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实体肿瘤内存在乏氧细胞,约占10%~50%,它们大多处于细胞周期的G0期,增殖缓慢或不增殖,对射线有较强的抗拒性而使肿瘤更具有侵袭性。乏氧细胞的放射敏感性只有常氧细胞的1/3,在放疗中这部分细胞不易被杀死,从而降低了肿瘤的放射敏感性,导致放疗效果不理想,也成为肿瘤放疗后复发和转移的潜在隐患[1]。因此,在肿瘤放疗增敏的相关研究中,建立合适的乏氧细胞模型,是一项非常重要的工作。目前国内外建立乏氧细胞模型常用的方法有两种,一种是在细胞或组织的培养液中加入二氯化钴或铁的螯合剂,阻断氧信号的转导,模拟乏氧信号传给细胞,也称作细胞乏氧[2];另一种是在低氧的环境中培养肿瘤细胞或组织,即使用特殊的实验设备如“乏氧培养箱”,也称作环境乏氧[3]。本研究主要探讨二氯化钴与环境乏氧诱导人卵巢癌细胞株SKOV3乏氧的特点及更适合用于放疗增敏研究中乏氧细胞模型的建立方法。
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不同浓度的二氯化钴作用不同时间点对SKOV3细胞的毒性作用如图 1所示。与正常对照组比较,相同时间内,50、100、150、200、250 μmol/L二氯化钴对SKOV3细胞的增殖具有抑制作用,并随二氯化钴的浓度增加,其抑制作用增强;相同浓度的二氯化钴作用24、48、72、96 h后,各时间点的细胞存活率差异均有统计学意义(50 μmol/L:F=102.555;100 μmol/L:F=168.357;150 μmol/L:F=2174.232;200 μmol/L:F=5620.296;250 μmol/L:F=23 076.373,P均<0.05),且作用时间越长,对SKOV3细胞的增殖抑制作用越强。100、150 μmol/L二氯化钴作用96 h后,SKOV3细胞的存活率分别为91.55%和70.79%,可见,150、200、250 μmol/L二氯化钴对SKOV3细胞产生了较强的毒性,因此选择100 μmol/L二氯化钴作为后续建立乏氧细胞模型的适宜浓度。
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由表 1各时间点的OD值可以看出,常氧对照组的细胞随培养时间的延长不断增殖(F=16 724.300,P<0.01),而环境乏氧组各时间点之间无显著性变化(F=1.202,P>0.05),即细胞在乏氧环境下,未出现明显的细胞增殖;相同时间点内,常氧对照组与环境乏氧组间差异均有统计学意义,前者的细胞增殖率远高于环境乏氧组。
组别 OD值 24 h 48 h 72 h 96 h 常氧对照组 0.3577±0.0054 0.4785±0.0034 0.6945±0.0070 0.9568±0.0033 环境乏氧组 0.2485±0.0035 0.2460±0.0064 0.2439±0.0052 0.2502±0.0082 t值 40.436 78.924 126.748 196.960 P值 <0.01 <0.01 <0.01 <0.01 表 1 各组培养不同时间后的OD值
(x±s, n=6) Table 1. The optical density of different groups at different times
(x±s, n=6) -
如图 2所示,与同组未照射细胞相比,常氧对照组与二氯化钴组接受2、4、6、8 Gy的X射线单次照射后,其细胞存活率均显著下降(F=2263.039、3672.044,P<0.01),且放射剂量越大,其细胞存活率越低,而环境乏氧组无显著性变化(F=1.412,P>0.05);与常氧对照组的同剂量照射细胞相比,除了2 Gy无显著性差异外(F=61.125,P>0.05),二氯化钴组其余各照射剂量下细胞存活率均升高(4 Gy:F=181.825;6 Gy:F=373.830;8 Gy:F=2425.510,P均<0.05),而与常氧对照组、二氯化钴组的同剂量照射细胞相比,环境乏氧组细胞存活率均显著升高(2 Gy:F=61.125;4 Gy:F=181.825;6 Gy:F=373.830;8 Gy:F=2425.510,P均<0.01)。
放疗增敏研究中人卵巢癌SKOV3乏氧细胞模型的建立方法
The method of establishing the hypoxic SKOV3 cellular model in the research of radiotherapy sensitizer
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摘要:
目的 探讨适用于放疗增敏研究中人卵巢癌SKOV3乏氧细胞模型的建立方法。 方法 将人卵巢癌细胞株SKOV3分为常氧对照组、二氯化钴组和环境乏氧组,各组均在X射线单次照射(0、2、4、6、8 Gy)后72 h,应用噻唑蓝法检测细胞增殖。 结果 与常氧对照组未照射细胞相比,二氯化钴组和环境乏氧组未照射细胞的细胞增殖率均显著降低(t=24.789、196.960,P均<0.01);与同组未照射细胞相比,常氧对照组与二氯化钴组接受不同剂量的X射线单次照射后,其细胞存活率均显著性下降(F=2263.039、3672.044,P均<0.01),且放射剂量越大,其细胞存活率越低,而环境乏氧组无显著性变化(F=1.412,P>0.05);与常氧对照组、二氯化钴组的同剂量照射细胞相比,环境乏氧组细胞存活率均显著升高(2 Gy:F=61.125;4 Gy:F=181.825;6 Gy:F=373.830;8 Gy:F=2425.510,P均<0.01)。 