Jiang Hui, Jin Wei-Lin, Jin Da-Di, Chen Jian-Ting
Department of Spinal Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
Zhonghua Yi Xue Za Zhi. 2011 Jul 5;91(25):1786-90.
To identify the dural activities of neuroprotection and penetrating blood-brain barrier (BBB) for TAT-BDNF (transactivating-brain-derived neurotrophic factor) fusion protein to explore an alternative treatment for the injury of central nerve system (CNS).
With molecular cloning techniques, a recombinant vector termed pTAT-BDNF was constructed to encode both TAT protein transduction domain and human BDNF. Purified TAT-BDNF fusion protein was generated from Escherichia coli BL21 (DE3). The injury model was established with in vitro cultured cortical neurons of neonatal rats. To observe the neuroprotective effects of TAT-BDNF fusion protein on glutamate-mediated excitotoxic insults, the contents of lactate dehydrogenase (LDH) were measured by spectrophotometry. Immunofluorescence and Hoechst 33342 analyses were used to observe the morphological changes. Immunocytochemical and Nissl stain analysis of TAT-BDNF content in CNS tissue were performed after an intravenous injection of TAT-BDNF fusion protein in normal or spinal cord injured rats.
During the study of glutamate-induced excitotoxic insults, as compared with the control group, TAT-BDNF could decrease the apoptotic ratio, reduce the leakage of LDH and enhance the survival of neurons (P < 0.05 ). As demonstrated by immunohistochemistry, TAT-BDNF fusion protein was efficiently delivered into rat brain and spinal cord tissues at 4 h post-injection. At Day 7 post-injury, Nissl stain show that the number and morphology of neurons in the TAT-BDNF group were better than those in the control group.
The synthetic neotype TAT-BDNF possess the dual biological effects of neuroprotection and penetrating BBB.
鉴定反式激活脑源性神经营养因子（TAT-BDNF）融合蛋白的神经保护及穿透血脑屏障（BBB）的作用，以探索一种治疗中枢神经系统（CNS）损伤的替代方法。
利用分子克隆技术构建重组载体pTAT-BDNF，用于编码TAT蛋白转导结构域和人BDNF。从大肠杆菌BL21（DE3）中产生纯化的TAT-BDNF融合蛋白。用新生大鼠体外培养的皮质神经元建立损伤模型。为观察TAT-BDNF融合蛋白对谷氨酸介导的兴奋性毒性损伤的神经保护作用，采用分光光度法测定乳酸脱氢酶（LDH）含量。采用免疫荧光和Hoechst 33342分析观察形态学变化。在正常或脊髓损伤大鼠静脉注射TAT-BDNF融合蛋白后，对CNS组织中的TAT-BDNF含量进行免疫细胞化学和尼氏染色分析。
在谷氨酸诱导的兴奋性毒性损伤研究中，与对照组相比，TAT-BDNF可降低凋亡率，减少LDH泄漏，提高神经元存活率（P<0.05）。免疫组织化学显示，注射后4小时，TAT-BDNF融合蛋白可有效递送至大鼠脑和脊髓组织。损伤后第7天，尼氏染色显示TAT-BDNF组神经元的数量和形态优于对照组。
合成的新型TAT-BDNF具有神经保护和穿透血脑屏障的双重生物学效应。
