新鲜肿瘤标本活细胞与固定细胞的DNA含量分析比较

DNA Content Comparison Analysis between Living Cells and Fixed Cells in Fresh Tumor Samples

目的:建立一种以新鲜细胞进行临床肿瘤DNA含量分析的方法。方法:血液肿瘤取肝素抗凝外固血或骨髓样本,用淋巴细胞分层液分离纯化单个核细胞;实体瘤组织则用机械法制成单细胞悬液。将细胞分成两份,一份酒精固定过夜,流式细胞术常规方法测定DNA含量,另一份直接用PIPESpiperazineNNbis2ethanesulfonicaciddisodiumsalt缓冲液配制的碘化丙锭(propidiumiodidePI)染色,以流式细胞术进行DNA含量测定,并与同期固定细胞的DNA含量检测结果进行比较。结果:同类型的肿瘤细胞,其固定前后G0/G1期细胞峰的变异系数(coefficientofvariationCV)没有明显差异(t检验,P>0.05)。重复实验结果显示,新鲜细胞DNA含量分析结果稳定。结论:用PIPES缓冲液配制的PI染液染色新鲜细胞,可以对临床肿瘤的细胞增殖状况和DNA倍性进行分析。

Objective:This study was designed to establish a method of analyzing DNA content in f resh tumor using fresh cells.Methods :Clinical blood tumor samples were se parated to obtain mononuclear cells by Ficoll-Hypaque,solid tumor tissues were made to be single c ells by using mechanical method.The purified mononuclear cells and single cells suspensions were divided into two portions.One portion was fixed by ethanol and placed overnight,then measu red DNA content by conventional flow cytome tric method.The other portion was stained directly by PI (propidium iodide)dissolved in PIPES(piperazine-Ν,Ν-bis[2-ethanesulfonic acid]disodium salt)buffer,then measured DNA content by flow cytometry,the DNA content re sults were compared with those of fix ed cells.Results:There was no obvious difference in the CV(coefficient of variation)of the peak of G 0 /G 1 cells between fresh and fixed tumor c ells from the same type tumor(t-test,P>0.05).Repetition test showed that the results of DNA content analysis using fr esh cells were stable.Conclusion:Fresh tumor cells stained by PI disso lved in PIPES buffer could be used for the analysis of clinical tumor propagation and DNA ploidy.