基质金属蛋白酶家族的表达对急性单核细胞白血病细胞体外侵袭力的影响  

Effect of TIMP-2, MT1-MMP and MMP-2 expression on the in vitro invasive capacity of acute monocytic leukemia SHI-1 cells

作　　者：王春玲[1] 陈子兴[1] 李振江[1] 岑建农[1] 

机构地区：[1]苏州大学附属第一医院、江苏省血液研究所,卫生部血栓与止血重点实验室,215006

出　　处：《中华血液学杂志》2010年第12期798-803,共6页Chinese Journal of Hematology

基　　金：国家自然科学基金（30670905）

摘　　要：目的 研究基质金属蛋白酶家族（TIMP-2、MT1-MMP、MMP-2）的表达对急性单核细胞白血病细胞体外侵袭力的影响.方法 以急性单核细胞白血病细胞株SHI-1细胞为研究对象,用定量PCR、Western blot法分别检测TIMP-2、MT1-MMP、MMP-2 mRNA和蛋白表达,并以NB4、K562、THP-1等人类其他白血病细胞株细胞为对照,进行比较.构建TIMP-2逆转录病毒载体,转染SHI-1细胞,G418筛选,有限稀释法挑选出单克隆S1、S2、S3细胞.RNA干扰（RNAi）法干扰SHI-1细胞TIMP-2、MT1-MMP、MMP-2表达.明胶酶谱法测定不同细胞和骨髓基质细胞（BMSC）共培养24 h后上清中MMP-2的表达,细胞侵袭实验测定SHI-1细胞侵袭人工基质膜的能力.结果 SHI-1细胞的TIMP-2、MT1-MMP、MMP-2 mRNA表达和蛋白表达均显著高于其他细胞（P＜0.05）.SHI-1细胞和BMSC共培养上清中的MMP-2酶原和活化的MMP-2含量及细胞体外侵袭率均高于其他细胞（P＜0.05）.单克隆S1、S2、S3细胞TIMP-2 mRNA表达水平分别是SHI-1细胞的3.0倍、2.0倍和1.5倍（P＜0.05）,蛋白表达水平分别上调2.6倍、1.5倍和1.3倍（P＜0.01）,体外侵袭率增加1.5～2.5倍（P＜0.05）,活化的MMP-2含量增加1.5～3.0倍（P＜0.01）.RNA干扰基因沉默效率为85%～98%.SHI-1细胞TIMP-2、MMP-2、MT1-MMP表达沉默后,细胞侵袭率分别下降60%～70%、50%～60%、40%～50%（P＜0.05）.RNA干扰后的细胞培养上清中检测不到活化的MMP-2.结论 SHI-1细胞在mRNA水平和蛋白水平均高表达TIMP-2、MT1-MMP、MMP-2 mRNA,SHI-1细胞和BMSC共培养后这些分子的高表达促进MMP-2的活化,增强细胞的体外侵袭力.TIMP-2表达增加1.5～2.5倍对SHI-1细胞MMP-2的活化和细胞的体外侵袭力发挥的是增强作用,而不是抑制作用.Objective To study the effect of matrix metalloproteinase 2 （ MMP-2）, membrane type 1 MMP （MT1-MMP） and tissue inhibitor of metalloproteinase 2 （TIMP-2） expressions on the in vitro invasive capacity of acute monocytic leukemia SHI-1 cells. Methods SHI-1, NB4, K562, M937 and THP-1 human leukemia cell lines were cultured in vitro. The mRNA and protein expressions of TIMP-2, MMP-2 and MT1-MMP in different cells were detected by quantitative RT-PCR and western blot. A retroviral vector carrying human TIMP-2 cDNA was constructed and transfected into SHI-1 cells. Three subclone cells （S1, S2 and S3） were screened by G418 and selected by limiting dilution. RNA interference （RNAi） was used to knock down the expression of MMP-2, MT1-MMP and TIMP-2. Cell invasion capacity was performed through a reconstituted human basement membrane assays. Zymography was used to analyze the expression of MMP-2 in the supernatant of co-culture. Results The expressions 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 amount of proMMP-2 and activated MMP-2 in the conditioned media from SHI-1 cells co-cultured with bone marrow stromal cells （BMSCs） was more than that from other cells （P 〈 0. 05 ）. The in vitro invasive capacity of SHI-1 cells were higher than that of other cells（ P 〈 0.05 ）. The mRNA levels of TIMP-2 were increased by about 3 fold, 2 fold and 1.5 fold in S1, S2 and S3 cells, respectively（ P 〈 0.05 ）, while the protein levels were by about 2.6 fold, 1.5 fold and 1.3 fold than that of SHI-1 cells, respectively（P 〈 0.01 ）. The invasion rates of subclone cells demonstrated a 1.5 - 2.5 fold＇ elevation （ P 〈 0.05 ） and activated MMP-2 from their supernatants increased by 1.5 -2.0 fold（P〈0.01 ）. The knock-down efficiency of siRNA was 85% to 98%. The down-regulation of TIMP-2, MMP-2 and MT1-MMP decreased the invasion rates of SHI-1 cells by 60% -70%, 50% - 60% and 40% - 50%, re

关 键 词：白血病 单核细胞 急性 白血病浸润 基质金属蛋白酶抑制剂2 基质金属蛋白酶类 

分 类 号：R686[医药卫生—骨科学]