APE1/Ref-1 siRNA抑制多发性骨髓瘤骨髓基质细胞IL-6及IL-8分泌的体外研究

APE1/Ref-1 siRNA inhibits IL-6 and IL-8 secretion by cultured bone marrow stromal cells isolated from multiple myeloma patients

目的体外通过APE1/Ref-1 siRNA敲低多发性骨髓瘤骨髓基质细胞(bone marrow stromal cells,BMSCs)APE1/Ref-1的表达,观察BMSCs的增殖及分泌细胞因子IL-6、IL-8的变化,初步探讨BMSCs APE1/Ref-1表达的功能特点。方法通过免疫细胞化学染色法定量检测35例初治、11例复发/难治多发性骨髓瘤患者及10例正常人BMSCs APE1/Ref-1的表达特点及其差异,经Adv5-APE1/Ref-1 siRNA感染BMSCs后,流式细胞仪检测BMSCs细胞周期的变化;ELISA法检测BMSCs分泌IL-6、IL-8的水平变化情况。结果多发性骨髓瘤BMSCs的APE1/Ref-1蛋白阳性表达率显著高于正常BMSCs APE1/Ref-1蛋白阳性表达率(P<0.05),且多发性骨髓瘤BMSCs的APE1/Ref-1呈细胞核及核浆共同表达方式。Adv5-APE1/Ref-1 siRNA感染敲低多发性骨髓瘤及正常BMSCs APE1/Ref-1的表达量呈进行性减少(P<0.01),同时发现APE1/Ref-1 siRNA对多发性骨髓瘤BMSCs抑制作用更明显。Adv5-APE1/Ref-1 siRNA感染BMSCs后对正常人及骨髓瘤患者BMSCs分泌细胞因子IL-6、IL-8的量有显著的抑制作用,特别是感染72h后,骨髓瘤患者及正常人的BMSCs分泌IL-6[初治患者(246.29±46.51)pg/ml,复发/难治患者(365.09±75.25)pg/ml]、IL-8[初治患者(118.77±18.08)pg/ml,复发/难治患者(188.71±33.76)pg/ml]的量最低,与其他时段比较差异有统计学意义(P<0.01)。结论多发性骨髓瘤BMSCs APE1/Ref-1的表达特点不同于正常BMSCs,可能导致其功能差异;APE1/Ref-1 siRNA敲低了MMBMSCs APE1/Ref-1的表达,同时明显抑制了其IL-6、IL-8的分泌,减少了对骨髓瘤细胞的促增殖和凋亡作用。

Objective To explore the possible roles of apurinic/apyrimidinic endonuclease 1/redox factor-1 （APE1/Ref-1） expression in the bone marrow stromal cells （BMSCs） form multiple myeloma （MM） patients. Methods The expression of APE1/Ref-1 protein in BMSCs isolated from the bone marrow samples of 35 initial cases, 11 relapse/refractory cases with MM and 10 cases health individuals were detected by immunocytoehemistry. Before and after the transfection of the adenoviral vector Adv5-APE1/Ref-1 siRNA, the concentrations of interleukin-6 （IL-6） and interleukin-8 （IL-8） in the supernatant of BMSCs were measured with ELISA, and the cell cycles proliferation of BMSCs were detected with flow cytometry. Results The expression of APE1/ Ref-1 protein in the cultured BMSCs was significantly higher than in normal BMSCs （P 〈 0.05）. APE1/Ref-1 siRNA obviously inhibited the expression of the protein in MM or normal BMSCs. Transfected with Adv5-APE1/ Ref-1 siRNA, the MM or normal BMSCs gradually decreased the levels of IL-6 and IL-8 secretion in a time course. In 72 h after the transfection, the amounts of IL-6 and IL-8 in the BMSCs supernatant reached the lowest levels as following, IL-6 （246.29 ±46.51 ） pg/ml and IL-8 （ 118.77 ± 18.08） pg/ml in initial MM group, and IL-6 （365.09 ± 75.25） pg/ml and IL-8 （ 188.71 ± 33.76） pg/ml in relapse/refractory group （P 〈 0.01）. Conclusion The abnormal expression of APE1/Ref-1 protein in MM BMSCs may lead to many changes of biological function. APE1/Ref-1 siRNA knocks down the expression of APE1/Ref-1 protein in BMSCs, and inhibits the production of IL-6 and IL-8 in MM BMSCs, thus indirectly reduces the proliferation or benefit apoptosis of myeloma cells.