下调PTEN基因对全脑缺血再灌注大鼠神经元损伤的保护作用研究

The protective effect of PTEN down-regulating on neuron injury of rats after global cerebral ischemia and reperfusion

目的探讨下调第10号染色体同源丢失性磷酸酶张力蛋白基因(PTEN)对全脑缺血再灌注大鼠神经元损伤的保护作用。方法 60只SD大鼠随机分为正常对照组(转染脂质体)、pshGFP组(转染pshGFP质粒)、pshRNApten治疗组(转染pshRNApten质粒),每组20只,各组分别于再灌注后3d(6只)、7d(7只)、14d(7只)三个时间点进行观察。正常对照组与pshGFP组分别注射脂质体与pshGFP质粒,pshRNApten治疗组于海马局部注射pshRNApten质粒。注射2d后,采用四动脉结扎法建立大鼠全脑缺血再灌注模型。利用RT-PCR和免疫组化法检测海马神经元PTEN基因的表达;采用HE和Nissl染色检测神经元损伤情况;采用TUNEL法检测神经元凋亡情况。结果 pshRNApten治疗组海马神经元PTEN基因的mRNA和蛋白表达量均较正常对照组明显下降(P<0.05)。pshRNApten治疗组海马神经元损伤和变性程度均明显轻于pshGFP组大鼠,海马TUNEL染色阳性细胞数也明显少于psh-GFP组大鼠(P<0.05)。结论下调PTEN能有效缓解由缺血再灌注所致的神经元损伤。

Objective To investigate the protective effect of down-regulation of phosphatase and tensin homology （PTEN） deleted on chromosome ten on neuron injury of rats after global cerebral ischemia and reperfusion.Methods Sixty SD rats were assigned randomly into three groups （20 each）：normal group （liposome trasnfected）,pshGFP group （isolated control） and pshRNApten group （pshRNApten transfected）.Rats in each group was inflicted with cerebral ischemia and reperfusion of the brains,and they were treated at three time points,i.e.3d （n=6）,7d （n=7） and 14d （n=6） after reperfusion.Plasmid pshRNApten was injected into hippocampus of rats in pshRNApten group,liposome and plasmid pshGFP were injected in normal group and pshGFP group,respectively.Two days after injection,global cerebral ischemia and reperfusion model was reproduced by four-artery ligation.PTEN expression in neuron of hippocampus was detected by RT-PCR and immunohistochemistry.The neuron injury was determined by HE and Nissl＇s staining,and the neuron apoptosis was detected by terminal deoxynucleotidy transferase UTP-nick end labeling （TUNEL）.Results The mRNA and protein expression of PTEN decreased obviously in pshRNApten group than in normal group （P〈0.05）.Nissl＇s and HE staining showed that the neuronal damage and degeneration in hippocampus after global ischemia and reperfusion （I/R） were more severe in PshGFP group than in pshRNApten group.There was a significant decrease in the number of TUNEL positive cells in pshRNApten group compared to that in PshGFP group （P〈0.05）.Conclusion PTEN down-regulation can effectively relieve the neuron injury induced by cerebral ischemia and reperfusion.