MiRNA-125a-5p对大鼠脊髓损伤后血脊髓屏障及运动功能的影响
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摘要
目的:通过干预大鼠脊髓中miRNA-125a-5p的表达量,探讨miRNA-125a-5p在脊髓损伤(SCI)后对血脊髓屏障(BSCB)的作用及对大鼠运动功能的影响。方法:SD大鼠随机分为假手术组(Sham组)、脊髓损伤组(SCI组)、miRNA空载组(NC组)和miRNA-125a-5pagomir鞘内注射组(miRNA组)。RT-PCR检测miRNA-125a-5p在各种大鼠脊髓中的表达,免疫印迹和免疫荧光实验检测脊髓微血管内皮细胞间紧密连接蛋白(ZO-1)表达,尼氏染色观察脊髓前角运动神经元的数量以及通过BBB评分评价大鼠运动功能恢复。结果:MiRNA组的miRNA-125a-5p的相对表达量明显高于SCI组,且ZO-1的mRNA的相对表达量和相对蛋白质表达量较SCI组和显著增加。MiRNA组的脊髓前角运动神经元相对数量明显多于SCI组。MiRNA组大鼠脊髓损伤后1 d、3 d、7 d、14 d、21 d和28 d运动功能恢复明显优于SCI组。结论:MiRNA-125a-5p可以有效保护脊髓损伤大鼠的血脊髓屏障功能并促进运动功能恢复。
Objective : To explore the effects of miRNA-125a-5p on the blood spinal cord barrier(BSCB) and the motor function by regulating the expression of miRNA-125a-5p after spinal cord injury(SCI) in rats. Methods : SD rats were randomly divided into sham group,SCI group,NC group and miRNA group(microRNA-125a-5p agomir administration by intrathecal injection). RT-PCR was used to detect the expression of microRNA-125a-5p in the spinal cord of rats. Western blotting and immunofluorescent staining were used to examine the expression of tight junction protein(ZO-1) in spinal microvascular endothelial cells. Nissl staining was used to determine the number of surviving motor neurons in the anterior horn of spinal cord after SCI. Basso-Beattie-Bresnahan(BBB) scores was used to evaluate the motor functional recovery at day 1,3,7,14,21 and 28 after SCI in rats. Results : Compared with SCI group(0.57±0.04),the relative expression of microRNA-125a-5p in miRNA group was significantly increased. The relative expression of ZO-1 mRNA and ZO-1 protein expression were significantly higher in miRNA group compared with SCI group and. Additionally,the relative number of surviving motor neurons in anterior horn of spinal cord in miRNA group was significantly higher than that in SCI group. And the functional recovery in miRNA group was significant improved at day 1,3,7,14,21 and 28 compared with SCI group. Conclusion : MiRNA-125a-5p can effectively protect the function of BSCB and promote the recovery of motor function after SCI in rats.
Objective : To explore the effects of miRNA-125a-5p on the blood spinal cord barrier(BSCB) and the motor function by regulating the expression of miRNA-125a-5p after spinal cord injury(SCI) in rats. Methods : SD rats were randomly divided into sham group,SCI group,NC group and miRNA group(microRNA-125a-5p agomir administration by intrathecal injection). RT-PCR was used to detect the expression of microRNA-125a-5p in the spinal cord of rats. Western blotting and immunofluorescent staining were used to examine the expression of tight junction protein(ZO-1) in spinal microvascular endothelial cells. Nissl staining was used to determine the number of surviving motor neurons in the anterior horn of spinal cord after SCI. Basso-Beattie-Bresnahan(BBB) scores was used to evaluate the motor functional recovery at day 1,3,7,14,21 and 28 after SCI in rats. Results : Compared with SCI group(0.57±0.04),the relative expression of microRNA-125a-5p in miRNA group was significantly increased. The relative expression of ZO-1 mRNA and ZO-1 protein expression were significantly higher in miRNA group compared with SCI group and. Additionally,the relative number of surviving motor neurons in anterior horn of spinal cord in miRNA group was significantly higher than that in SCI group. And the functional recovery in miRNA group was significant improved at day 1,3,7,14,21 and 28 compared with SCI group. Conclusion : MiRNA-125a-5p can effectively protect the function of BSCB and promote the recovery of motor function after SCI in rats.
引文
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[2]Yuan Q,Su H,Chiu K,et al.Contrasting neuropathology and functional recovery after spinal cord injury in developing and adult rats.[J].Neurosci Bull,2013,29(4):509-516.
[3]刘明明,程建,马勇,等.脊髓损伤后血脊髓屏障病变机制研究进展[J].安徽医科大学学报,2015,50(12):1827-1830.
[4]李禾,邢更彦,杨传铎,等.大鼠脊髓损伤后血脊髓屏障通透性变化的观察[J].中国脊柱脊髓杂志,2001,11(4):216-218.
[5]Abbott N J,Patabendige A A,Dolman D E, et al.Structure and function of the blood-brain barrier[J].Neurobiol Dis,2010,37(1):13-25.
[6]Yi F,Fuhua C.MicroRNA-125a-5p regulates cancer cell proliferation and migration through NAIF1 in prostate carcinoma[J].OncoTargets Therapy,2015,17(8):3827-3835.
[7]Nakkeeran S,Kavitha K,Chandrasekar G,et al.Induction ofplant defence compounds by Pseudomonas chlororaphis PA23 and Bacillus subtilis BSCBE4 in controlling damping-off of hot pepper caused by Pythium aphanidermatum.[J].Biocontrol Sci Technol,2006,16(4):403-416.
[8]Carthew R W,Sontheimer E J.Origins and mechanisms of miRNAs and siRNAs[J].Cell,2009,136(4):642-655.
[9]Bartanusz V,Jezova D,Alajajian B,et al.The blood-spinal cord barrier:morphology and clinical implications[J].Ann Neurol,2011,70(2):194-206.
[10]张永涛,高渊涛,李宵,等.miR-126促进脊髓损伤模型神经干细胞的生长增殖和分化[J].解剖学杂志,2018,41(3):297-302.
[11]Bartanusz V,Jezova D,Alajajian B,et al.The blood-spinal cord barrier:morphology and clinical implications[J].Ann Neurol,2011,70(2):194-206.
[12]Kuntner C,Bankstahl J P,Bankstahl M,et al.Dose-response assessment of tariq uidar and elacridar and regional quantification of P-glycoprotein inhibition at the rat blood-brain barrier using(R)-[11C]verapamil PET[J].Eur J Nuclear Med Mol Imag,2010,37(5):942-953.
[13]Zheng B,Ye L,Zhou Y,et al.Epidermal growth factor attenuates blood-spinal cord barrier disruption via PI3K/Akt/Racl pathway after acute spinal cord injury[J].J Cell Mol Med,2016,20(6):1062-1075.
[14]Wu L,Fan J,Belasco J G.MicroRNAs direct rapid deadenylation of mRNA[J].Proc Nat Acad Sci U S A,2006,103(11):4034-4039.
[15]Reijerkerk A,Lopez-Ramirez M A,van Het Hof B,et al.MicroRNAs regulate human brain endothelial cell-barrier function in inflammation:implications for multiple sclerosis[J].J Neurosci,2013,33(16):6857-6863.
[16]Che P,Liu J,Shan Z,et al.miR-125a-5p impairs endothelial cell angiogenesis in aging mice via RTEF-1 downregulation[J].Aging Cell,2014,13(5):926-934.