【摘要】 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。更多还原

【Abstract】 Objective To study the influence of up regulating the expression of tissue inhibitor of metalloproteinase-2(TIMP-2) on the invasive capacity of SHI-1 cells.Methods The mRNA and protein expressions of TIMP-2,MMP-2 and MT1-MMP in SHI-1 cells were detected by quantitative RT-PCR and Western blot.A retroviral vector carrying human TIMP-2 cDNA was constructed and transfected into the SHI-1 cells.After transfection,three subclone cells(S1,S2 and S3 cells) were selected by limiting dilution.Cell invasion through a barrier of reconstituted human basement membrane assays was performed to measure the invasive capacity of different cells.The leukemia model of nu/nu mice was made and then pathological section of different organ was done by routine method to survey the infiltration of leukemia.The leukemic cells engrafted in the mice organs were tracked by the expression of the MLL/AF6 fuse gene detected by RT-PCR.Results The expression of MMP-2,MT1-MMP and TIMP-2 in SHI-1 cells were higher than that in other leukemic cells at both mRNA and protein levels(P<0.05).The in vitro invasive capacity of SHI-1 cells was also higher than that of other cells(P<0.05).The invasion rates of subclone cells demonstrated a 1.5~2.5 folds’ elevation(P<0.05).The time of disease onset in mice of S1,S2 and S3 group were all earlier than that in SHI-1 group and the survival time of S1,S2 and S3 group were all shorter than that in the SHI-1 group(P<0.05).Histopathological evidences demonstrated severally leukemic infiltration in many organs of each group.RT-PCR detection showed that many organs expressed MLL/F6 fuse gene,moreover,the mounts of organs which were infiltrated by leukemic cells in S1,S2 and S3 group were all more than that in the SHI-1 group(P<0.05).Conclusion SHI-1 cells strongly express constitutive MMP-2,MT1-MMP and TIMP-2 at both mRNA and protein levels.Up-regulating the expression of TIMP-2 in SHI-1 cells not only enhances cell in vitro invasion but also increases SHI-1 cell in vivo infiltration in the nu/nu mice.更多还原