rate of the sorted cells were measured by flow cytometry and trypan blue dyeing, respectively. The expression of several NK cells receptors on hepatic NK cells were analyzed by flow cytometry. Results ： The purity of hepatic NK （ CD3^ - DX5^＋ ） cells reached （ 94. 8 ± 1.0 ） % with the survival rate of 92% - 95%. The percentages of hepatic NK cells expressing NKP46, NKG2D, NKG2A/C/E and Ly49A/C/I were （97.9±0.7） %, （93.2 ± 1.7） %, （57.2 ± 0. 9） % and （63.3 ± 1.9） %, respectively. Conclusion： A efficient method for isolating and purifying murine hepatic NK cells is established successfuly, the expression of several important NK receptors including NKG2D and NKP46 is analyzed, which provides the basis for subsequent studies of hepatic NK cells founction and microray analyse of hepatic NK cell receptors in the mouse models of viral hepatitis.

关 键 词：自然杀伤细胞 受体 密度梯度离心 免疫磁珠分选 

分 类 号：R392[医药卫生—免疫学]