Wang Qiang, Gou Xingchun, Jin Weilin, Xiong Lize, Hou Lichao, Chen Shaoyang, Zhang Hui, Zhu Xiaoling, Xu Lixian
Department of Anesthesiology, Xijing Hospital, Fourth Military Medical University, Xi'an, China.
Cell Mol Neurobiol. 2009 Feb;29(1):97-108. doi: 10.1007/s10571-008-9301-2. Epub 2008 Aug 29.
Nogo extracellular peptide 1-40 (NEP1-40), a Nogo-66 antagonistic peptide, is one of the potential candidates for therapeutic intervention after central nervous system injury. This study is focused on the generation of TAT-NEP1-40 fusion protein and its transducible effects and biological activity.
TAT-NEP1-40 fusion protein was expressed in vitro. Transducible effects of TAT-NEP1-40 were analyzed by using immunofluorescence staining or Western blot in vitro and in vivo. The biological activity of TAT-NEP1-40 was assessed by its effects against oxygen and glucose deprivation (OGD)-induced PC12 cell damages.
Our results showed that the TAT-NEP1-40 fusion protein was successfully expressed, purified, and refolded. Western blot analysis and immunofluorescence staining confirmed the delivery of TAT-NEP1-40 protein into PC12 cells and rat brains. OGD caused cell apoptosis or death, decreased cell viability, increased lactate dehydrogenase release in medium and the Bax/Bcl-2 ratio, all of which were prevented by the TAT-NEP1-40 fusion proteins when added exogenously to culture medium. In addition, TAT-NEP1-40 promoted neurite outgrowth of PC12 cells exposed to OGD.
These results demonstrate that the TAT-NEP1-40 can be successfully generated and efficiently transduced into PC12 cells and rat brains. The TAT-NEP1-40 can protect PC12 cells against OGD and promote neurite outgrowth. This finding suggests that the transducible TAT-NEP1-40 fusion protein offers a possibility of the development of novel therapy for cerebral injuries via delivery of the biologically active TAT-NEP1-40 fusion protein into injured sites.
Nogo细胞外肽1 - 40（NEP1 - 40）是一种Nogo - 66拮抗肽，是中枢神经系统损伤后治疗干预的潜在候选物之一。本研究聚焦于TAT - NEP1 - 40融合蛋白的生成及其转导效应和生物学活性。
体外表达TAT - NEP1 - 40融合蛋白。通过体外和体内免疫荧光染色或蛋白质印迹分析TAT - NEP1 - 40的转导效应。通过其对氧和葡萄糖剥夺（OGD）诱导的PC12细胞损伤的作用评估TAT - NEP1 - 40的生物学活性。
我们的结果表明，TAT - NEP1 - 40融合蛋白成功表达、纯化和复性。蛋白质印迹分析和免疫荧光染色证实TAT - NEP1 - 40蛋白进入了PC12细胞和大鼠脑内。OGD导致细胞凋亡或死亡，降低细胞活力，增加培养基中乳酸脱氢酶释放及Bax/Bcl - 2比值，当向培养基中外源添加TAT - NEP1 - 40融合蛋白时，所有这些均受到抑制。此外，TAT - NEP1 - 40促进了暴露于OGD的PC12细胞的神经突生长。
这些结果表明，TAT - NEP1 - 40能够成功生成并有效转导至PC12细胞和大鼠脑内。TAT - NEP1 - 40可保护PC12细胞免受OGD损伤并促进神经突生长。这一发现表明，可转导的TAT - NEP1 - 40融合蛋白通过将具有生物活性的TAT - NEP1 - 40融合蛋白递送至损伤部位，为脑损伤新疗法的开发提供了可能性。
