摘要
目的探讨阻抑骨髓基质细胞衍生因子(SDF1)表达对与其共培养的Jurkat细胞增殖、凋亡的影响。方法脂质体介导SDF1特异性RNA干扰质粒转染培养的急性白血病骨髓基质细胞,G418筛选阳性克隆(A组),采用ELISA法检测SDF1表达变化;与Jurkat细胞共培养,绘制生长曲线并计算倍增时间,用流式细胞术检测细胞周期,末端脱氧核苷酸转移酶介导的dUTP缺失末端标记(TUNEL)法检测细胞凋亡,免疫细胞化学检测增殖细胞核抗原(PCNA)、Bcl2、Bax、Fas、FasL表达。以未转染急性白血病骨髓基质细胞(B组)及正常骨髓基质细胞(C组)作为对照。结果A组骨髓基质细胞培养上清SDF1含量为(384±41)pg/ml,较B组[(2474±271)pg/ml]、C组[(1324±154)pg/ml]明显降低。与之共培养的Jurkat细胞,A组与B组、C组比较,细胞增殖速度减缓,倍增时间延长(A组42h,B组29h,C组33h);细胞周期分析G0/G1期细胞比例增多[A组(28.47±2.39)%,B组(19.43±2.80)%,C组(27.15±2.07)%],S期细胞减少[A组(25.57±1.90)%,B组(74.48±3.23)%,C组(60.99±2.33)%],G2/M期细胞增多[A组(45.96±3.24)%,B组(6.09±1.96)%,C组(11.86±1.98)%];细胞凋亡率增加[A组(15.2±0.8)%,B组(5.4±0.7)%,C组(9.5±0.4)%];PCNA、Bcl2、Fas表达减少,Bax、FasL表达增多。结论阻抑SDF1表达在一定程度上抑制与骨髓基质细胞共培养的Jurkat细胞的增殖活性,并促进其凋亡。
Objective To study the effects of RNA interference inhibiting stromal cell derived factor-1 (SDF-1) expression on the proliferation and apoptosis of co-cultured Jurkat cells. Method Inhibition of SDF-1 expression by RNA interference (RNAi) was achieved by transfering SDF-1 specific short hairpin RNA(shRNA) expressing plasmid into cultured human acute leukemic bone marrow stromal cells. Resistant clones were obtained by G418 selection (group A). The concentration of SDF-1 protein in culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). The population double time (PDT), cell cycles,apoptosis rates and the expressions of PCNA, Bcl-2/Bax, Fas/FasL of co-cultured Jurkat cells were detected by cells counting, flow cytometry. TdT-mediated dUTP nick-end labelling (TUNEL) and immunocytochemistry (ICC), respectively. The untransfected acute leukemic(group B) and normal(group C) bone marrow stromal cells were taken as controls. Results The content of SDF-1 protein in supernatant of group A was significantly lower than that in group B or group C. As group A compared with group B and group C, the PDT of co-cultured Jurkat cells was prolonged (group A: 42 h, vs group B:29 h, group C:33 h), and G_0/G_1 stage cells increased [group A: ( 28.47±2.39)%, vs group B: (19.43±2.80)%,group C: (27.15±2.07)%], S stage cells decreased [group A: (25.57±1.90)%, vs group B: (74.48±3.23)%, group C: (60.99±2.33)%], G_ 2/M stage cells increased [group A: (45.96±3.24)%, vs group B: (6.09±1.96)%, group C: (11.86±1.98)%], the apoptosis rate increased [group A: (15.2±0.8)%, vs group B: (5.4± 0.7)%, group C: (9.5±0.4)%], and the expressions of PCNA, Bcl-2, Fas decreased; whereas the expressions of Bax and FasL were increased.Conclusion The inhibition of SDF-1 expression in bone marrow stromal cells inhibits the proliferation and promotes the apoptosis of co-cultured Jurkat cells.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2005年第7期421-424,共4页
Chinese Journal of Hematology
基金
国家自然科学基金资助项目(30170396)
