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目的研究上调SHI-1细胞金属蛋白酶组织抑制剂(TIMP)-2的表达对SHI-1细胞侵袭力的影响。方法实时定量PCR检测SHI-1细胞的TIMP-2、MT1-MMP、MMP-2的mRNA表达,Western blot检测蛋白表达。构建TIMP-2逆转录病毒载体,转染SHI-1细胞,有限稀释法挑选出单克隆细胞S1、S2、S3。跨膜侵袭实验观察细胞侵袭力。建立裸鼠白血病模型,留取各脏器病理切片,观察人白血病细胞在各脏器中的浸润;RT-PCR检测裸鼠各脏器中人白血病细胞MLL/AF6融合基因表达。结果 SHI-1细胞TIMP-2、MT1-MMP、MMP-2的mRNA表达和蛋白表达以及它的体外侵袭率均高于其他细胞株(均P<0.05)。单克隆细胞S1、S2、S3体外侵袭率增加了1.5~2.5倍(P<0.05)。S1、S2和S3组裸鼠较SHI-1组发病时间提前、生存期缩短(P<0.05)。各组裸鼠多脏器病理切片均出现明显的白血病细胞浸润。各组裸鼠体内多脏器均扩增出MLL/AF6融合基因,且S1、S2和S3组裸鼠脏器累及多于SHI-1组(P<0.05)。结论 SHI-1细胞在mRNA和蛋白水平高表达MMP-2、MT1-MMP、TIMP-2;上调TIMP-2增强了SHI-1细胞体内外的侵袭力。
Objective To study the effect of up-regulating the expression of tissue inhibitor of metalloproteinase-2 (TIMP-2) in SHI-1 cells on invasiveness of SHI-1 cells. Methods The mRNA expressions of TIMP-2, MT1-MMP and MMP-2 in SHI-1 cells were detected by real-time PCR. The protein expression was detected by Western blot. TIMP-2 retroviral vector was constructed and transfected into SHI-1 cells. Monoclonal cells S1, S2 and S3 were selected by limiting dilution. Transmembrane invasion assay to observe cell invasiveness. The model of nude mice leukemia was established. The pathological sections of various organs were collected to observe the infiltration of human leukemia cells in various organs. The expression of MLL / AF6 fusion gene in human leukemia cells was detected by RT-PCR. Results The mRNA and protein expressions of TIMP-2, MT1-MMP and MMP-2 in SHI-1 cells were higher than those in other cell lines (all P <0.05). The invasive rates of S1, S2 and S3 in vitro increased by 1.5-2.5 folds (P <0.05). The onset time of S1, S2 and S3 nude mice was shorter than that of SHI-1 group (P <0.05). Multiple groups of nude mice in each pathological sections showed significant leukemia cell infiltration. MLL / AF6 fusion gene was amplified from multiple organs in nude mice in each group, and more organs were found in S1, S2 and S3 groups than in SHI-1 group (P <0.05). Conclusion SHI-1 cells express high levels of MMP-2, MT1-MMP and TIMP-2 at mRNA and protein levels. Up-regulation of TIMP-2 enhances the invasiveness of SHI-1 cells both in vitro and in vivo.