【作者】 饶佳；

【导师】 张荣艳；

【作者基本信息】 南昌大学 ， 内科血液病学， 2007， 硕士

【摘要】 目的:探讨人重组可溶性TRAIL(rsTRAIL)单独和联合阿糖胞苷(Ara-c)诱导HL-60细胞凋亡作用及其机制,为其应用于临床治疗急性白血病提供实验依据。方法:在体外以人类急性白血病细胞株HL-60细胞为研究对象,分成5组进行细胞培养:对照组、Ara-c组、rsTRAIL组、同时给药组(Ara-c+rsTRAIL)、先后给药组(Ara-c→rsTRAIL)。MTT比色法测定HL-60细胞生长抑制率;Annexin V/PI染色、流式细胞术检测HL-60细胞凋亡率;流式细胞术检测不同浓度Ara-c对HL-60细胞表面DR5表达水平的影响;流式细胞术检测rsTRAIL单药组及先后给药组对HL-60细胞表面DR5表达水平和HL-60细胞内caspase8活性的影响。结果:(1)MTT实验结果显示各组HL-60细胞生长抑制率为:对照组5.20±0.65;5mg/L Ara-c组11.53±0.51;rsTRAIL组(50、100、200μg /L):16.45±1.35、22.86±0.81、29.79±1.88;不同浓度的同时给药组:27.78±2.63、33.36±2.01、36.35±3.27;不同浓度的先后给药组:34.08±3.44、41.21±5.98、44.45±4.55。与对照组比较,50、100、200μg /L浓度的rsTRAIL能抑制HL-60细胞生长,在50-200μg /L浓度范围内生长抑制率呈现剂量依赖性(P<0.001)。同时给药组HL-60细胞生长抑制率大于单用同等浓度的rsTRAIL组和Ara-c组(P<0.001)。先后给药组细胞生长抑制率大于相应浓度的同时给药组,有统计学差异(P<0.05)。(2)流式细胞术分析显示各组HL-60细胞凋亡率:对照组13.41±1.52;Ara-c组61.67±2.79;rsTRAIL组(50、100、200 ug/L)25.78±2.21、36.76±0.62、44.50±2.78;不同浓度的同时给药组74.45±5.06、77.39±5.34、80.66±6.27;不同浓度的先后给药组85.05±3.77、86.33±4.83、87.71±4.78。与对照组比较,50、100、200ug/L浓度的rsTRAIL能诱导HL-60细胞凋亡,在50-200ug/L浓度范围内诱导凋亡率呈现剂量依赖性(P<0.001)。同时给药组HL-60细胞凋亡率大于相应浓度的单用同等浓度的rsTRAIL组和Ara-c组(P<0.05)。先后给药组HL-60细胞凋亡率大于同时给药组(P<0.05)。(3)Ara-c(2.5mg/L、5mg/L、10mg/L )作用24小时后,HL-60细胞表面DR5表达水平分别为:1.22±0.18、3.02±0.03、3.56±0.45。对照组HL-60细胞表面DR5表达水平为1.20±0.16。10mg/L Ara-C组细胞表面DR5表达水平最高,5mg/LAra-C组次之,2.5mg/L Ara-c组与对照组最低(P<0.001)。(4)流式细胞术检测HL-60细胞表面DR5表达水平结果显示:不同浓度的rsTRAIL组分别为0.61±0.05、0.91±0.02、1.29±0.04。不同浓度的先后给药组分别为0.90±0.03、1.31±0.06、3.69±0.09。先后给药组HL-60细胞表面DR5表达水平大于单用同等浓度的rsTRAIL组(P<0.001)。(5)流式细胞术检测HL-60细胞内caspase-8表达水平结果显示:不同浓度的rsTRAIL组分别为74.86±1.57、78.26±1.30、83.69±1.42。先后给药组分别为83.31±1.24、87.46±2.43、91.08±2.29。先后给药组HL-60细胞内caspase-8的活性水平大于单用同等浓度的rsTRAIL组(P<0.05)。结论:(1)rsTRAIL能抑制HL-60细胞生长,诱导HL-60细胞凋亡,在50-200ug/L浓度范围内,诱导细胞凋亡率呈现剂量依赖性。(2)Ara-c能上调HL-60细胞表面DR5表达水平,增强rsTRAIL诱导HL-60细胞凋亡的作用。(3)Ara-c和rsTRAIL联合用药时,先加Ara-c、再加rsTRAIL诱导HL-60细胞凋亡的效果比同时给药效果好。(4)Ara-c增强rsTRAIL诱导HL-60细胞凋亡的机制可能由Ara-c上调HL-60细胞表面DR5表达水平和/或增强细胞内caspase8的活性实现的。更多还原

