左氧氟沙星对大鼠骨髓间充质干细胞的毒性作用研究

The cytotoxic effects of levofloxacin on rat bone marrow mesenchymal stem cells

目的观察左氧氟沙星对大鼠骨髓间充质干细胞是否具有细胞毒性作用。方法采用大鼠骨髓间充质干细胞，设立正常对照组及梯度浓度的左氧氟沙星处理组（5、10、20、40、80 μg/ml）分别作用24、48 h。以噻唑蓝（MTT）法检测细胞活性，流式细胞膜联蛋白V（Annexin V）-异硫氰酸荧光素（FITC）/碘化丙锭（PI）双染法检测细胞凋亡情况，反转录-聚合酶链反应（RT-PCR）检测对细胞基质促降解的基质金属蛋白酶-3（MMP-3）、基质金属蛋白酶-13（MMP-13）及抑制降解的基质金属蛋白酶抑制因子-1（TIMP-1）、基质金属蛋白酶抑制因子-13（TIMP-3）的mRNA水平的表达。结果左氧氟沙星（5～80 μg/ml）作用48 h后，骨髓间充质干细胞细胞数分别减少至对照组的92.39%、88.26%、84.11%、78.71%和71.90% Annexin V-FITC/PI双染流式细胞检测示，左氧氟沙星（5～80 μg/ml）作用24 h后，细胞凋亡细数呈药物浓度依耐性升高（与对照组比较其P值分别为0.022、0.013、0.007、0.004、0.002）。RT-PCR检测结果示，左氧氟沙星作用细胞24 h后，显著增高MMP-3（与对照组比较，P值分别为0.025、0.021、0.014、0.002、0.001）和显著增高MMP-13（与对照组比较，P值分别为0.015、0.009、0.004、0.003、0.001）表达，显著降低TIMP-1表达，并且该作用具有药物浓度依耐性（与对照组比较，P值分别为0.027、0.015、0.007、0.005、0.001）。结论左氧氟沙星对大鼠骨髓间充质干细胞具有明确的细胞毒性作用。

ObjectiveTo study the effects of levofloxacin on rat bone marrow mesenchymal stem cells （BMSCs）.MethodsRat BMSCs were treated with levofloxacin at different concentrations （0, 5, 10, 20, 40, 80 μg/ml）. Toxic effects of levofloxacin on cell viability was assessed by methyl thiazol tetrazolium （MTT） assay. The cell apoptosis was detected by double staining. Reverse transcription-polymerase chain reaction （RT-PCR） analysis were used to detect the expression of matrix metalloproteinase-3 （MMP-3）, matrix metalloproteinase-13 （MMP-13）, tissue inhibitors of metalloproteinase-1 （TIMP-1） and tissue inhibitors of metalloproteinase-3 （TIMP-3） in the treated cells.ResultsAfter being treated with a concentration of levofloxacin （5-80 μg/ml） for 48 h, the cell viabilities were decreased to 92.39%, 88.26%, 84.11%, 78.71%, and 71.90% of that in the control. Levofloxacin, with concentrations ranging from 5 to 80 μg/ml, induced dose-dependent BMSCs apoptosis （vs. control P values were 0.022, 0.013, 0.007, 0.004, 0.002）. Moreover, levofloxacin significantly increased the mRNA expression of MMP-3 （vs. control P values were 0.025, 0.021, 0.014, 0.002, 0.001） and MMP-13 （vs. control P values were 0.015, 0.009, 0.004, 0.003, 0.001）and decreased the expression of TIMP-1 （vs. control P values were 0.027, 0.015, 0.007, 0.005, 0.001）in a concentration-dependent manner.ConclusionLevofloxacin has cytotoxic effects on BMSCs characterized, which suggest a potential adverse effect of fluoroquinolones.