蛋白激酶信号级联Akt在大鼠脊髓损伤后的诱导表达

Induced expression of protein kinase signaling cascade Akt after spinal cord injury in rats

目的研究蛋白激酶信号级联蛋白激酶B(Akt)在大鼠脊髓损伤(SCI)后的表达变化,以及对SCI后运动功能恢复的影响,从而为SCI的修复提供分子学机制依据。方法采用改良Allen′s打击器制成大鼠SCI模型。大鼠分为3组:假手术组(Sham组)、干预组(Int组)、对照组(Con组)。分别于术后1、7、14d采用免疫组织化学、蛋白免疫印迹法(Western blot)和实时荧光定量-聚合酶链反应(RT-PCR)检测Akt mRNA和蛋白的表达,并采用Basso-Beattie-Bresnahan(BBB)运动功能评分评价大鼠SCI后运动恢复情况。结果 Sham组在Akt蛋白和mRNA水平上呈低水平表达,SCI后Akt的表达水平明显提高;Akt上游磷脂酰肌醇3-激酶(PI3K)的抑制剂LY294002可明显抑制Akt的表达,并阻止SCI后自发性运动功能恢复。结论 SCI后激活的蛋白激酶信号Akt可能是参与SCI后神经保护和修复的重要干预环节。

Objective To examine the expression of protein kinase signaling cascade protein kinase B （Akt）-which plays an im-portant role in a number of key biological functions in cellular processes after spinal cord injury （SCI） in rats ,and its effects on SCI induced motor function defects ,and to provide the molecular mechanism for repairing SCI .Methods SCI was produced by extra-dural contusion using modified Allen′s stall with damage energy .The rats were divided into three groups ：sham-operated group （Sham） ,interference group （Int） ,and control group （Con） .Using immunostaining studies ,Western blot analyses and real-time qualitative RT-PCR analyses ,we detected the changes of Akt expression at the protein and mRNA level in spinal cord tissues at 1 , 7 ,and 14 d after surgery ,and evaluated the presence and extent of neurological impairment after SCI by the BBB locomotor rating scale .Results The sham operated groups exhibited low expression of the Akt signaling at the protein and mRNA level ,and the ex-pressions increased following SCI .Compared to the animals in the sham operated groups ,prominently elevated level of Akt signaling was detected in the injured spinal cords of SCI group .LY294002 ,an inhibitor of phosphatidylinositol 3-kinase （PI3K） that initiates Akt phosphorylation ,prominently inhibited the expression of Akt produced by SCI and the spontaneous recovery of motor function after SCI .Conclusion Activated protein kinase signaling cascade Akt might be the important intervention aspects of involving in neuroprotective and repair process after SCI .