新型化疗药物对视网膜母细胞瘤的体外药效实验
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摘要
一、目的：
1、研究肿瘤干细胞在视网膜母细胞瘤化疗耐药中的作用；
2、开发更有效的新型化疗药物并探索更科学的化疗方案
二、方法：
选用Y79细胞株及本课题组前期工作中自行建立的、具有肿瘤干细胞样特性的细胞株FD-RB作为研究的对象。用CCK-8的方法检测新型化疗药物拓扑替康、DG-2在不同浓度下对Y79细胞和FD-RB细胞的抑制作用,并与Rb传统化疗药物卡铂及长春新碱的体外作用做对照,用t检验的方法比较两株细胞在各种药物不同浓度下的存活率差异,同时比较新型化疗药物与传统化疗药物的半数抑制浓度(IC50),筛选出体外杀伤作用最强的化疗药物；将不同浓度的DG-2与拓扑替康、卡铂进行两两组合,检测联合用药下Y79和FD-RB的细胞存活率,用多重线性回归分析联合用药与单药使用的效用差异,并分析药物间的相互作用。
三、结果
FD-RB细胞的体外耐药性低于Y79细胞,两者的差异具有统计学意义(P<0.05)；在四种检测药物中,拓扑替康的体外细胞杀伤作用仅次于长春新碱,它对Y79细胞的IC50约为6.52umol/L,对FD-RB细胞的IC50约为0.026umol/L; DG-2在有氧状态下也能有效杀伤两株细胞；拓扑替康与DG-2联合应用时可能会相互削弱的彼此的效用；卡铂和DG-2的体外联合用药未发现交互作用,两者的联合效应只是两者单独效应的叠加。
四、结论
从本研究的数据来看,肿瘤干细胞不一定是导致肿瘤耐药、化疗失败的唯一因素,其自身的耐药性也受多方面的影响。拓扑替康在体外对Y79及FD-RB细胞均有良好的杀伤作用,对于治疗复发转移的Rb具有潜在的临床应用价值；DG-2在正常氧供状态下也可抑制Y79及FD-RB细胞的生长,结合其降低缺氧相关的化疗耐药作用,DG-2在Rb中的治疗前景令人期待。拓扑替康与DG-2联合应用体内疗效有待进一步实验证实。
Purpose:
1、To investigate the role of cancer stem cells in retinoblastoma chemoresistance.
2、To identify novel active drugs with higher efficacy and to develop better chemotherapeutics.
Methods:
We have established a new continuous cell line named FD-RB, and demonstrated its characteristics as tumor stem-like cells in our previous work. Y79 is another cell line chosen in this study. Using Cell Counting Kit-8, we tested the in vitro efficacy of novel chemotherapeutics (including a topoisomerase I inhibitor named Topotecan and a glycolytic inhibitor named 2-deoxy-D-glucose) to both Y79 and FD-RB. The efficacy of tradition chemotherapeutics (including carboplatin and vincristine) is used as matched control. Cell viabilities of Y79 and FD-RB under every concentration was analysed by t-test. Then we compared the 50% inhibiting concentration of each drug. After selecting the most effective drug in vitro, we tested the efficacy of topotecan combined with DG-2 and of DG-2 combined with carboplatin for the treatment of Y79 and FD-RB, and assessed the cell viabilities under different drug combination. The effect of combination chemotherapeutics is compared with the effect of single chemotherapeutics. Then we investigated the interaction between these combined agents.
Results:
The drug tolerance of FD-RB in vitro is inferior to that of Y79, the difference has statistics significance (P<0.05); Among four tested drugs, topotecan has a good cell-killing effect second only to vincristine, its IC50 Y79 is about 6.52umol/L,and its IC50 ED-RB is about 0.026umol/L. DG-2 can effectively kill both Y79 and FD-RB even under normoxia. Combined topotecan and DG-2 may attenuate their efficacy. No interaction between DG-2 and Carboplatin was determined. The combined effect of this two agents is simply accumulated.
Conclusion:
Cancer stem cells may not be the only reason that leads to chemoresistance and treatment failure. The resistance of CSCs can also be affected by many aspects. Topotecan has good cell-killing effect to both Y79 and FD-RB in vitro, and it has a potential value in treating recurrent or metastasis Rb. DG-2 can inhibit the growth of Y79 and FD-RB in vitro even under normoxia condition, while it can also reduce the hypoxia-related chemoresistance. So it may have a good prospect in Rb treatment. The efficacy of topotecan combined with DG-2 in vivo must be further confirmed.
