神经营养素3修饰间充质干细胞对颅脑损伤大鼠神经保护作用

Neuroprotective effect of neuronutrient 3-modified mesenchymal stem cells on traumatic brain injury in rats

目的探讨神经营养素3(NT-3)修饰间充质干细胞对颅脑损伤大鼠的神经保护作用及其机制。方法随机选取1只SD大鼠分离骨髓间充质干细胞(MSCs),并构建NT-3过表达间充质干细胞。采用蛋白质印迹法(Western blot)检测pLent-GFP-NC和pLent-GFP-NT-3细胞中NT-3蛋白的表达水平。60只SD大鼠成功制备颅脑损伤模型后,随机分为颅脑损伤组、间充质干细胞组、NT-3修饰间充质干细胞组。采用改良神经功能缺损评分法(mNSS)评价各组大鼠神经功能恢复情况。采用原位末端标记法(TUNEL)细胞凋亡检测试剂盒测定大鼠脑组织中细胞凋亡。采用天冬氨酸特异性半胱氨酸蛋白酶(Caspase)-3、Caspase-8、Caspase-9活性测定试剂盒(比色法)分析大鼠脑组织中Caspase-3、Caspase-8、Caspase-9的活性,组间比较采用t检验。结果pLent-GFP-NT-3细胞(1.323±0.020)NT-3蛋白的表达水平明显高于pLent-GFP-NC细胞(0.357±0.018),差异有统计学意义(t=62.670,P<0.05)。NT-3修饰间充质干细胞组大鼠(7.700±0.733)神经功能评分明显低于颅脑损伤组(10.250±0.716)和间充质干细胞组(8.850±0.587),差异有统计学意义(t=11.130、5.478,P<0.05)。NT-3修饰间充质干细胞组大鼠[(16.808±1.280)%]脑组织细胞凋亡指数明显低于颅脑损伤组[(33.524±2.337)%]和间充质干细胞组[(24.930±2.230)%],差异有统计学意义(t=28.050、14.130,P<0.05)。NT-3修饰间充质干细胞组大鼠[(53.563±4.380)U/mg prot]脑组织中Caspase-3的活性明显低于颅脑损伤组[(148.943±10.937)U/mg prot]和间充质干细胞组[(90.860±5.312)U/mg prot],差异有统计学意义(t=36.210、24.230,P<0.05)。NT-3修饰间充质干细胞组[(43.800±4.835)U/mg prot]Caspase-8的活性明显低于颅脑损伤组[(124.530±8.603)U/mg prot]和间充质干细胞组[(79.262±5.749)U/mg prot],差异有统计学意义(t=36.570、21.110,P<0.05)。NT-3修饰间充质干细胞组[(34.575±3.241)U/mg prot]Caspase-9的活性明显低于颅脑损伤组[(88.640±4.178)U/mg prot]和间充质干细胞组[(65.624±5.119)U/mg prot],差异有统计学意义(t=45.730、22.920,P<0.05)。结论NT-3修饰间充质干细胞可显著减少颅脑损伤大鼠受损组织的细胞凋亡,起到改善并修复大鼠神经功能的作用。

Objective To investigate the neuroprotective effect of neurotrophin-3(NT-3)-modified mesenchymal stem cells(MSCs)on traumatic brain injury in rats and the mechanism.Methods Bone marrow MSCs were isolated from a randomly selected SD rat,and the neurotrophin-3(NT-3)-overexpressed MSCs were constructed.Western blotting was used to detect the expression of NT-3 protein in pLent-GFP-NC and pLent-GFP-NT-3 cells.A total of 60 SD rats were randomly divided into traumatic brain injury group,MSCs group and NT-3-modified MSCs group.Neurological function recovery in each group was evaluated by modified neurological severity scores(mNSS).Cell apoptosis in rat brain tissue was determined by TdT-mediated dUTP nick-end labeling(TUNEL)apoptosis detection kit.The activity of cysteine aspartate-specific protease(Caspase)-3,Caspase-8 and Caspase-9 in the rat brain was analyzed with the Caspase-3,Caspase-8 and Caspase-9 activity assay kit(colorimetric method).Results The expression of NT-3 protein in pLent-GFP-NT-3 cells(1.323±0.020)was significantly higher than that in pLent-GFP-NC cells(0.357±0.018,t=62.670,P<0.05).The neural function scores of NT-3-modified MSCs group(7.700±0.733)were significantly lower than those of traumatic brain injury group(10.250±0.716)and MSCs group(8.850±0.587,t=11.130,5.478,P<0.05).The apoptosis index of MSCs[(16.808±1.280)%]in NT-3 modified MSCs group was significantly lower than that in traumatic brain injury group[(33.524±2.337)%]and MSCs group[(24.930±2.230)%,t=28.050,14.130,P<0.05].The activity of Caspase-3 in brain tissue of NT-3-modified MSCs group[(53.563±4.380)U/mg prot]was significantly lower than that in traumatic brain injury group[(148.943±10.937)U/mg prot]and MSCs group[(90.860±5.312)U/mg prot,t=36.210,24.230,P<0.05].The Caspase-8 activity of NT-3-modified MSCS group[(43.800±4.835)U/mg prot]was significantly lower than that of traumatic brain injury group[(124.530±8.603)U/mg prot]and MSCs group[(79.262±5.749)U/mg prot,t=36.570,21.110,P<0.05].The Caspase-9 activity of NT-3-modified MSCs group[(34.575±3.241)U/mg prot]was significantly lower than that of traumatic brain injury group[(88.640±4.178)U/mg prot]and MSCs[(65.624±5.119)U/mg prot,t=45.730,22.920,P<0.05].Conclusion NT-3-modified MSCs can significantly reduce the apoptosis of damaged tissues in rats with craniocerebral injury,thereby improving the neural function injury in rats.