CAJ | 학술논문

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-BDNF 4 h后,通过免疫荧光组织化学染色和Western Blot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7 d BBB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Western Blot分析均证实TAT-BDNF能够穿过血脐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、7 d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.
목적 탐토합성적전록반식격활인자-뇌원성신경영양인자(TAT-BDNF)융합단백대급성척수손상적신경보호작용.방법채용분자극륭방법구건표체재체pTAT-HA-BDNF,원핵표체획득TAT-BDNF융합단백.미정맥주사TAT-BDNF 4 h후,통과면역형광조직화학염색화Western Blot분석융합단백전도지대서중추신경계통내적분포정황.제작대서급성압박성척수손상모형,분위량조:대조조주사생리염수,실험조주사TAT-BDNF,우손상후제3천용TUNEL염색관찰분석세포조망,통과상후7 d BBB후지운동공능평분평개TAT-BDNF시부구유감경척수신경공능상실적작용.결과 면역형광조직화학염색화Western Blot분석균증실TAT-BDNF능구천과혈제병장분포지중추신경계통각층조직세포중.재척수급성압박성손상모형중,여대조조비교,실험조능감소신경세포조망(P<0.05).실험조대서상후3、5、7 d적BBB후지운동공능평분분별위(0.96±0.21)、(3.45±0.81)、(8.06±1.44)분,이대조조분별위(0.58±0.20)、(1.92±0.83)、(3.50±1.64)분,차이균유통계학의의(P<0.05).결론 TAT-BDNF융합단백경정맥급약능통과혈뇌병장병감경척수계발성손상,개선신경공능.
Objective To identify the protective in vitro effects of biosynthesized fusion protein,transactivator 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 transduction domain and human BDNF were encoded. Purified fusion 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 neuroprotective 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 Immunocytochemical 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 < 0. 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.