转录反式激活因子-源性神经营养因子融合蛋白对大鼠急性脊髓损伤的神经保护作用研究

Intravenous injection of fusion protein TAT-BDNF relieves acute spinal cord injury in rats

目的探讨合成的转录反式激活因子－脑源性神经营养因子（TAT-BDNF）融合蛋白对急性脊髓损伤的神经保护作用。方法采用分子克隆方法构建表达载体pTAT—HA—BDNF，原核表达获得TAT—BDNF融合蛋白。尾静脉注射TAT—BDNF4Ii后，通过免疫荧光组织化学染色和WesternBlot分析融合蛋白转导至大鼠中枢神经系统内的分布情况。制作大鼠急性压迫性脊髓损伤模型，分为两组：对照组注射生理盐水，实验组注射TAT-BDNF，于损伤后第3天用TUNEL染色观察分析细胞凋亡，通过伤后7dBBB后肢运动功能评分评价TAT—BDNF是否具有减轻脊髓神经功能丧失的作用。结果免疫荧光组织化学染色和WesternBlot分析均证实TAT．BDNF能够穿过血脑屏障分布至中枢神经系统各层组织细胞中。在脊髓急性压迫性损伤模型中，与对照组比较，实验组能减少神经细胞凋亡（P〈0．05）。实验组大鼠伤后3、5、7d的BBB后肢运动功能评分分别为（0．96±0．21）、（3．45±0．81）、（8．06±1．44）分，而对照组分别为（0．58±0．20）、（1．92±0．83）、（3．50±1．64）分，差异均有统计学意义（P〈0．05）。结论TAT—BDNF融合蛋白经静脉给药能通过血脑屏障并减轻脊髓继发性损伤，改善神经功能。

Objective To identify the protective in vitro effects of biosynthesized ihsion protein, transaetivator of transcription/brain-derived neurotrophic factor （TAT-BDNF）, on acute spinal cord injury in rats. Methods The recombinant vector termed pTAT-BDNF was constructed by molecular cloning. Both TAT protein transduetion domain and human BDNF were encoded. Purified fllsion protein TAT-BDNF was generated from Escherichia coli BL21 （DE3） . Immunocytochemistry and Western Blot were conducted to analyze the TAT-BDNF content in central nervous system tissue 4 hours after intravenous injection of fusion protein TAT-BDNF. Sprague Dawley rats were divided into a saline control group and a TAT-BDNF group. An animal model of acute compressive spinal cord injury was used to analyze neuroprotective effect of TAT-BDNF on cell apoptosis 3 days later through TUNEL staining. The neuroproteetive effect of TAT-BDNF was also evaluated by testing movements of the hind limb according to the blood-brain barrier （BBB） scales 7 days after the injury. Results hnmunocytochemical and Western Blot analyses of the central nervous system tissue revealed that intravenous TAT-BDNF penetrated BBB throughout the central nervous system. TAT-BDNF significantly decreased the apoptosis ratio in vivo after acute spinal cord injury at 3 days （ n = 6, P 〈 O. 05）. According to the BBB scales, evaluation of hind limb movements for TAT-BDNF treated rats was significantly better than that for TAT-BDNF non-treated ones （P 〈 0. 05） . Conclusion Intravenous fusion protein TAT-BDNF may penetrate BBB and thus relieve acute spinal cord injury.