骨髓成纤维样基质细胞系对急性髓细胞白血病细胞系增殖与分化影响的研究

Effect of human bone marrow fibroblastoid stromal cell line on the proliferation and differentiation in acute myeloid leukemia cell lines

目的:以急性髓细胞白血病(acute myeloid leukaemia,AML)细胞系为研究对象,观察正常人骨髓成纤维样细胞系(human bone marrow fibro-blastoid stromal cell line,HFCL)对急性单核细胞白血病细胞系U937、急性粒细胞白血病敏感细胞HL-60和多药耐药细胞HL-60/VCR增殖和分化的影响。方法:建立U937、HL-60和HL-60/VCR细胞与HFCL细胞的共培养模型,实验分对照组、直接接触组和transwell组。采用台盼蓝拒染法测定生长曲线;硝基四氮唑蓝(NBT)确定细胞分化;流式细胞仪检测细胞周期和CD11b、CD13、CD14和CD33细胞表面抗原进一步鉴定细胞分化;West-ernblot检测增殖细胞核抗原(PCNA)和P-糖蛋白(P-gp)的表达。结果:与HFCL细胞共培养96h后,U937、HL-60和HL-60/VCR细胞的生长受抑,且与HFCL细胞直接接触组的抑制作用大于用tr-answell组,P<0.01。同时发现AML细胞系与HFCL细胞共培养后,G0/G1期细胞比例均增高,而S期细胞均减少,P<0.01;尤其是直接接触组CD11b和CD14表达也均增高(P<0.01),CD13和CD33变化不大,NBT阳性细胞轻度增高,且差异有统计学意义。Western blot检测结果显示,3种AML细胞系PCNA表达下调;以直接接触组为甚。提示介导着白血病细胞和骨髓基质细胞间的相互作用的整合素β1(VLA-4)和β2(LFA-1),在HFCL细胞对AML细胞生长作用影响中起着不可忽视的作用。结论:HFCL对3种具有代表性的AML细胞系U937、HL-60和HL-60/VCR有增殖抑制和诱导分化作用,除了能抑制AML细胞的生长,抑制PCNA的表达,出现G0/G1期阻滞外,还能诱导其部分向单核细胞分化。

OBJECTIVE： To investigate the effects of human bone marrow fibroblastoid stromal cell line （HFCL） on the proliferation and differentiation of AML sensitive U937, HL-60 cell line and multidrug-resistant （MDR） HL-60/VCR cell line. METHODS：The coculture systems of U937, HL-60 or HL-60/VCR cells in direct contact with HFCL cells, and with HFCL cells separated by transwell were established. The cell growth curves were measured by cell counting and the cell cycle analysis was measured by flow cytometry （FCM）. Cell differentiation was determined by morphologic observation of the ability of NBT cells and flow cytometric detection of expression of CDllb, CD14, CD13 and CD33. The expression of PCNA and P-gp were determined by Western blot. RESULTS： Compared with leukemic cells alone, the proliferation of U937, HL-60 and HL-60/VCR cells cocultured with HFCL cells was inhibi-ted and the inhibition of direct contact group was higher than that of transwell group. The percentage of G1 phase cells of AML cells cocultured with HFCL cells was higher than that without HFCL cells, and the percentage of S phase cells was lower, P〈0.01. The NBT positive cells and the expressions of CDllb and CD14 increased, P〈0.01. The results of Western blot showed that the expres-sion of PCNA was lower. As the adhesion of AML blasts with stromal cells was mediated by the interaction of β1 （VLA-4）and β2（LFA-1） integrin and stromal cells, it was inferred that β1 （VLA-4）and i32（LFA-1） integrins might play an indelible important role. CONCLUSION： Coculture with HFCL stromal cells could inhibit the proliferation and induce the differentiation of U937 HL-60 and HL-60/VCR cells.