bcl-2基因修饰神经干细胞移植修复大鼠脊髓损伤  

Bcl-2 gene-modified neural stem cell transplantation for spinal cord injury in rats

作　　者：张梅[1] 王跃新[1] 侯晓华[1] 洪军[1] 殷胜春[1] 李岩[1] 刘庆阳 

机构地区：[1]唐山市工人医院,河北唐山063000 [2]煤炭总医院,北京100028

出　　处：《中国比较医学杂志》2016年第7期35-41,共7页Chinese Journal of Comparative Medicine

基　　金：唐山市科技计划项目(15130254a)

摘　　要：目的探讨bcl-2基因修饰神经干细胞移植对脊髓损伤大鼠损伤神经功能恢复的影响。方法体外培养大鼠神经干细胞,经Ad-EGFP为载体介导端B淋巴细胞瘤-2基因(bcl-2)基因转染神经干细胞,分为3组:对照组、阴性转染组、bcl-2转染组。Western-blot检测神经干细胞在转染前后bcl-2蛋白的表达。成年雌性SD大鼠85只,造模成功72只,随机分为对照组,NSCs组,bcl-2-NSCs组,24只/组,按照改良的Allen打击法建立大鼠急性脊髓损伤模型。通过BBB评分、斜板试验进行运动功能评定。造模后7 d通过RT-PCR及Western-blot检测检测脊髓损伤区周围HSP27、c-fos基因的表达,TUNEL法检测细胞凋亡情况。造模后4周取材行病理切片HE染色及荧光显微镜观测EGFP标记的NSC存活及分布情况,通过SEP和MEP观察大鼠神经电生理恢复情况。结果 bcl-2基因转染大鼠神经干细胞后,bcl-2转染组与对照组、阴性转染组相比bcl-2基因和蛋白水平均有表达(P<0.05);大鼠下肢运动功能评价bcl-2-NSCs组优于NSCs组,NSCs组优于对照组。造模后72 h,bcl-2-NSCs组细胞凋亡数均明显低于对照组和NSCs组(P<0.05)。造模后7 d,与对照组和NSCs组相比,bcl-2-NSCs组HSP27基因和蛋白的表达均较显著升高(P<0.05),bcl-2-NSCs组c-fos基因和蛋白的表达较显著降低(P<0.05)。造模后4周,HE染色对照组可见脊髓组织缺失及脊髓空洞形成,无神经轴索通过。NSCs组损伤区可见少量神经轴索样结构,脊髓空洞较小,bcl-2-NSCs组可见较多神经轴索样结构,未见脊髓空洞。EGFP标记的阳性细胞数:bcl-2-NSCs组最多,NSCs组次之,对照组未见,且各组之间差异有显著性(P<0.05)。造模后4周,SEP和MEP的潜伏期:bcl-2-NSCs组<NSCs组<对照组,且各组之间差异有显著性(P<0.05);波幅:bcl-2-NSCs组>NSCs组>对照组,且各组之间差异有显著性意义(P<0.05)。结论通过Ad-EGFP为载体介导端B淋巴细胞瘤-2基因(bcl-2)基因转染使神经干细胞能够促进�Objective To investigate the bcl-2 gene modification on neurological function recovery in rats withspinal cord injury in neural stem cell transplantation. Methods Cultured rat neural stem cells by Ad-EGFP as vector- mediated side B-cell lymphoma 2 gene （bcl-2） gcne transfection of neural stem cells were divided into 3 groups： control group, negative transfeetion group, bcl-2 transfection group. Use western-blot to detect the expression of bcl-2 protein in neural stem cells before and after transfection. 85 adult female SD rats, successful model 72, were randomly divided into control group, NSCs group, bcl-2-NSCs groups, 24/group, rat acute spinal cord injury model in accordance with a modified Allen＇ s method. Assess the motor function by BBB rating and the swash plate test. 7 days after modeling by RT- PCR and Western blot detection of spinal cord injury around HSP27, c - los gene expression, TUNEL assay apoptosis. Four weeks after model drawn line HE staining and fluorescence microscopy EGFP-labeled NSC survival and distribution of the rats neurophysiological recovery by SEP and MEP. Results bcl-2 gene transfection of rat neural stem cells, bcl-2 transfection group and control group, negative transfection group compared to bcl-2 mRNA and protein levels were expressed （P 〈 0.05）; lower extremity motor function in rats evaluation of bcl-2-NSCs group than NSCs group, NSCs group than the control group. 72 hours after modeling, bcl-2-NSCs number of apoptotic cells were significantly lower than the control group and NSCs group （P 〈 0.05）. 7 days after modeling, compared with the control group and NSCs group, bcl-2-NSCs group HSP27 gene and protein expression was significantly higher than that （P 〈 0.05） , bcl-2-NSCs group c- fos mRNA and protein expression was significantly reduced compared （P 〈 0.05）. 4 weeks after modeling, HE staining control group showed spinal cord tissue loss and the formation of syringomyelia, no axonal through. NSCs group danaage zone few of neuraxis-like struc

关 键 词：脊髓损伤 BCL-2基因 修饰 神经干细胞 移植 修复 大鼠 神经功能 

分 类 号：R332[医药卫生—人体生理学]