研究自然杀伤(natural killer, NK)细胞表面杀伤细胞免疫球蛋白样受体(killer immunoglobulinlike receptor，KIR)和HLACw配体不相合的异基因NK细胞对乳腺癌细胞的体外杀伤作用；分析杀伤活化受体NKG2D及其MICA配体表达水平与NK细胞对乳腺癌细胞杀伤敏感性之间的关系。方法：取10例健康人及5例乳腺癌患者外周血10 ml,采用MACS系统NK细胞免疫磁珠分选试剂盒负向分选高纯度NK细胞。取乳腺癌细胞(MCF7、MDAMB435s和SKBr3)和NK细胞各1×10 5个，碱裂解法抽提DNA，PCRSSP方法分别检测HLACw位点、KIR位点表达。MTT法检测KIR相合与不相合组的NK细胞对乳腺癌细胞的杀伤率。流式细胞术检测NK细胞NKG2D表达水平以及RTPCR方法检测乳腺癌细胞MICA表达。结果：MACS系统分选出的NK细胞，经流式细胞术检测，其纯度在90%以上；KIR与 HLACw相合组的NK细胞对乳腺癌细胞株的杀伤率明显高于不相合组，G1组和G2组NK细胞对MDAMB435s（G 1组）杀伤率分别为（73.2±14.5）%和（34.2±76）%，对SKBr3（G 1组）杀伤率为（67.3±12.5）%和（36.5±7.7）%，而对MCF7（G 2组）杀伤率分别为（36.7±85）%和（76.5±11.7）%。结果还显示,3株乳腺癌细胞均表达MICA分子；NK细胞与MCF7细胞共培养，可上调NK细胞表面NKG2D的表达。结论：NK细胞对乳腺癌细胞的杀伤并非由KIR的不相合机制介导；乳腺癌细胞表面表达的MICA分子可上调NK细胞表面NKG2D的表达,激发NK细胞对乳腺癌细胞的细胞毒效应。

To study the effects of natural killer cells with KIR(killer immunoglobulinlike receptor)/HLACw incompatibility on breast cancer cell lines in vitro, and to analyze the relationship between NKG2D (on NK cells) and MICA (on tumor cells) expression with NK cells alloreactivity against breast cancer.Methods: Monocytes were derived from 10 ml peripheral blood of healthy donors and patients of breast cancer by FicollHypaque. NK cells were purified by MASC separation system with NK Cell Isolation Kit. The polymerase chain reactionbased sequencespecific primer (PCRSSP) was used for KIR typing of NK cells and HLACw typing of tumor cells. NK cell cytotoxicity was assessed by MTT assays. Flow cytometry was used to investigate the expression level of NKG2D in NK cells and RTPCR was used to investigate the expression MICA in tumor cells. Results: The purity of enriched NK cells was more than 90% as determined by flow cytometry. The cytotoxicity of NK cells in KIR/HLACw compatible group was greatly higher than that of incompatible group. The cytotoxicity rates of NK cells of G1 and G2 group against MDAMB435s（G1 group）were (84.6±16.7)% and (25.4±8.9)%, respectively; against SKBr3（G1 group） were (70.2±11.8)% and (29.5±6.8)%, respectively; against MCF7（G2 group）were (33.1±5.7)% and (81.4±10.3)%; respectively. MICA expression was noticed in MCF7, MDAMB435s, and SKBr3 breast cancer cell lines. The expression of NKG2D on the NK cells was increased when cocultured with MCF7 cells. Conclusion: The cytotoxicity of NK cells against breast cancer cells is not mediated by KIR/HLACw incompatibility. MICA might increase the sensitivity of the breast cancer to NK cells by activating NKG2D on the NK cells.

R730.3

福建省科技重点项目（No.2006Y0011）；福建省自然科学基金（No.C0310035）；福建省卫生厅创新课题项目（No.2003CX16）