可穿越血脑屏障的新型神经生长因子TAT-BDNF神经保护作用研究

目的 研究合成的新型神经生长因子TAT-BDNF融合蛋白兼具穿越血脑屏障及神经保护双重活性,为使用功能蛋白质治疗中枢神经损伤提供研究基础.方法 采用分子克隆方法构建表达载体胡AT.HA-BDNF,原核表达获得TAT-BDNF融合蛋白.利用体外培养的大鼠大脑皮层神经元谷氨酸兴奋性损伤模型,通过检测培养液中乳酸脱氢酶(LDH)漏出量,免疫荧光染色观察TAT-BDNF的神经保护作用.尾静脉注射TAT-BDNF后,通过免疫组化检测其穿透血脑屏障的活性;Nissl染色验证TAT.BDNF急性脊髓压迫损伤神经保护作用.结果 TAT·BDNF融合蛋白由重组质粒能有效表达.在神经元谷氨酸损伤模型中,使用TAT-BDNF后各组间培养液中LDH的漏出量差异有统计学意义(F=24 27,P＜0 05).形态学观察显示,TAT.BDNF可改善神经元的存活状态,减少谷氨酸诱导的细胞凋亡坏死比例(t=4.59,P:0 001).大鼠体内实验显示TAT-BDNF能有效透过血脑屏障,分布于脑与脊髓组织.脊髓损伤7 d后Nissl染色显示TAT-BDNF注射组髓内神经元存活状态优于对照组.结论 合成的新型神经生长因子TAT-BDNF具有穿透血脑屏障活性及神经保护作用.

Abstract：

Objective 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). Methods 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 Hoechst33342 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. Results 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. Conclusion The synthetic neotype TAT-BDNF possess the dual biological effects of neuroprotection and penetrating BBB.

Neuroproctection of a neotype transactivating-brain-derived neurotrophic factor fusion protein with penetrating activity of blood-brain barrier

Objective 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). Methods 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 Hoechst33342 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. Results 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. Conclusion The synthetic neotype TAT-BDNF possess the dual biological effects of neuroprotection and penetrating BBB.