论文部分内容阅读
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入小鼠黑色素瘤B16F1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。
Objective To investigate the role of cystatin (sv-cystatin) in the invasion and metastasis of melanoma cells. Methods The eukaryotic expression plasmid pcDNA3.1 / sv-cystatin was constructed. The recombinant plasmid was transfected into mouse B16F1 cells by lipofectamine and the resistant clone was screened by G418. Western blotting and RT-PCR were used to identify the expression of sv-cystatin in melanoma The expression of sv-cystatin was detected by MTT assay in vitro. The invasion and migration of tumor cells in vitro and the experimental lung metastasis model were used to analyze the expression of sv-cystatin in melanoma cells , The impact of external invasion. Results Stable transfection of sv-cystatin gene significantly decreased the invasiveness and motor ability of B16F1 cells. The number of transmembrane cells was significantly lower than that of B16F1 / pcDNA3.1 vector and untransfected cells (P <0.01) The expression of cystatin can inhibit the formation of pulmonary metastases in C57BL / 6 mice. The proliferation and adhesion of cystatin did not change significantly in vitro. Conclusion The sv-cystatin gene transfection can inhibit the in vitro and in vivo invasion and metastasis of melanoma B16 cells.