【摘要】 目的研究NF-κB特异性抑制剂吡咯烷二硫代氨基甲酸(pyrrolidine dithiocarbamate,PDTC)对Jurkat细胞凋亡及基质金属蛋白酶-9(matrx metallo preteinases-9,MMP-9)基因表达的影响,探讨转录因子NF-κB与MMP-9在白血病发生发展及浸润转移过程中的作用及2者之间的关系。方法取处于对数生长期的Jurkat细胞随机分为6组,包括对照组(培养基中不含PDTC),试验组(培养基中分别含有50、100、150μmol/L PDTC、1μg/m L VCR、1μg/m L VCR+150μmol/L PDTC);MTT法测定PDTC对Jurkat细胞增殖的抑制作用;使用流式细胞术(flow cytometry,FCM)检测PDTC联合长春新碱(vincristine oncovin,VCR)诱导Jurkat细胞凋亡的情况;使用半定量RT-PCR检测Jurkat细胞中MMP-9基因的表达。结果不同浓度的PDTC(50、100、150μmol/L)作用不同时间(12~48 h)后对Jurkat细胞的增殖抑制率随PDTC浓度的增高及作用时间的延长而逐渐增加(P<0.05),但在PDTC处理48 h后,细胞增殖抑制率无明显增加。在不同浓度的PDTC分别处理细胞48 h后,随着PDTC浓度增高,Jurkat细胞凋亡率逐渐增加,各实验组凋亡率显著高于对照组,差异均有统计学意义(P<0.05)。与单纯VCR组相比,VCR+PDTC组Jurkat细胞凋亡率显著增高,差异有统计学意义(P<0.05),显示PDTC可以增强VCR诱导的Jurkat细胞凋亡作用。不同浓度的PDTC分别处理各组细胞48 h,RT-PCR结果显示各实验组细胞MMP-9基因表达水平与对照组相比明显降低,差异有统计学意义(P<0.05)。结论白血病细胞中存在MMP-9基因高表达,导致细胞基底膜及细胞外基质降解,促进了白血病的浸润和转移,并引起化疗耐药,而这一过程可被NF-κB抑制剂PDTC所抑制,为临床上白血病的治疗探索了一条新的途径。更多还原

【Abstract】 Objective To investigate the effect of PDTC,a specific inhibitor of NF-κB,on cell apoptosis of Jurkat cell and expression of MMP-9in Jurkat cell, and investigate the relationship between NF-κB and MMP-9 in genesis, development, infiltration and metastasis of leukemia.Methods Jurkat cells in logarithmic growth phase were divided into six groups randomly,including control group in which the culture medium was free of PDTC,and experimental groups in which Jurkat cells were treated respectively with 50,100,150 μmol / L of PDTC,1 μg / m L of VCR,1 μg / m L of VCR and 150 μmol / L of PDTC. MTT assay was performed to evaluate the inhibition effect of PDTC on the proliferation of Jurkat cells. The apoptosis rate of Jurkat cells induced by the PDTC with VCR were detected by FCM. The expression of MMP-9 in Jurkat cells were detected by semi-quantitative RT-PCR. Results After being treated with different concentrations of PDTC,the proliferation inhibition rate of PDTC on Jurkat cell increased with the raise of PDTC concentration and time extension. However,after being treated with PDTC for 48 hours,there was no obvious growth of proliferation inhibition rate,which showed the inhibitory effect of PDTC depending on time and dose within a certain range of concentration and time. The experimental group with high dose of PDTC presented highest inhibitory effect. After being treated with PDTC for 48 hours,apoptosis rate of Jurkat cell increased with the raise of PDTC concentration. Apoptosis rate of experimental groups were significantly higher than control group(P < 0. 05).Compared with VCR group,the apoptosis rate of VCR + PDTC was significantly higher than VCR group(P < 0. 05),which showed PDTC could enhance induction of VCR in the aspect of promoting apoptosis. After being treated with different concentrations of PDTC,levels of MMP-9 mRNA were detected by RT-PCR. The results showed that levels of MMP-9 mRNA in experimental groups were significantly lower than control group(P < 0. 05).Conclusion This study explores a new way for the clinical treatment of leukemia. The high expression of MMP-9 gene existed in leukemia cells leads to degradation of cell basement membrane and extracellular matrix,promotes leukemic infiltration and metastasis,causes chemotherapy resistance,and this process can be inhibited by PDTC. This discovery is worthy of reference for treatment of children with ALL.更多还原