1、研究肿瘤干细胞在视网膜母细胞瘤化疗耐药中的作用；
2、开发更有效的新型化疗药物并探索更科学的化疗方案
二、方法：
选用Y79细胞株及本课题组前期工作中自行建立的、具有肿瘤干细胞样特性的细胞株FD-RB作为研究的对象。用CCK-8的方法检测新型化疗药物拓扑替康、DG-2在不同浓度下对Y79细胞和FD-RB细胞的抑制作用,并与Rb传统化疗药物卡铂及长春新碱的体外作用做对照,用t检验的方法比较两株细胞在各种药物不同浓度下的存活率差异,同时比较新型化疗药物与传统化疗药物的半数抑制浓度(IC50),筛选出体外杀伤作用最强的化疗药物；将不同浓度的DG-2与拓扑替康、卡铂进行两两组合,检测联合用药下Y79和FD-RB的细胞存活率,用多重线性回归分析联合用药与单药使用的效用差异,并分析药物间的相互作用。
三、结果
FD-RB细胞的体外耐药性低于Y79细胞,两者的差异具有统计学意义(P<0.05)；在四种检测药物中,拓扑替康的体外细胞杀伤作用仅次于长春新碱,它对Y79细胞的IC50约为6.52umol/L,对FD-RB细胞的IC50约为0.026umol/L; DG-2在有氧状态下也能有效杀伤两株细胞；拓扑替康与DG-2联合应用时可能会相互削弱的彼此的效用；卡铂和DG-2的体外联合用药未发现交互作用,两者的联合效应只是两者单独效应的叠加。
四、结论
从本研究的数据来看,肿瘤干细胞不一定是导致肿瘤耐药、化疗失败的唯一因素,其自身的耐药性也受多方面的影响。拓扑替康在体外对Y79及FD-RB细胞均有良好的杀伤作用,对于治疗复发转移的Rb具有潜在的临床应用价值；DG-2在正常氧供状态下也可抑制Y79及FD-RB细胞的生长,结合其降低缺氧相关的化疗耐药作用,DG-2在Rb中的治疗前景令人期待。拓扑替康与DG-2联合应用体内疗效有待进一步实验证实。
Purpose:
1、To investigate the role of cancer stem cells in retinoblastoma chemoresistance.
2、To identify novel active drugs with higher efficacy and to develop better chemotherapeutics.
Methods:
We have established a new continuous cell line named FD-RB, and demonstrated its characteristics as tumor stem-like cells in our previous work. Y79 is another cell line chosen in this study. Using Cell Counting Kit-8, we tested the in vitro efficacy of novel chemotherapeutics (including a topoisomerase I inhibitor named Topotecan and a glycolytic inhibitor named 2-deoxy-D-glucose) to both Y79 and FD-RB. The efficacy of tradition chemotherapeutics (including carboplatin and vincristine) is used as matched control. Cell viabilities of Y79 and FD-RB under every concentration was analysed by t-test. Then we compared the 50% inhibiting concentration of each drug. After selecting the most effective drug in vitro, we tested the efficacy of topotecan combined with DG-2 and of DG-2 combined with carboplatin for the treatment of Y79 and FD-RB, and assessed the cell viabilities under different drug combination. The effect of combination chemotherapeutics is compared with the effect of single chemotherapeutics. Then we investigated the interaction between these combined agents.
Results:
The drug tolerance of FD-RB in vitro is inferior to that of Y79, the difference has statistics significance (P<0.05); Among four tested drugs, topotecan has a good cell-killing effect second only to vincristine, its IC50 Y79 is about 6.52umol/L,and its IC50 ED-RB is about 0.026umol/L. DG-2 can effectively kill both Y79 and FD-RB even under normoxia. Combined topotecan and DG-2 may attenuate their efficacy. No interaction between DG-2 and Carboplatin was determined. The combined effect of this two agents is simply accumulated.
Conclusion:
Cancer stem cells may not be the only reason that leads to chemoresistance and treatment failure. The resistance of CSCs can also be affected by many aspects. Topotecan has good cell-killing effect to both Y79 and FD-RB in vitro, and it has a potential value in treating recurrent or metastasis Rb. DG-2 can inhibit the growth of Y79 and FD-RB in vitro even under normoxia condition, while it can also reduce the hypoxia-related chemoresistance. So it may have a good prospect in Rb treatment. The efficacy of topotecan combined with DG-2 in vivo must be further confirmed.
引文
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