荷人鼻咽癌裸鼠骨髓细胞DNA合成及凋亡相关基因表达的生物节律的初步研究

Circadian Rhythms of DNA Synthesis a nd Apoptosis Correlated Gene Expression in Bone Marrow Cells of Nude Mice Bearing Human Nasopharyngeal Carcinoma

背景与目的:有关鼻咽癌的生物节律特征尚未见文献报道。本文拟探索荷人鼻咽癌裸鼠骨髓细胞的DNA合成及几种凋亡相关基因表达的生物节律,为临床制定鼻咽癌时辰化疗方案提供必要的参数。方法:BALB/C裸小鼠69只,置于程控的独立光照系统(12h光照,12h黑暗)同步化至少3周。然后双侧腋窝皮下接种人鼻咽低分化鳞癌细胞(CNE-2)裸小鼠移植瘤,成瘤后按6个不同时间点分批处死小鼠,收集骨髓细胞,固定、染色后流式细胞仪测DNA含量,用ANOVA法检验各期细胞在6个时间点差异的显著性,Cosinor法考察G1、S、G2/M期细胞在24h的分布是否符合余弦函数。部分骨髓细胞经裂解、蛋白定量后用WesternBlot法检测不同时间点p53、p21和Bcl-2三种基因蛋白表达的变化情况。结果:G1、S、G2/M期细胞在3、7、11、15、19、23HALO(HoursAfterLightOnset即灯亮后第3、7、11、19、23h)的分布随时间变化有显著性差异,G1、G2/M期细胞24h变化符合余弦节律,高峰值分别位于10.8HALO和1.8HALO。骨髓细胞p53和Bcl-2蛋白表达24h强度不同。p53在11HALO最强,在7HALO最弱;Bcl-2在15HALO最强,在19HALO最弱,p21蛋白未见表达。结论:荷人鼻咽癌裸小鼠骨髓细胞DNA合成随昼夜交替呈节律变化,p53和Bcl-2蛋白表达随昼夜时间变化而规律性波动。

Background &Objective:No report was found on research of cir cadian rhythms of nasopharyngeal carcinoma (NPC).This study was designed to investig ate the circadian rhythms of DNA synt hesis and apoptosis correlated gene expression in bone marrow cells of nude mice bearing NPC and to collect necessary data for mak ing clinical chrono-chemotherapy schedule of NPC.Methods:Sixty-nine BALB /C nude mice were synchronized with an a lternative lighting regimen with 12hours in light and 12hours in d ark(LD 12:12)for at least 3weeks.Human nasopharyngeal poorly differentiated squamous cell carcinoma (CNE-2)cells were implanted into each mouse.Ten days after transplan tation,the mice were sacrificed,bone marrow cells were collected in the3,7,11,15,19,and 23hours after light onset(HALO).Single cell suspension was obtained and stained with propidium iodide.The cellular DNA content was measured by flow cytometry.Data were analyzed w ith analysis of variance(ANOVA)and Cosinor analysis.Proteins were extracted f rom bone marrow cells and determined;Bcl-2,p53,and p21expression were tested using Western blot analysis.Results:The proportion of bone marrow cells i n phase G 1 ,S,and G 2 /M varied according to circadian sampling time with statistical sign ificance(ANOVA).Moreover,such variation in G 1 and G 2 /M was statistically valid by Cosinor analysis with an acrophase l ocated at 10.8HALO and 1.8HALO,resp ectively.The distribution curves o f phase G 1 and G 2 /M fit to Cosinor changes but not for S phase cells.The expression of p53an d Bcl-2protein level in bone marrow cells varied in 24h time scale.No p21protein expression was found in this experiment.Conclusion:DNA synthesis of bone marrow cells in nude mice bearing hum an nasopharyngeal poorly different iated squamous cell carcinoma (CNE-2)cells varies according to circadian rhyth m.The expression of p53and Bcl-2pro tein level in bone marrow cells varie s in 24h time scale.