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目的一氧化氮（ＮＯ）供体细胞系的建立及其对肿瘤细胞凋亡的诱导效应研究。方法采用３Ｈ－胍氨酸转换法进行ＮＯ合成酶（ＮＯＳ）活性测定；亚硝酸盐检测；ＤＮＡ片段的提取及凝胶电泳分析；凋亡细胞的ＤＡＰＩ染色观察；Ｗｅｓｔｅｒｎｂｌｏｔ分析。结果将ｉＮＯＳ真核表达质粒、ｐＣＭＶ／ｉＮＯＳ转染至Ｓｐ２／０骨髓瘤的变异株中，并获得能稳定表达ｉＮＯＳ和合成ＮＯ的重组细胞系（ＳＰｍｔ／ｉＮＯＳ），表达ｉＮＯＳ的效率为每升培养物含４００μｇ蛋白。利用该细胞作ＮＯ细胞性供体，成功地证实ＮＯ能诱导Ｓｐ２／０骨髓瘤细胞发生凋亡。结论所建细胞性ＮＯ供体应用于ＮＯ的生物学功能研究具有ＮＯ合成稳定；更能模拟体内细胞间ＮＯ的信息传递过程；不需任何外源刺激即可合成ＮＯ等独特优点。所建ＮＯ供体细胞可以用于肿瘤细胞凋亡的研究。
Objective To establish a nitric oxide (NO) donor cell line and its effect on the induction of tumor cell apoptosis. Methods The activity of NO synthase (NOS) was determined by 3H-proline conversion method; nitrite was detected; DNA fragments were extracted and analyzed by gel electrophoresis; DAPI staining of apoptotic cells was observed; Western blot analysis was performed. Results The iNOS eukaryotic expression plasmid and pCMV/iNOS were transfected into the mutant strains of Sp2/0 myeloma, and the recombinant cell line (SPmt/iNOS) which can stably express iNOS and synthesize NO was obtained. The efficiency of expressing iNOS was 1%. The culture contained 400 μg of protein. Using this cell as a NO cell donor, it was confirmed that NO can induce apoptosis in Sp2/0 myeloma cells. CONCLUSION: The study of the biological function of the established cellular NO donor in NO has the advantages of stable NO synthesis; it can simulate the information transfer process of NO between cells in vivo; and it can synthesize NO without any exogenous stimulation. The constructed NO donor cells can be used for the study of tumor cell apoptosis.