特异性RNA干扰阻抑骨髓基质细胞SDF-1表达对Jurkat细胞黏附及药物敏感性的影响

Effects of inhibiting SDF-1 expression by RNA interference on adhesion and drug sensitivity of Jurkat cells co-cultured with bone marrow stromal cells

目的观察特异性RNA干扰(RNA i)阻抑急性白血病骨髓基质细胞衍生因子-1(SDF-1)表达对共培养的急性白血病细胞系Jurkat细胞黏附及药物敏感性的影响。方法脂质体介导SDF-1特异性RNA i质粒转染培养的急性白血病患者骨髓基质细胞,G418筛选阳性混合克隆,ELISA法检测SDF-1表达;Jurkat细胞与之共培养,通过细胞计数计算黏附率,MTT法检测对阿霉素的药物敏感性。以未转染急性白血病骨髓基质细胞及正常骨髓基质细胞作为对照。结果转染阳性克隆组、未转染急性白血病组及正常对照组骨髓基质细胞培养上清SDF-1水平分别为每周(1920±205)pg/105细胞、(12 370±1355)pg/105细胞和(6620±770)pg/105细胞;与之共培养的Jurkat细胞黏附率分别为(28.8±2.6)%,(57.4±3.8)%和(45.2±4.0)%,阿霉素50%抑制浓度分别为585,6162,1758 nmol/L。结论RNA i阻抑SDF-1表达的骨髓基质细胞使Jurkat细胞黏附减少,对阿霉素的敏感性增加。

Objective To observe the effects of inhibiting stromal cell derived factor-1(SDF-1) expression by RNA interference(RNAi) on adhesion and drug sensitivity of Jurkat cells co-cultured with bone marrow stromal cells. Methods SDF-1 specific short hairpin RNA(shRNA) expressing plasmid was transfered into cultured human acute leukemic bone marrow stromal cells, positive clones were isolated by screening G418 resistance (Group A),SDF-1 protein level in culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). The adhesion rates to bone marrow stromal cells layer and the drug sensitivity to doxorubicin of co-cultured Jurkat cells were detected by cell counting and MTT assay, respectively. The untransfected bone marrow stromal cells of acute leukemia patient (Group B) or normal subject (Group C) were taken as control. Results The level of secreted SDF-1 protein(pg/10^(5) cells/week) in the supernatants of Group A, B and C were 1920±205, 12 370±1355 and 6620±770, respectively. Of co-cultured Jurkat cells in Group A, B and C, the adhesion rates after 24 h co-culturing were (28.8±2.6)%, (57.4±3.8)% and (45.2±4.0)%, respectively, and the IC_(50) values of doxorubicin were 585, 6162 and 1758 nmol/L, respectively. Conclusion Down-regulating SDF-1 expression of bone marrow stromal cells by RNAi reduces adhesion rates and enhances drug sensitivity to doxorubicin of their co-cultured Jurkat cells.