基于八腔室玻片的染色体畸变初筛方法的建立与验证

目的：为简化传统体外染色体畸变试验工序并减少受试物用量,建立基于八腔室玻片的染色体畸变初筛方法。方法：首先利用环磷酰胺（CP）和丝裂霉素C（MMC）作为阳性模式药物,分别建立CP作用6 h（＋S_9）和MMC作用24 h（-S_9）的八腔室玻片的染色体畸变试验方法。再用于检测5个已知遗传毒性物质苯并芘[B（a）P]、2-氨基蒽（2-AA）、甲基磺酸甲酯（MMS）、乙基磺酸甲酯（EMS）、依托泊苷（Eto）和2个非遗传毒性药物氨苄青霉素钠（AS）和氯化钠（SC）在±S_9两个条件下的染色体畸变效应,验证该法的灵敏度和特异性。结果：CP和MMC分别在±S_9活化系统中染色体结构畸变率呈浓度依赖性增加,与溶剂对照组相比,差异均具有统计学意义（P〈0.05或P〈0.01）。在验证试验中,B（a）P和2-AA在＋S_9系统中呈染色体畸变阳性,与溶剂对照组相比,差异均具有统计学意义（P〈0.05或P〈0.01）;MMS、EMS和Eto的染色体畸变率呈浓度依赖性增加（±S_9结果均为阳性）,与溶剂对照组相比,差异均具有统计学意义（P〈0.05或P〈0.01）;而2个已知非遗传毒性药物（AS和SC）在±S_9系统中均呈阴性结果。结论：通过上述物质的验证表明,八腔室玻片染色体畸变试验是一种简单、快速、有效的遗传毒性初筛方法。

Establishment and validation of a chromosomal aberration screening test using eight-well chamber slides

OBJECTIVE:A chromosomal aberration（CA） screening test was established using eight-well chamber slides without adopting complicated procedures and chemical agents in the traditional chromosomal aberration test in vitro.The validation of the protocol was conducted using several genotoxic chemicals with various mechanisms of action and non-genotoxic compounds.METHODS:Optimized conditions for the test procedure in which cells were treated with cyclophosphamide（CP） for 6 h（for the +S₉ condition） and mitomycin C（MMC） for 24 h（for the -S₉ condition） were investigated.And then five known genotoxic chemicals including benzo（a）pyrene[B（a）P],2-aminoanthracene（2- AA）,methyl methanesulphonate（MMS）,ethyl methansulfonate（EMS）,etoposide（Eto） and two non-genotoxic chemicals including ampicilin sodium salt（AS） and sodium chloride（SC） were used in the sensitive and specific validation research under these conditions.RESULTS:The CA percentage of CP（6 h,+S₉） and MMC（24 h,-S₉） showed dose-dependent accumulation.In the validation tests,B（a）P and 2-AA were positive only in（6 h,+S₉） condition while MMS,EMS and Eto showed positive results in both +/-S₉ conditions.AS and SC were negative in both +/-S₉ conditions.CONCLUSION: The chromosomal aberration test using eight-well chamber slides could be considered as a simple,rapid and effective method,which could be used in genotoxicity screening in the early research phase of drug development.