BUdR标记BUdR/DNA双参数流式细胞仪测定肿瘤潜在倍增时间的方法学研究

The study of methods for BUdR incorporation and BUdR/DNA bivariated flow cytometry(FCM) in determining potential doubling time(Tpot)

目的探讨溴脱氧尿嘧啶核苷（ＢＵｄＲ）标记ＢＵｄＲ／ＤＮＡ双参数流式细胞仪（ＦＣＭ）测定肿瘤潜在倍增时间Ｔｐｏｔ的方法，比较ＢＵｄＲ在体（ｉｎｖｉｖｏ）、离体（ｉｎｖｉｔｒｏ）标记结果的差异。材料与方法实验采用人鼻咽癌裸小鼠移植瘤模型分别进行ＢＵｄＲ在体、离体标记；离体标记时分别将样本制成组织块和细胞悬液在０．５ｍＭ、０．１ｍＭＢＵｄＲ的含２０％胎牛血清ＲＰＭＩ１６４０培养基、５％ＣＯ２、３７℃、ＣＯ２孵箱中开放培养不同时间后收集样本。在体标记：腹腔注射ＢＵｄＲ０．１ｍｇ／ｇ体重，６小时后处死小鼠取瘤剪碎，在７０％的冷乙醇固定冰箱保存；全部样本用ＦＩＴＣＰＩ染色后行ＦＣＭ测定。结果（１）本实验室的ＢＵｄＲ离体培养条件下，组织块标记较理想。（２）用ＢＵｄＲ在体标记法测定不同大小的肿瘤（直径０．３～１．２ｃｍ），结果表明：直径＜０．５ｃｍ的肿瘤的标记效果比较理想。（３）相同体积的肿瘤在体（ｎ＝９）、离体（ｎ＝７）标记两种不同标记方法的结果显示，相同体积的肿瘤用ＢＵｄＲ离体标记法时ＬＩ比在体标记低５３％，其它各项细胞动力学参数两者均有明显差异，但离体标记法的组内变异很小。结论本实验室成功建立了在体ＢＵｄＲ标记ＢＵ?

Purpose To investigate the method for BUdR incorporation and BUdR/DNA bivariated FCM in determing Tpot,and to compare BUdR incorporation in vitro and in vivo method for Tpot measurement. Materials and Methods Carcinoma of nasopharyngeal xenografts in nude mice were used for Tpot measurement in vivo and in vitro BUdR incorporation method.In vitro experiment ,the tissue fragments (in 1mm3 blocks with 0.1mM BUdR) and the cell suspension(0.05,0.1mM BUdR) were incubated in RPMI 1640 medium containing 20% fetal calf serum at 37℃ and 5% CO2 in CO2 incubator for different durations;in vivo experiment ,the nude mice were injected i.p. with 0.1mg/g BUdR and sacrificed six hours later.The samples were fixed in 70% ethanol and stored in dark at -20℃.All samples were prepared as routine for FITCPI staining and measured by Coulter EPICS751 FCM.Results (1)In vitro experiment,better results were obtained from the tissue fragments labelling than cell suspension with BUdR.(2)The results were better when the tumour diameter was about 0.5cm with in vivo BUdR incorporation for six hours.(3) Through comparing in vitro(n=7) BUdR incorporation method for Tpot measurement,the labelling index (LI) in vitro incorpation method was 53% lower than in vivo method and the differences also existed in other cell kinetic parameters while interdeviation of in vitro method showed quite small deviation.Conclusion The method for measuring of Tpot is successfully set up.Appropriate culture condition in vitro labeling method is obtained.There is obvious difference between the results of in vivo labeling and in vitro labeling.