结论 环境乏氧组的细胞对射线产生了强烈的抵抗性,即放射敏感性显著性降低,射线对其杀伤力减弱,且所获得的乏氧细胞模型明显优于二氯化钴组,因此环境乏氧比二氯化钴更适合作为放疗增敏研究中人卵巢癌SKOV3乏氧细胞模型的建立方法。 Abstract:Objective To investigate the suitable method of establishing the hypoxic SKOV3 cellular model in the research of radiotherapy sensitizer. Methods Human ovarian cancer cell line SKOV3 were divided into three groups:normoxic control group, cobalt chloride group and environmental hypoxia group. Cell proliferation of each group 72 h after single X-ray irradiation(0, 2, 4, 6, 8 Gy)were detected by MTT assay. Results Cell proliferation of non-irradiated cells in cobalt chloride group and environmental hypoxia group were significantly lower than that in normoxic control group(t=24.789, 196.960, both P < 0.01). Cell viability of the cells in normoxic control group and cobalt chloride group were significantly decreased after receiving a single X-ray irradiation of different doses compared with non-irradiated cells in the same group(F=2263.039, 3672.044, both P < 0.01) and their cell viability were decreased with the increase of radiation dose, while environmental hypoxia group had no significant change(F=1.412, P > 0.05). The cell viability of irradiated cells in environmental hypoxia group were significantly higher than the irradiated cells with the same radiation in normoxic control group and cobalt chloride group(2 Gy:F=61.125;4 Gy:F=181.825;6 Gy:F=373.830;8 Gy:F=2425.510, all P < 0.01). Conclusions Cells in environmental hypoxia group were very resistant to radiation. Their radiation sensitivity strikingly decreased and the killing effect of radiation on them was weak. The hypoxic cellular model of environmental hypoxia group was obviously superior to cobalt chloride group. Environmental hypoxia method was more suitable to establish the hypoxic SKOV3 cellular model in the research of radiotherapy sensitizer compared with cobalt chloride. -
Key words:
- Breast neoplasms /
- SKOV3 cell line /
- Hypox cell /
- Radiotherapy sensitizer
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表 1 各组培养不同时间后的OD值
(x±s, n=6) Table 1. The optical density of different groups at different times
(x±s, n=6) 组别 OD值 24 h 48 h 72 h 96 h 常氧对照组 0.3577±0.0054 0.4785±0.0034 0.6945±0.0070 0.9568±0.0033 环境乏氧组 0.2485±0.0035 0.2460±0.0064 0.2439±0.0052 0.2502±0.0082 t值 40.436 78.924 126.748 196.960 P值 <0.01 <0.01 <0.01 <0.01 -
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