蛇毒半胱氨酸蛋白酶抑制剂的表达对黑色素瘤B16细胞侵袭与转移的作用

目的探讨蛇毒半胱氨酸蛋白酶抑制剂cystatin（sv-cystatin）在黑色素瘤细胞侵袭与转移中的作用。方法构建真核表达质粒pcDNA3．1／sv-cystatin，采用脂质体法将重组质粒导人小鼠黑色素瘤B16FI细胞。G418筛选抗性克隆，Western blotting法和RT-PCR鉴定sv—cystatin在黑色素瘤细胞中的表达，四甲基偶氮唑盐（MTT）法检测肿瘤细胞体外生长和黏附能力的变化，体外侵袭、运动实验和小鼠实验性肺转移模型分析sv-cystatin表达对黑色素瘤细胞体内、外侵袭力的影响。结果sv-cystatin基因稳定转染后B16F1细胞体外侵袭与运动能力明显降低，其穿膜的细胞数显著低于B16F1／pcDNA3．1空载体组和未转染细胞组（P〈0．01）；sv—cystatin的表达可抑制C57BL／6小鼠肺转移瘤灶的形成；其体外增殖及黏附能力未见明显改变。结论sv—cystatin基因转染可抑制黑色素瘤B16细胞的体内、外侵袭与转移能力。

The Effect of Cysteine Protease Inhibitor of Snake Venom(Sv-Cystatin )on Mous Melanoma Cells Invasion in vitro and in vivo

Objective To investigate the effect of cysteine protease inhibitor of snake venom(sv-cystatin) on invasiveness and metastasis of melanoma cells.Methods The recombinant plasmid pcDNA3.1/sv-cystatin was constructed and transferred into mouse melanoma cell line B16F1 by lipofection technology.The positive clones were screened by G418 and the sv-cystatin expression was detected by RT-PCR and Western blotting.The ability of tumor cell invasion was identified by Boyden chamber in vitro and tumor invasion animal model in vivo.The ability of tumor cell proliferation and adhension was also determined by MTT assay.Results Expression of sv-cystatin was detected in B16F1/sv-cys cells after gene-transfection.Transfection of sv-cystatin significantly decreased the invasion and migration of B16F1/sv-cys cells along with obviously inhibited the experimental pulmonary metastasis in vivo,but the proliferation and adhension capacity of B16F1/sv-cys cells had no change.Conclusion Transfection of sv-cystatin gene into mouse melanoma cell line results in the suppression of the invasion potential in vitro and in vivo.