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目的探讨原核表达的人源基质金属蛋白酶2的C端结构域(MMP-2-PEX)对黑色素瘤细胞A375与内皮细胞黏附的影响。方法利用RT-PCR方法从人成纤维肉瘤细胞HT1080中克隆MMP-2 C端结构域,构建原核表达载体pET-His-PEX;转化大肠杆菌BL21(DE3),IPTG诱导表达;盐酸胍裂解包涵体,经镍琼脂糖凝胶柱纯化,透析复性后获得MMP-2-PEX蛋白,随后对其进行Western印迹和质谱鉴定。采用明胶酶谱法检测MMP-2-PEX的活性;采用平行板流动小室系统检测在流动状态下MMP-2-PEX对A375细胞与脐静脉内皮细胞HUVEC黏附的影响,通过小鼠实验性肺转移模型检测MMP-2-PEX对A375细胞在小鼠肺内癌栓形成的影响。结果 Western印迹和质谱鉴定原核表达的MMP-2-PEX蛋白正确,明胶酶谱检测MMP-2-PEX能够明显抑制MMP-2的活性。表达的MMP-2-PEX蛋白能够在流动状态下抑制A375细胞与HUVEC细胞的黏附,其黏附抑制率与MMP-2-PEX蛋白浓度增加成正相关,MMP-2-PEX浓度为12.5,25和50μg/ml时抑制率分别为32.5%,41.4%和53.9%。用MMP-2-PEX处理后,A375细胞在小鼠肺内癌栓的形成与对照组相比降低了26.4%。结论原核表达的人MMP-2-PEX在体外流动状态下可抑制A375细胞与内皮细胞的黏附,并抑制A375细胞在小鼠肺内癌栓的形成。
Objective To investigate the effect of prokaryotic expression of human MMP-2-PEX on adhesion of melanoma A375 cells to endothelial cells. METHODS: The C-terminal domain of MMP-2 was cloned from human fibroblastic sarcoma cell line HT1080 by RT-PCR and the prokaryotic expression vector pET-His-PEX was constructed. The recombinant plasmid was transformed into E. coli BL21 (DE3) and induced by IPTG. , Purified by Ni-Sepharose column, dialyzed and renatured to obtain MMP-2-PEX protein, followed by Western blot and mass spectrometry. The activity of MMP-2-PEX was detected by gelatin zymography. The effect of MMP-2-PEX on the adhesion of A375 cells to HUVEC in umbilical vein endothelial cells was detected by flow cytometry. The effect of MMP-2-PEX on the formation of tumor thrombus in the lungs of mice was investigated by the model. Results The prokaryotic expression of MMP-2-PEX was identified by Western blotting and mass spectrometry. The detection of MMP-2-PEX by gelatin zymography significantly inhibited the activity of MMP-2. The expression of MMP-2-PEX protein could inhibit the adhesion of A375 cells to HUVECs in a flowing state. The adhesion inhibition rate was positively correlated with the increase of MMP-2-PEX protein concentration, and the MMP-2-PEX concentrations were 12.5, 25 and 50μg / ml when the inhibition rates were 32.5%, 41.4% and 53.9%. After treated with MMP-2-PEX, the formation of tumor thrombi in the lungs of A375 cells decreased by 26.4% compared with the control group. Conclusion The prokaryotic expression of human MMP-2-PEX can inhibit the adhesion of A375 cells to endothelial cells and inhibit the formation of tumor thrombus in the lungs of mice in vitro.