癌变·畸变·突变
癌變·畸變·突變
암변·기변·돌변
CARCINOGENSES,TERATOGENSIS AND MUTAGENESIS
2015年
1期
39-43
,共5页
欧红梅%周长慧%涂宏刚%黄鹏程%常艳
歐紅梅%週長慧%塗宏剛%黃鵬程%常豔
구홍매%주장혜%도굉강%황붕정%상염
CHO-K1细胞%体外微核试验%流式细胞术%96孔板
CHO-K1細胞%體外微覈試驗%流式細胞術%96孔闆
CHO-K1세포%체외미핵시험%류식세포술%96공판
CHO-K1 cells%in vitro micronucleus assay%flow cytometric%96-well microplate
目的:建立96孔板流式细胞术体外微核自动化检测的方法,并探讨其用于药物早期遗传毒性筛选和遗传毒性评价的可能性。方法:试验分为+S9短时处理组(4 h)和-S9持续处理组(24 h),分别选择3个不同浓度的环磷酰胺和丝裂霉素C处理CHO-K1细胞,24 h后收获细胞。采用EMA和SYTOX Green双色标记,流式细胞仪分析96孔板的微核率,并与常规标准平皿培养、细胞分裂阻滞法的双核微核结果进行比较。结果:在有或无S9处理条件下,不同浓度的环磷酰胺、丝裂霉素C诱导产生的微核率较溶剂对照组均显著增加(P均<0.05),剂量-效应关系明显。两种微核检测方法的Spearman相关系数( rs)为1.000。结论:流式细胞术检测环磷酰胺、丝裂霉素C作用于CHO-K1细胞的微核试验结果均为阳性,与文献报道一致,因而本试验初步建立了流式细胞术体外微核检测方法。流式细胞术检测微核的方法与人工阅片的方法相关性好,提示该方法用于化合物早期遗传毒性筛选和遗传毒性评价具有良好的前景。
目的:建立96孔闆流式細胞術體外微覈自動化檢測的方法,併探討其用于藥物早期遺傳毒性篩選和遺傳毒性評價的可能性。方法:試驗分為+S9短時處理組(4 h)和-S9持續處理組(24 h),分彆選擇3箇不同濃度的環燐酰胺和絲裂黴素C處理CHO-K1細胞,24 h後收穫細胞。採用EMA和SYTOX Green雙色標記,流式細胞儀分析96孔闆的微覈率,併與常規標準平皿培養、細胞分裂阻滯法的雙覈微覈結果進行比較。結果:在有或無S9處理條件下,不同濃度的環燐酰胺、絲裂黴素C誘導產生的微覈率較溶劑對照組均顯著增加(P均<0.05),劑量-效應關繫明顯。兩種微覈檢測方法的Spearman相關繫數( rs)為1.000。結論:流式細胞術檢測環燐酰胺、絲裂黴素C作用于CHO-K1細胞的微覈試驗結果均為暘性,與文獻報道一緻,因而本試驗初步建立瞭流式細胞術體外微覈檢測方法。流式細胞術檢測微覈的方法與人工閱片的方法相關性好,提示該方法用于化閤物早期遺傳毒性篩選和遺傳毒性評價具有良好的前景。
목적:건립96공판류식세포술체외미핵자동화검측적방법,병탐토기용우약물조기유전독성사선화유전독성평개적가능성。방법:시험분위+S9단시처리조(4 h)화-S9지속처리조(24 h),분별선택3개불동농도적배린선알화사렬매소C처리CHO-K1세포,24 h후수획세포。채용EMA화SYTOX Green쌍색표기,류식세포의분석96공판적미핵솔,병여상규표준평명배양、세포분렬조체법적쌍핵미핵결과진행비교。결과:재유혹무S9처리조건하,불동농도적배린선알、사렬매소C유도산생적미핵솔교용제대조조균현저증가(P균<0.05),제량-효응관계명현。량충미핵검측방법적Spearman상관계수( rs)위1.000。결론:류식세포술검측배린선알、사렬매소C작용우CHO-K1세포적미핵시험결과균위양성,여문헌보도일치,인이본시험초보건립료류식세포술체외미핵검측방법。류식세포술검측미핵적방법여인공열편적방법상관성호,제시해방법용우화합물조기유전독성사선화유전독성평개구유량호적전경。
OBJECTIVE:Establish the flow cytometric 96-well microplate-basedin vitro micronucleus assay in CHO-K1 cells,and explore the possibility of this method for early genetic toxicity screening during drug discovery. MEHTODS:The test included treatment with and without metabolic activation. For the treatment with metabolic activation,CHO-K1 cells were treated with three different concentrations of cyclophosphamide in the S9 mixmedium for 4 h,then incubated with S9-free fresh medium for 20 h. For the treatment without metabolic activation,cells were incubated with three different concentrations of mitomycin C continuously for 24 h. In all cases,after a total of 24 h since initiation of the treatment,cells were processed for microscopic scoring or flow cytometric MN analysis. A flow cytometric method for scoring MN used EMA and SYTOX Green to label the cells in 96-well microplate,and then compared with cytokinesis-block micronucleus assay in cell culture disks based on microscopy.RESULTS:Mitomycin C and cyclophosphamide at different concerntrations caused statistically significant and dose-dependent increasess in micronucleus assay . Non-parametric Spearman's coefficients (rs) is 1.000.CONCLUSION:Similar to literature published,mitomycin C and cyclophosphamide induced positive results in flow cytometric based in vitro micronucleus assay. So the method of flow cytometric 96-well microplate-based in vitro micronucleus assay in CHO-K1 cells was established. The concordance between microscopic scoring and flow cytometricwas good,therefore this method is promising for screening and evaluating genetic toxicity of chemicals.