脐血CD_3 AK细胞培养上清液对白血病细胞株HL-60细胞的影响

Effects of supernatant of cord blood CD_3 AK cells on proliferation, differentiation and apoptosis of HL-60 cells

目的在体外实验中,研究脐血CD3AK细胞培养上清液(cord blood CD3 AK supernatant,CS)对HL-60细胞增殖、分化和凋亡的影响。方法以不同浓度的CS(10%CS、15%CS、20%CS)作用于不同时间(3d,6d和9d)的HL-60细胞为实验对象,应用细胞计数法结合锥虫蓝拒染法研究CS诱导的HL-60增殖情况。应用流式细胞仪分析CS作用前后细胞周期及细胞表面分化标志CD11b的变化,光学显微镜下观察细胞形态变化,氯化硝基唑蓝(NBT)还原实验研究细胞的NBT还原阳性百分率,从而研究HL-60细胞的分化。应用电子显微镜观察细胞凋亡,原位末端标记法研究CS作用前后HL-60细胞的凋亡变化。结果随CS作用时间的延长和剂量的增加,细胞增殖速度逐渐减慢。细胞周期分析显示,CS作用后G0期和G1期显著增多,S期显著减少,G2期和M期无明显变化,说明CS使细胞阻滞在G0和G1期。随CS作用时间的延长,细胞体积逐渐增大,3d后被诱导的细胞表面开始表达分化标志物CD11b并出现细胞核型变化,NBT阳性细胞增多,随CS浓度的增加和作用时间的延长,这些变化越明显。20%CS诱导72h后,被诱导的细胞出现凋亡,随诱导时间延长,凋亡细胞增多。结论CS能抑制HL-60细胞的增殖,诱导HL-60细胞分化和凋亡。

Objective The study was to investigate the impacto of cord blood CD3AK cell culture supernatant （CS） on proliferation, differentiation and apoptosis of HL-60 cells. Methods HL-60 cells were treated with different concentrations of CS （10%, 15% , 20% ） for 3 days, 6 days and 9 days, and the same cells of control group were not treated with CS. The growth of induced cells was assessed with Trypan blue staining and cell counting with cytometer. The differentiation marker CD11b on the cell surface and cell-cycle was analyzed by flow cytometry （FCM）, cell morphology （Wright-Giemsa staining） and NBT test to determine the extent of differentiation. Meanwhile, the changes of the apoptosis of the cells induced by 20% CS at different time points （3, 6 and 9 days） were analyzed by TUNNEL-POD, and the apoptotic characteristics of cells were observed. Results The growth of HL-60 cell was inhibited as CS-inducing time and the dose of CS increased. At the same time, but HL-60 cell number in G0/G1 phase of cell-cycle increased, but HL-60 cell number in S phase decreased compared with untreated group. The HL-60 cells induced by 20% CS for 9 days showed that （52.7 ± 1.8 ） % of cells were at Go ＋ G1 phase and （43.8 ± 1.1 ） % were at S phase （P 〈 0.05）, which demonstrated that HL-60 cells induced by 20% CS underwent G0/G1 phase cell-cycle arrest. The volume of the differentiated cells was enlarged gradually as CS-inducing time prolonged. After 3 days the differentiating cells began to express differentiating marker CD11b on the cell surface and the nuclei morphology of the differentiated cells was also changed and NBT-stained cells increased in number with the increased dose of CS increased. Three days after induction by 20% CS, the induced ceils began to show signs of apoptosis and the apoptotic percentage of induced ceils gradually increased with CS-induction time. The rate of apoptosis of cells was （ 33.3 ± 2.3 ） % at 9 days （ P 〈 0.01 ）. Conclusion CS could not only inhibit the growth of HL-60 cells but also induce the differentiation and apoptosis in HL-60 cells.