Flavopiridol对大鼠局灶性脑缺血后神经元细胞周期蛋白依赖蛋白激酶-4蛋白表达的影响

目的　观察大鼠局灶性脑缺血后神经元内细胞周期蛋白依赖蛋白激酶 4 (cdk4 )的蛋白表达与神经细胞凋亡的关系以及cdk4阻滞剂———Flavopiridol对其的影响。方法　采用线栓法大鼠大脑中动脉持续栓塞模型 ,应用免疫组织化学和原位末端标记 (TUNEL)染色方法观察缺血组、Flavopiridol治疗组 [根据剂量多少又分为FH(多剂量 )组和FL(少剂量 )组 ]和假手术组神经元阳性细胞染色数量和分布情况。结果　cdk4蛋白和凋亡细胞自缺血后 12h开始表达 ,前者缺血后 4 8h达高峰 ,后者缺血后 72h达高峰 ;FH组和FL组各相应时间点cdk4蛋白表达均明显减少 (P<0 .0 1) ;FL组各相应时间点凋亡细胞明显减少 (P <0 .0 1) ,FH组仅在缺血 4 8h凋亡细胞明显减少。相邻切片可见cdk4蛋白表达和凋亡细胞染色区域基本相同。结论　cdk4的蛋白表达可能诱发缺血神经细胞的凋亡 ,Flavopiri dol通过抑制cdk4的表达而减少缺血后神经细胞的损害 ,但同时Flavopiridol本身也可能引起神经细胞凋亡。

Effects of Flavopiridol on the expression of cdk4 in neurons after focal cerebral ischemia in rats

Objective To investigate the effects of flavopiridol on expression of cdk4 protein in neurons and neuronal apoptosis after permanent focal cerebral ischemia in rats.Methods A model of middle cerebral artery occlusion (MCAO) in rats was established with intraluminal filament occlusion. The rats of Flavopiridol groups (Flavopiridol concentration: 1 mmol/L. FH group: 200 μl/kg;FL group: 100 μl/kg) were treated with Flavopiridol 30 minutes before and 2, 3 days after the operation. The immunohistochemistry and TUNEL staining were used respectively to observe the distribution and quantities of cdk4 protein and neuronal apoptosis in the brain tissues following the sham operation and permanent MCAO for 3 h, 6 h, 12 h, 24 h, 48 h, 72 h and 7 d.Results A significant increase in cdk4 immunostaining and TUNEL positive staining began to be detected at 12 h after MCAO in the neurons of the ischemia penumbra. The immunoreactivity of cdk4 was most marked at 48 h,while the TUNEL staining was at 72 h. The difference of immunoreactivity of cdk4 between ischemic group and FH or FL group was significant (P<0.01). The difference of TUNEL staining between ischemic group and FL group was also significant(P<0.01).TUNEL staining in FH group decreased remarkably only at 48 h after MCAO (P<0.01). Conclusions The induction of cdk4 protein at the ischemia penumbra indicated that it might be involved in the mechanism of neuronal apoptosis. Flavopiridol reduced the neuronal apoptosis through down-regulating cdk4 expression,but Flavopiridol itself might induce neuronal apoptosis.