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目的:探讨bFGF是否通过PI3K/PKB途径调节p21~(WAFl)的表达。方法:~(32)P掺入法检测 PKB酶活性,RT-PCR、Western blot检测不同处理组Bel-7402细胞的p21~(WAFl)表达,流式细胞术分析细胞周期。结果:25 μg/L bFGF刺激细胞10min,就可使胞液和膜性PKB酶活性达高峰.p21~(WAFl)mRNA表达水平在1h达高峰,比对照升高了5.5倍.p21~(WAFl)蛋白表达在2 h达高峰,比对照升高了2.2倍.wortmannin预处理后,PKB活性在各时间点均明显降低(P<0.05),p21~(WAFl)mRNA表达及p21~(WAFl)蛋白表达无明显变化.流式细胞术分析显示,bFGF处理组与对照组相比 GI期细胞减少(0.65 ± 0.01→0.49 ± 0.02,P<0.01),S期细胞增多(0.14 ± 0.01→0.28 ± 0.01,P<0.01),wortmannin能抑制此促增生作用(GI:0.58±0.01;S:0.22 ±0.01,P<0.01)。结论:PI3K/PKB途径可介导bFGF对Bel-7402细胞的促增生作用,但是bFGF对p21~(WAFl)表达的调节作用不依赖PI3K/PKB途径。
Objective: To investigate whether bFGF regulates the expression of p21 WAF1 through the PI3K / PKB pathway. Methods: PKB enzyme activity was detected by ~ (32) P incorporation method. The expression of p21 WAF1 in different treatment groups was detected by RT-PCR and Western blot. The cell cycle was analyzed by flow cytometry. Results: After stimulated with 25 μg / L bFGF for 10 min, the activity of cytosolic and membranous PKB reached a peak, and the expression level of p21 WAF1 reached its peak at 1 h, which was 5.5 times higher than that of the control.p21 WAF1 ) Peaked at 2 h, which was 2.2 times higher than that of the control.Protein kinase activity was significantly decreased at wortmannin pretreatment at all time points (P <0.05) Flow cytometry analysis showed that compared with the control group, the number of cells in the bFGF group decreased (0.65 ± 0.01 → 0.49 ± 0.02, P <0.01) and the number of S phase cells increased (0.14 ± 0.01 → 0.28 ± 0.01, P <0.01). Wortmannin could inhibit the proliferation (GI: 0.58 ± 0.01; S: 0.22 ± 0.01, P <0.01). CONCLUSION: The PI3K / PKB pathway mediates the pro-proliferation effect of bFGF on Bel-7402 cells. However, the regulatory effect of bFGF on p21 WAF1 expression does not depend on the PI3K / PKB pathway.