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为了观察正常人骨髓成纤维样基质细胞系HFCL对白血病敏感细胞HL-60和多药耐药细胞HL-60／VCR 凋亡易感性的影响,先建立HL-60或HL-60／VCR细胞与HFCL细胞共培养体系;采用瑞氏-吉姆萨染色和吖啶橙／ 溴化乙啶(AO／EB)染色,分别在光镜和荧光显微镜下进行形态学观察;TUNEL检测晚期凋亡细胞,流式细胞术检 测细胞周期、凋亡峰和annexin V阳性的早期凋亡细胞;Western blot检测Bcl-2、活化的胱冬蛋白酶(caspase)-3蛋 白和P糖蛋白(Pgp)的表达变化。结果表明,经topotecan(TPT)处理后的HL-60和HL-60／VCR细胞,在光镜和荧光 显微镜下均有典型的凋亡细胞形态学改变,这些改变具有时间和剂量依赖性;annexin V染色后能检测到早期凋亡 细胞;细胞周期显示:G1期细胞比例增高,S期减低,并有明显凋亡峰;TUNEL能检测到许多阳性细胞;同时出现活 化的caspase-3,伴有Bcl-2的表达下调,但与HFCL细胞共培养后,经TPT处理的HL-60和HL-60／VCR细胞中早期 和晚期凋亡细胞有所减少,凋亡峰减低,而且活化的Caspase-3表达减弱,Bcl-2蛋白表达上调,且以直接接触组为 甚。结论:正常骨髓成纤维样基质细胞HFCL能轻度降低白血病HL-60和HL-60／VCR细胞对TPT的凋亡易感性, 并有caspase-3和Bcl-2重要信号传导分子的参与。
In order to observe the effect of HFCL on the susceptibility of HL-60 and HL-60 / VCR cells to HL-60 / VCR induced by normal human bone marrow fibroblastoid stromal cell line HL-60 or HL-60 / VCR, HL- HFCL cells co-culture system; Wright-Giemsa staining and acridine orange / ethidium bromide (AO / EB) staining, respectively, under light microscope and fluorescence microscopy morphological observation; TUNEL detection of late apoptotic cells, flow Cell cycle, apoptosis peak and annexin V positive early apoptotic cells were detected by flow cytometry. The expressions of Bcl-2, caspase-3 and Pgp were detected by Western blot. The results showed that morphological changes of apoptotic cells were observed in both HL-60 and HL-60 / VCR cells treated with topotecan (TPT), both in time and in dose-dependent manner under light and fluorescence microscopes. Annexin The apoptotic cells were detected by V staining. The cell cycle showed that the proportion of cells in G1 phase increased, the S phase decreased, and the apoptotic peak was obvious. TUNEL could detect many positive cells. At the same time, activated caspase-3 accompanied with Bcl-2 expression was down-regulated in HFCL cells. However, the number of apoptotic cells in HL-60 and HL-60 / VCR cells treated with TPT decreased and the apoptotic peak decreased in co-cultured HFCL cells. The activated Caspase- 3 expression weakened, Bcl-2 protein expression increased, and in direct contact group is even. CONCLUSIONS: HFCL in normal bone marrow fibroblast-like stromal cells can slightly reduce the susceptibility of TPT to leukemia HL-60 and HL-60 / VCR cells, and involve in important signal transduction molecules of caspase-3 and Bcl-2.