化疗药物诱导视网膜母细胞瘤细胞凋亡模型的建立

Establishment of a Chemotherapeutic Agents-Induced Apoptosis Model of Retinoblastoma

目的:检测不同类型肿瘤化疗药物对视网膜母细胞瘤(retinoblastoma RB)细胞系 HXO-RB_(44) 的诱导凋亡作用,建立该细胞系的细胞凋亡模型,作为今后研究化疗药物诱导RB细胞凋亡机理的工作基础。方法:将长春新碱、阿糖胞苷、氨甲喋呤、环磷酰胺、噻替派、米托蒽醌、阿克拉霉素、吡喃阿霉素、顺铂、卡铂、丝裂霉素、足叶乙甙等十二种化疗药物分别以10^(-9) 、10^(-8)、10^(-7)、10^(-6)、10^(-5)、10^(-4)mol·L^(-1)的浓度加入RB细胞中培养24小时,利用DNA凝胶电泳技术,检测RB细胞在不同化疗药物、不同浓度作用下的凋亡情况;将其中能产生明显凋亡梯带的化疗药物按最适浓度加入RB细胞中分别培养4、8、16、24、48、72小时,检测在不同时间的作用下,HXO-RB_(44)细胞系的细胞凋亡现象,并运用透射电子显微镜技术从形态学上观察凋亡细胞的存在。结果:10^(-6)～10^(-5)mol·L^(-1)长春新碱及10^(-5)mol·L^(-1)阿克拉霉素作用于HXO-RB_(44)细胞系24小时后,DNA凝胶电泳出现典型的限制性降解片段梯带,其余十种化疗药物作用后均未见该特征性表现。取10^(-5)mol·L^(-1)长春新碱作用于RB细胞,8小时开始出现DNA电泳梯带,24小时最明显,48小时后减弱。透射电镜可观察到大量具有明显形态学特征的凋亡RB细胞。结论:长春新碱和?

Objective: To determine the apoptotic effects of different chemotherapeutic agents on the retinoblastoma(RB) cell line HXO-RB44 and to establish a drug-induced apoptosis model of RB in vitro as the basis for further research on the mechanism of drug-induced apoptosis and spontaneous regression of RB. Methods: Twelve chemotherapeutic agents , including vincristine, cytarabin, metho-trexate, cyclophosphamide, thiotepa, mitoxantrone, aclanomycin, pirarubicin, cispla-tin, carboplatin, mitomycin, etoposide, of different concentration (10-9, 10 -8, 10 -7, 10-6,10-5,10-4 mol - L-1) were employed into HXO-RB44 cell line respectively for 24 hours, then apoptotic effects on it were decided by observing the DNA ladders on agarose gel electrophoresis. After that, one chemotherapeutic agent with the most evident DNA ladders was applied into HXO-RELw cell line for 4, 8, 16, 24, 48 and 72 hours respectively, and the DNA ladders on agarose gel were also surveyed. Apoptotic cells were identified with transmission electron microscopy. Results: Typical DNA ladders were shown on agarose gel after HXO-RB44 cell line were exposed to 10-6 - 10-5mol - L-1 vincristine and 10-5mol - L-1 aclanomycin for 24 hours and the former were much clearer. No DNA ladders did emerge when the RB cells were treated by the rest ten chemotherapeutic agents. The DNA ladders began to appear when dealt with 10-5mol - L-1 vincristine for 8 hours, to be most obvious for 24 hours, and abated for 48 hours then disappeared for 72 hours. Amount of apoptotic RB cells were observed by transmission electron microscopy. Conclusion: Both vincristine and aclanomycin have the effect of inducing apoptosis on HXO-RB44 cell line, but that of vincristine is more forceful, which is time and concentration dependent. So vincritine is an ideal agent for establishing an apoptosis model of HXO-RB44 cell line. Eye Science 1999; 15, 207 - 211.