MMP-2-PEX原核表达纯化及其对黑色素瘤细胞与内皮细胞黏附的影响

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。

Prokaryotic expression and purification of MMP-2-PEX and the effect on adhesion of melanoma cells A375 to endothelial cells

Objective To investigate the effect of recombinant hemopexin-like domain of human matrix metalloproteinase 2（rMMP-2-PEX） on the adhesion of melanoma cells A375 to endothelial cells.Methods The cDNA of C terminal of MMP-2 was cloned from human fibroblast sarcoma cells HT1080 by RT-PCR,and then cloned into the prokaryotic expression vector pET-His.E.coli BL21（DE3） was transformed with the recombinant vector,and the expression of the foreign protein was induced by IPTG.The rMMP-2-PEX was obtained by GuHCl lysis of the inclusion bodies followed by immobilized Ni2＋ absorption chromatography and a dialysis procedure.Western blot and mass spectrometry（MS） analysis were performed to confirm the recombinant protein.Gelatin zymography was used to measure the activity of rMMP-2-PEX.The function of rMMP-2-PEX on the adhesion of A375 to HUVEC was investigated by a parallel-plate flow chamber.An experimental lung metastasis assay was performed to find out whether MMP-2-PEX was involved in the formation of lung metastatic tumor foci of A375 in mice.Results Western blot analysis and MS results showed that the rMMP-2-PEX expressed by E.coli was correct.The fact that rMMP-2-PEX could significantly inhibit the activity of MMP-2 was confirmed by gelatin zymography.The rMMP-2-PEX could inhibit the adhesion between A375 and HUVEC under flow conditions.The addition of 12.5,25 and 50 μg/ml rMMP-2-PEX reduced A375 cells adhered to HUVEC by 32.5%,41.4% and 53.9%.The number of cancer thrombus in the lung was also reduced by 26.4% after rMMP-2-PEX treatment.Conclusion rMMP-2-PEX can inhibit the adhesion of A375 to endothelial cells under flow in vitro and decrease the experimental lung metastasis in vivo.