表皮神经嵴干细胞移植对大鼠损伤脊髓组织中脑源性神经营养因子表达的影响

Effects of epidermal neural crest stem cell transplantation on expression of brain-derived neurotrophical factor in rats with spinal cord injury

摘要:

目的：观察表皮神经嵴干细胞（epidermal neural crest stem cells，EPI-NCSCs）移植治疗大鼠脊髓损伤（spinal cord in－jury，SCI）的作用以及SCI组织中脑源性神经营养因子（brain-derived neurotrophical factor，BDNF）表达的变化，从神经营养因子的角度探讨EPI-NCSCs移植修复SCI的机制。方法：分离培养绿色荧光蛋白（green fluorescent protein，GFP）转基因大鼠的EPI-NCSCs，备移植用。60只SD大鼠均暴露T10脊髓并在NYU-Ⅱ撞击机（10 g 25 mm致伤力）挫伤脊髓制作SCI模型，随机分为空白损伤组（A组）、培养基移植组（B组）、EPI-NCSCs移植组（C组）。SCI模型大鼠损伤1周后将EPI-NCSCs细胞悬液移植入C组大鼠SCI处，B组用单纯DMEM/F12培养基代替，空白损伤组不做处理。伤后每周进行一次运动功能评分，移植后1、3、6周取材行酶联免疫分析、免疫组织化学染色、免疫荧光染色以及Western blot检测BDNF蛋白的表达。结果：移植术后第2周起C组运动功能评分高于同期A组（2、3、4、5、6周，P值分别为0.003、0.000、0.000、0.000、0.000）和B组（2、3、4、5、6周，P值分别为0.044、0.000、0.006、0.000、0.008）。酶联免疫分析显示C组BDNF蛋白在移植后3周表达高于A组（P=0.009）和B组（P=0.013），移植后6周表达量显著高于A组（P=0.003）和B组（P=0.003）。免疫组化和免疫荧光结果显示移植后3周和6周，C组BDNF表达高于同期A组和B组。Western blot结果显示移植后6周，C组BDNF表达高于同期A组（P=0.000）和B组（P=0.000）。结论：EPI-NCSCs移植能促进SCI内BDNF的表达，改善局部微环境，从而有助于大鼠损伤脊髓的修复。

Abstract:

Objective：To explore the role of epidermal neural crest stem cells（EPI-NCSCs） in the treatment of spinal cord injury（SCI） of rats and changes in expression of brain-derived neurotrophical factor（BDNF） and to investigate the mechanism of EPI-NCSCs re－pairing SCI from the perspective of neurotrophic factors. Methods：EPI-NCSCs were isolated from green fluorescent protein（GFP） transgenic rats for transplantation. Rat models of SCI were made by NYU-Ⅱ impactor（10 g 25 mm injuries force） at T10 level. Then 60 SD rats were randomly divided into blank injury group（group A），medium transplantation group（group B），epidermal neural crest stem cell transplantation group（group C）. EPI-NCSCs were transplanted into the injured region in group C at 1 week after establishing SCI model. Group B used the DMEM/F12 to take place of EPI-NCSCs. Nothing was done in blank injury group. Then locomotor function score was appraised at every week after the transplantation. Expression of BDNF in three groups was detected by ELISA，immunohistochemistry，immunofluoresence and Western blot respectively at 1，3，6 weeks after the transplantation. Results：After 2 weeks of transplantation，locomotor function score was higher in group C than in group A（2，3，4，5，6 weeks：P=0.003，0.000，0.000，0.000，0.000） and group B（2，3，4，5，6 weeks：P=0.440，0.000，0.006，0.000，0.008）. Based on the results of ELISA，expression of BDNF was higher in group C than in group A（P=0.009） and group B（P=0.013） at 3 weeks after the transplantation；expression of BDNF was higher in group C than in group A（P=0.003） and group B（P=0.003） at 6 weeks after the transplantation. Expression of BDNF was higher in group C than in group A and group B at 3 weeks and 6 weeks after the transplan－tation based on the results of immunohistochemistry and immunofluoresence. Expression of BDNF was higher in group C than in group A（P=0.000） and group B（P=0.000） at 6 weeks after the transplantation based on the results of Western blot. Conclusions：EPI-NC－SCs transplantation can repair SCI by increasing expression of BDNF to improve local microenvironment.