穿透肽介导的铜-锌超氧化物歧化酶1融合蛋白的抗氧化作用及对脂肪干细胞移植修复脊髓损伤的促进作用

PEP - 1 - SOD1 improve the therapeutic potential of transplanted adipose - derived mesenchymal stem cells in spinal cord injury by its antioxidant effects

目的探讨穿透肽介导的铜-锌超氧化物歧化酶1融合蛋白（PEP-1-SOD1）在脊髓损伤早期的抗氧化作用及对脂肪干细胞（ADSCs）移植修复脊髓损伤的促进作用。方法体外分离培养SD大鼠ADSCs，并以携带绿色荧光蛋白（GFP）基因的腺病毒感染ADSCs。取成年SD大鼠96只，以改良Allen法制作脊髓损伤模型，随机分为3组：盐水治疗组、ADSCs治疗组、PEP-1-SOD1联合ADSCs治疗组（联合治疗组），术后1、3、7 d各取8只大鼠分别行超氧化物歧化酶（SOD）活性检测及丙二醛（MDA）含量检测，术后7 d各取4只大鼠行细胞凋亡检测；术后1、2、3、4周行Basso-Beattie-Bresnahan（BBB）评分，术后4周BBB评分后处死大鼠行神经元特异性核蛋白（NeuN）表达阳性细胞检测，对ADSCs治疗组及联合治疗组行脊髓损伤区域绿色荧光蛋白（GFP）阳性细胞检测。 结果术后7 d ADSCs治疗组SOD活性大于盐水治疗组（t＝3.882，P＝0.030），MDA含量小于盐水治疗组（t＝10.392 ，P＝0.002），联合治疗组SOD活性大于ADSCs治疗组（t＝4.690，P＝0.018），MDA含量小于ADSCs治疗组（t＝3.464，P＝0.031）。术后7 d联合治疗组细胞凋亡少于ADSCs治疗组（t＝13.416，P＝0.000），ADSCs治疗组细胞凋亡多于盐水治疗组（t＝12.000，P＝0.000）。术后4周，联合治疗组BBB评分为（18.59±1.54）分大于ADSCs治疗组[（11.90±1.72）分，t＝8.430，P＝0.000]；联合治疗组NeuN阳性细胞百分比为（61.65±12.65）%大于ADSCs治疗组的（31.45±5.76）%（t＝154.214，P＝0.000）；联合治疗组GFP阳性细胞数大于ADSCs治疗组（t＝64.325，P＝0.000）。 结论与单纯ADSCs移植比较，联合应用PEP-1-SOD1进一步增加损伤局部SOD活性，降低MDA含量，改善运动功能，PEP-1-SOD1发挥了协同效果，这可能与减少移植细胞凋亡有关。

ObjectiveTo investigate the antioxidant effects of PEP-1-SOD1 and its positive role in spinal cord injury （SCI） with adipose-derived mesenchymal stem cells. MethodsAdipose-derived stem cells （ADSCs） of SD rat were isolated, cultured in vitro, and cells of passage 3 were transfected with adenovirus vector [Adv-green fluorescent protein （GFP）]. 96 SD rats were randomly divided into three groups： saline group, ADSCs group, PEP-1-SOD1-ADSCs group. 1, 3, 7 days post surgery, 8 rats of each group in were killed to observe superoxide dismutase （SOD） activities and malondialdehyde （MDA） levels. 7 days post surgery 4 rats of each group were killed to observe cell apoptosis. 1, 2, 3, 4 weeks post surgery, the neurological functional changes were evaluated by Basso-Beattie-Bresnahan （BBB） scale; neuron-specific nuclear protein （NeuN） positive cells and the GFP positive cells in injured area were also be detected and analyzed 4 weeks post surgery. Results7 days post surgery, the SOD activities of ADSCs group was stronger than saline group （t=3.882, P=0.030） , the MDA levels of ADSCs group was less than saline group （t=10.392, P=0.002）, the SOD activities of PEP-1-SOD1-ADSCs group was stronger than ADSCs group （t=4.690, P=0.018）; the MDA levels of PEP-1-SOD1-ADSCs group was less than ADSCs group（t=3.464, P=0.031）. 7 days post surgery, there was less apoptotic cells in PEP-1-SOD1-ADSCs group than ADSCs group （t=13.416, P=0.000）, apoptotic cell numbers in ADSCs group was more than saline group（t=12.000, P=0.000）. 4 weeks after surgery the BBB score of PEP-1-SOD1-ADSCs group was 18.59±1.54 which was more than that of ADSCs group（11.90±1.72; t=8.430, P=0.000）, the percentage of NeuN positive cells in PEP-1-SOD1-ADSCs group was （61.65±12.65）% which was more than that in ADSCs group [（31.45±5.76）%; t=154.214, P=0.000], the GFP positive cells of PEP-1-SOD1-ADSCs group was higher than that of ADSCs group （t=64.325, t=0.000）. ConclusionCompared with using ADSCs alone the combination of PEP-1-SOD1 significantly increased the SOD activities, decreased the MDA level and improved the motor behavior after SCI. Combination using PEP-1-SOD1 play a synergistic effect which may be related to its antioxidant effects to reduce cell apoptosis.