【摘要】 目的探讨基质金属蛋白酶9(MMP-9)在粒细胞集落刺激因子(G-CSF)诱导的造血干/祖细胞(HSPC)动员中的作用。方法应用酶联免疫吸附试验(ELISA)、免疫组织化学、逆转录聚合酶链反应(RT-PCR)等方法测定了健康供者稳态及G-CSF动员第5天骨髓、外周血MMP-9蛋白及mRNA水平的变化,比较富含MMP-9的细胞系HT1080无血清培养上清孵育前后CD34+细胞表面CXCR4表达及对基质细胞衍生因子1(SDF-1)的迁移能力的变化,将rhMMP-9与rhSDF-1孵育后应用Western印迹检测MMP-9对SDF-1的降解作用,观察注射MMP-9化学抑制剂(MPI)ο-邻二氮杂菲对G-CSF动员BALB/c小鼠外周血成熟白细胞计数(WBC)和干/祖细胞的数量的影响。结果G-CSF动员第5天,骨髓上清MMP-9浓度增加了6.6倍(P<0.01),骨髓组织表达MMP-9的中性粒细胞明显增多。动员后骨髓单个核细胞MMP-9mRNA表达水平上调(P<0.05)。rhSDF-1与rhMMP-9短期孵育后可被后者降解。HT1080无血清培养上清液能够上调脐血和动员外周血富集的CD34+细胞的CXCR4表达水平(均P<0.05)及增强CD34+细胞向SDF-1浓度梯度迁移能力(均P<0.05),该效应可被MPI阻断(P<0.05)。与G-CSF共注射MPI后BALB/c小鼠外周血成熟WBC(12.3×106/L±1.2×106/L)和造血祖细胞集落形成单位(69U/2×105MNC±3U/2×105MNC)数量显著低于对照组(P<0.05)。结论MMP-9可能通过裂解SDF-1、上调CD34+细胞表面SDF-1特异性受体CXCR4的表达及增强CD34+细胞迁移能力在G-CSF介导的HSPC动员中发挥重要作用。更多还原

【Abstract】 Objective To investigate the effects of matrix metalloproteinase-9 (MMP-9) on granulocyte colony stimulation factor (G-CSF)-induced hematopoietic stem/progenitor cell (HSPC) mobilization in healthy donors of hematopoietic stem cells. Methods Peripheral blood (PB) samples and bone marrow (BM) blood samples were collected from 12 healthy donors of hematopoietic stem cell before and 5 days after G-CSF-induced mobilization. CD34+ cells were isolated and purified. ELISA was used to detect the protein expression of MMP-9 in the peripheral blood and BM blood of the healthy donors. The protein expression of MMP-9 in the BM blood was detected by ELISA and immunohistochemistry, and the stromal cell-derived factor-1 (SDF-1) level in the BM blood was detected by ELISA. The mRNA expression of MMP-9 in the BM blood samples was detected by RT-PCR. HT1080 cells rich in MMP-9 were cultured. CD34+ cells were co-cultured with the supernatant of HT1080 cell culture fluid. CD34+ cells cultured in Iscove′s modified Dulbecco′s medium were used as control group. Fluorescence-activeted cell sorter was used to detect the CXCR4 expression on the suface of the CD34+ cells. In the transwell experiment CD34+ cells were divided into 4 groups: control group, o-phenanthroline (MMP-9 chemical inhibitor , MPI) group, HT1080 sup group, and HT1080+MPI group to be co-cultured with buffer, o-phenanthroline, supernatant of culture fluid of HT1080 cells, or supernatant of culture fluid of HT1080 cells Flow cytometry was used to calculate the cell migration capacity. Results The MMP-9 level of BM and PB of the healthy donors 5 days after G-CSF mobilization were 278 ng/ml±34 ng/ml and 392 ng/ml±284 ng/ml respectively, both significantly higher than those before G-CSF mobilization (42 ng/ml±17 ng/ml and 27 ng/ml±12 ng/ml respectively (P<0.01 and P<0.05). Western blotting showed that the SDF-1 level in the supernatant 5 days after G-CSF mobilization was 5.9 ng/ml±1.0 ng/ml, significantly lower than that before G-CSF mobilization (7.2 ng/ml±0.7 ng/ml, P<0.05 ).The CXCR4 levels of the CD34+ cell from both PB and BM blood were up-regulated after co-culture with the supernatant of HT1080 cells (both P<0.05). The migration capacity of CD34+ cells cultured in the supernatant of HT1080 cells was increased significantly (P<0.05), however, this effect could be inhibited by MIP (P<0.05). The PB WBC numbers of the G-CSF group and G-CSF+MPI group were 14.9×106/L±4.3×106/L and 12.3×106/L±1.2×106/L respectively, the PB WBC numbers of the G-CSF+MPI group was significantly lower than that of the G-CSF group (P<0.05), however, significantly higher than that of the negative control group (6.8×106/L±2.5×106/L, P<0.05). The CFU of the G-CSF group was (84±10) U/2×105 MNC, significantly higher than that of the G-CSF+MPI group, (69±3) U/2×105 MNC (P<0.05). The BM MNC number of the G-CSF group was 12.7×106/L±0.7×106/L, not significantly different from that of the G-CSF+MPI groups (13.1×106/L±1.3×106/L;P>0.05). Conclusion MMP-9 probably facilitates HSPC mobilization by degrading SDF-1,up-regulating CXCR4 expression on the CD34+ cells, and increasing the migration ability of CD34+ cells.更多还原