过量胸苷对细胞周期素表达的影响

Effects of excessive thymidine on the expression of cyclin

目的 :探讨过量胸苷干扰细胞周期素细胞周期时相表达的特点 ,为进一步研究细胞周期转化以及细胞周期素与DNA合成的关系奠定基础 .方法 :应用二次过量胸苷阻断法进行新生猪肾小管上皮细胞体外同步化培养 ,采用流式细胞术进行鉴定 ;然后分别进行G1/S,S及G2 期细胞CyclinA ,B ,D1mAb免疫细胞化学染色并进行图像分析 .结果 :二次过量胸苷阻断法可获取G1/S界面细胞为 (99 5± 0 8) % ,S期细胞为(90 5± 0 7) % ,G2 期细胞为 (93 5± 2 2 ) % ,G2 /G1=2 2 0 .G1/S ,S,G2 细胞CyclinA表达特点为G2 期大于G1/S和S期 (目标灰度值 :13 6 3± 2 7,14 5 0± 1 4 ,14 7 5± 1 8,P <0 0 1) ,CyclinB表达特点为G1/S期小于S和G2 期 (目标灰度值 :15 5 6± 1 6,14 8 8± 2 2 ,13 6 6± 2 6,P <0 0 1) ,CyclinD1的表达特点为G2期大于G1/S期 (目标灰度值 :13 2 .6± 1.9,14 6.7± 1.3 ,P <0 .0 1) ,S期小于G1/S期 (目标灰度值 :15 8 8± 1 1,14 6 7± 1 3 ,P<0 0 5 ) .结论

AIM: To explore the effects of excessive thymidine on the expression of cyclin A, B, D1 in each phases of cell cycle by studying the relationship between cyclin and DNA synthesis and the mechanism of cell cycle transition. METHODS: Neonatal pigs renal tubular epithelial cells were synchronized by the excessive thymidine block technique and the distribution of G 1, S, G 2 phases were determined by flow cytometry. The expression of cyclin A, B, D 1 were detected by immunocytochemistry. RESULTS: Flow cytometry analysis revealed that (99.5±0.8)% of the neonatal renal tubular epithelial cells were at G 1/S transition, (90.5± 0.7) % in S phase and (93.5±2.2)% in G 2 stage respectively by the method of the excessive thymidine block technique. The expression of cyclin A at G 2 phases was higher than that of G 1/S and S phases (average of gray level: 136.28 ± 2.65 , 145.04±1.35, 147.52±1.84,P<0.01). The expression of cyclin B was G 1<S<G 2 phases(average of gray level:155.55±1.62, 148.76±2.18, 136.60±2.64,P<0.01)and that of cyclin D 1 was G 2>G 1/S>S (average of grey level: 132.60±1.92, 146.69±1.32,158.84±1.07, P<0.05). CONCLUSION: The block of DNA synthesis with double excessive thymidine may be associated with the change of cyclin expression.