【Abstract】 Objective:To investigate the combined effect of rsTRAIL with Ara-c or alone on HL-60 leukemia cell lines and its mechanism,and to provide experimental basis for treatment of acute leukemia.Methods: Human leukemia cell line HL-60 was cultured in vitro.HL-60 cell was separated into 5 groups:control group, Ara-c group,rsTRAILgroup, Ara-c+rsTRAIL group, Ara-c followed by rsTRAIL group(Ara-c→rsTRAIL group). The cytotoxic effect were meatured by MTT assay;Cell apoptosis rate was determined by flow cytometry after Annexin V/PI staining; HL-60 cell was treated with Ara-c of different concentration for 24 hours, expression level of DR5 on surface of HL-60 cells was determined by flow cytometry. Expression level of DR5 on surface of HL-60 cells and caspase-8 activition in HL-60 cells of rsTRAIL group、Ara-c→rsTRAIL group was determined by flow cytometry.Results:(1)The result by MTT assay: control group 5.20±0.65;Ara-c group 11.53±0.51;rsTRAIL group16.45±1.35、22.86±0.81、29.79±1.88,respectively;Ara-c+rsTRAIL group27.78±2.63、33.36±2.01、36.35±3.27,respectively;Ara-c→rsTRAIL group34.08±3.44、41.21±5.98、44.45±4.55,respectively. 50-200ug/L rsTRAIL could inhibit the proliferation of the HL-60 cell in concentration-dependent manners(P<0.001).The effects presented by cotreatment with Ara-c and rsTRAIL were significantly higher than Ara-c or rsTRAIL used alone(P<0.001). The effects presented by sequential treatment of HL-60 cells with Ara-c followed by rsTRAIL were significantly higher than cotreatment with rsTRAIL and Ara-c(P<0.05).(2)The result of percentage of apoptisis cells: control group 13.41±1.52;rsTRAIL group 25.78±2.21、36.76±0.62、44.50±2.78,respectively;Ara-c+rsTRAIL group74.45±5.06、77.39±5.34、80.66±6.27,respectively;Ara-c→rsTRAIL group 85.05±3.77、86.33±4.83、87.71±4.78,respectively. The apoptosis rate of rsTRAIL group was higher than that of control group,The apoptosis-induction of rsTRAIL was concentration-dependen(tP<0.05). The apoptosis rate of cotreatment with Ara-c and rsTRAIL was higher than that of Ara-c group and rsTRAIL group alone(P<0.001). The apoptosis rate of sequential treatment of HL-60 cells with Ara-c followed by rsTRAIL was higher than that of cotreatment with Ara-c and rsTRAIL(P<0.05)(.3)After treatment with Ara-c(2.5 mg/L、5 mg/L、10mg/L)for 24 hours,the expression level of DR5 on HL-60 was 1.22±0.18、3.02±0.03、3.56±0.45,respectively. The expression level of DR5 of control group was 1.20±0.16。The expression level of DR5 in 10mg/L Ara-c group was the highest, the expression level of DR5 in 5mg/L Ara-c group was the second, the expression level of DR5 in 2.5mg/L Ara-c group and control group was the lowes(tP<0.001)(.4)The expression level of DR5 on HL-60 of rsTRAIL group was 0.61±0.05、0.91±0.02、1.29±0.04。The expression level of DR5 on HL-60 of Ara-c→rsTRAIL group was 0.90±0.03、1.31±0.06、3.69±0.09,respectively. The expression level of DR5 was significant higher in sequential treatment of HL-60 cells with Ara-c followed by rsTRAIL than that in rsTRAIL group alone(P<0.001)(.5)The expression level of caspase-8 of rsTRAIL group was 74.86±1.57、78.26±1.30、83.69±1.42。The expression level of caspase-8 of Ara-c→rsTRAIL group was 83.31±1.24、87.46±2.43、91.08±2.29,respectively. The expression level of caspase-8 was significant higher in HL-60 with Ara-c followed by rsTRAIL than that in rsTRAIL group alone(P<0.05).Conclusion: (1)RsTRAIL could inhibit the proliferation of HL-60 cells, rsTRAIL could induced apoptosis of HL-60 cells.The apoptosis-induction of rsTRAIL was concentration-dependent.(2)Ara-c could upregulated DR5 expression on the surface of HL-60 cells ,and enhance effect of rsTRAIL-inducing apoptosis(.3)Sequential treatment of HL-60 cells with Ara-c followed by rsTRAIL induced significantly more apoptosis than cotreatment with rsTRAIL and Ara-c(.4)Ara-c and rsTRAIL has a synergistic inhibitory effect on growth of HL-60 cells,the mechanism may correlate with the up-regulation the expression level of DR5 and/or caspase-8 of HL-60 cells.更多还原