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目的观察选择性细胞周期抑制剂 olomoucine 对胶质细胞增殖和瘢痕形成及神经元凋亡的影响。方法建立光化学法诱导大鼠局灶性脑缺血模型,随机分为假手术组、对照组和干预组,采用 MRI 显示梗死灶并计算其梗死灶体积;应用免疫荧光化学法检测胶质纤维酸性蛋白(GFAP)的表达及通过 TUNEL 方法检测神经元凋亡;免疫印迹法观察光照侧皮质 GFAP、增殖细胞核抗原(proliferation cell nuclear antigen,PCNA)、周期索蛋白 A 和周期素蛋白 B1蛋白的表达。结果缺血后3、7、30 d 对照组(5.10%±0.35%,4.60%±0.26%,3.96%±0.28%)梗死灶体积占全脑体积百分比值的平均值明显大于干预组(2.27%±0.28%,1.87%±0.19%,1.08%±0.18%,P<0.05);缺血后各时间点组 GFAP 表达明显增强,对照组明显强于干预组,并且7、30 d 对照组梗死灶周边可见明显的胶质瘢痕形成,以30 d 最为显著;缺血后3 d 梗死灶周围可见大量 TUNEL 阳性染色细胞,对照组[(41±11)个/高倍视野]数量明显多于干预组[(26±8)爪/高倍视野,P<0.05];干预组大鼠(22.44%±2.17%)NeuN+TUNEL 双标阳性表达明显弱于对照组大鼠(34.41%±3.10%,P<0.05);3、7、30 d 的 GFAP、PCNA、周期素蛋白 A 和周期素蛋白 B1蛋白量表达,对照组明显高于干预组。结论通过对细胞周期的调控,可部分抑制胶质细胞的活化增殖及瘢痕形成,同时减小脑梗死体积及减少神经元凋亡。
Objective To observe the effects of olomoucine, a selective cell cycle inhibitor, on glial cell proliferation, scar formation and neuronal apoptosis. Methods A rat model of focal cerebral ischemia induced by photochemical method was established and randomly divided into sham operation group, control group and intervention group. The infarct size was calculated by MRI and the volume of infarction area was calculated. Immunofluorescence staining was used to detect the glial fibrillary acidic (GFAP) and neuronal apoptosis by TUNEL method. The expression of GFAP, proliferating cell nuclear antigen (PCNA), cyclin A and cyclin B1 were detected by Western blotting. Results The percentage of infarct volume to total brain volume in the control group (5.10% ± 0.35%, 4.60% ± 0.26%, 3.96% ± 0.28%) at 3, 7 and 30 days after ischemia was significantly higher than that in the intervention group (2.27% ± 0.28%, 1.87% ± 0.19%, 1.08% ± 0.18%, P <0.05). The expression of GFAP at each time point after ischemia increased significantly, while the control group was significantly stronger than the intervention group Significant glial scar formation was seen in the periphery, most notably in 30 days. A large number of TUNEL-positive stained cells were observed around the infarct 3 days after ischemia, while the number of [(41 ± 11) / high-power fields in the control group was significantly more than that in the intervention group [ (26 ± 8) paw / high power field, P <0.05]. NeuN + TUNEL double positive expression in the intervention group was significantly weaker than that in the control group (34.41% ± 3.10%, P <0.05 ). The protein expressions of GFAP, PCNA, cyclin A and cyclin B1 were significantly increased at 3, 7 and 30 days in control group, which were significantly higher than those in intervention group. Conclusion The regulation of the cell cycle can partially inhibit the activation and proliferation of glial cells and scar formation, reduce the volume of cerebral infarction and reduce the apoptosis of neurons.