组织蛋白酶L反义基因转染对高转移人骨肉瘤细胞生物学行为的影响

Inhibitory effect of cathepsin L antisense nucleotide on biological behavior of human highly metastatic osteosarcoma cell

目的构建人组织蛋白酶L(CATL)基因的正、反义真核表达载体。观察反义CATL核酸转染后对高转移人骨肉瘤细胞体内、外侵袭特性的抑制效应。方法采用RT-PCR的方法,从人骨肉瘤组织中扩增出1001bp的CATL全长cDNA片段,以BamHⅠ及XbaⅠ双酶切,定向克隆到真核表达载体pcDNA 3.0中。对重组质粒进行限制性内切酶酶切分析及DNA序列测定。应用脂质体介导的基因转染技术将重组质粒导入高转移人骨肉瘤细胞中,观察转染后细胞的生长、体外侵袭能力及裸鼠体内成瘤及自发转移能力等指标的变化。结果正、反义真核表达载体成功构建并转染入高转移人骨肉瘤细胞中。基因转染对细胞的体外生长无明显影响。反义载体转染后细胞的体外侵袭能力和裸鼠体内成瘤性及自发转移能力均受到一定程度抑制。结论反义CATL基因可显著抑制骨肉瘤细胞的体内、外侵袭力。

Objective To set up sense/antisense eukaryotic expression vectors for human cathepsin L（CATL） gene, and to study the biological effects on human highly metastatic osteosarcoma cell line MG-63AI after transfection. Methods A 1001bp cDNA fragment was amplified by RT-PCR method from total RNA of human osteosarcoma tissue. The fragment was cloned into pcDNA 3.0 plasmid with sense/antisense direction respectively. The recombinant plasmids were confirmed by double digestion with restriction endonucleases BamH Ⅰ and Xba Ⅰ and DNA sequencing. The recombinant plasmids were transfected into MG-63A1 cells. Changes of cell proliferation, cell invasion ability and tumorigenecity and spontaneous metastasis in nude mice were detected. Results The sense/antisense recombinant eukaryotic expression vectors were obtained. Sequence analysis further confirmed that the sense recombinent plasmid contained a fragment which is identical to the cDNA sequence of CATL gene, while antisense recombinent plasmid contained a fragment which is in opposite orientation to the cDNA sequence of CATL gene. The vivo/ vitro invasion ability was remarkably decreased after antisense transfection compared with that of control cells. Conclusions The vivo/vitro invasion ability of osteosarcoma cells can be remarkably inhibited by antisense CATL.