定向胞内释放NO的半乳糖缀合物及其对HeLa细胞的抑制作用

Targeted intracellular release and inhibition effect on HeLa cells of a galactosylated NO donor

目的研究糖基化NO供体-二醇二氮烯翁(d iazen ium-d iolate,NONOate)与β-半乳糖(β-galactose)的缀合物β-Gal-NONOate(β-galactosyl-d iazen iumd iolate)在肿瘤细胞中的NO释放及其对癌细胞的抑制效应。方法采用阳离子脂质体介导法,将真核表达载体pcDNA3-LacZ转染到HeLa细胞中;经G418(neomyc in)筛选,以邻-硝基苯-β-D-半乳糖苷(or-tho-n itrophenyl-beta-D-galactopyranoside,ONPG)法进行β-半乳糖苷酶(β-galactosidase)活性检测,获得稳定表达β-半乳糖苷酶的HeLa细胞株;通过G riess方法比较β-Gal-NONOate与NONOate在HeLa/LacZ+细胞和HeLa细胞中NO的释放量,根据细胞克隆形成率、细胞计数和MTT法比较β-Gal-NONOate与NONOate对细胞的作用。结果β-Gal-NONOate在HeLa/LacZ+细胞中NO的释放量和细胞毒性明显高于NONOate(P<0.05),而在未转染LacZ基因的HeLa细胞中比较稳定(P>0.05)。结论β-Gal-NONOate是一个成功的定向胞内释放的NO供体,可作为研究中的新型探针,并可能在抗肿瘤治疗中发挥作用。

Aim To study NO release inside the tumor cells and the antitumor effect of glycosylated NO donor β-Gal-NONOate, conjugate of NONOate and galactose. Methods Through cationic liposome - mediated transfection, the eukaryotic expression vector pcDNA3-LacZ was transferred into Hela cells; after G418 screening and ONPG detecting the enzyme activity, HeLa cell line steadily expressing β-galactosidase was obtained; Griess assay was used to evaluate NO levels discharged from β-Gal-NONOate and NONOate in HeLa/LaeZ^＋ cells. Cell counting, cell colony formation and MTT were used to estimate the toxicity of β-Gal-NONOate and NONOate to the two cell lines above, respectively. Results The levels of NO released from β-Gal-NONOate in HeLa/LacZ^＋ cells were clearly higher than that from NONOate （ P 〈 0. 01 ）, and its toxicity to HeLa/ LacZ^＋ cells were also obviously more powerful （P 〈 0. 05）. β-Gal-NONOate was stable inside HeLa cells without LacZ gene. Conclusion β-Gal-NONOate is a successful targeted intracellular NO donor, which could be used as a probe in researches and prospective application in anticancer therapy.