bcl-2基因修饰神经干细胞移植修复大鼠脊髓损伤
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唐山市科技计划项目(15130254a)。


Bcl-2 gene-modified neural stem cell transplantation for spinal cord injury in rats
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    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体外培养的大鼠神经干细胞增殖。bcl-2基因修饰神经干细胞移植可促进脊髓损伤大鼠神经突触的再生,升高脊髓损伤区HSP27表达,降低脊髓损伤区bcl-2基因的表达和神经细胞凋亡,改善大鼠的肢体运动功能和电生理功能。

    Abstract:

    Objective To investigate the bcl-2 gene modification on neurological function recovery in rats with spinal 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) gene transfection of neural stem cells were divided into 3 groups:control group, negative transfection 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-fos 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 damage zone few of neuraxis-like structures, syringomyelia smaller, bcl-2-NSCs group showed more nerve axon-like structure, no syringomyelia. EGFP-positive cells labeled:bcl-2-NSCs group the most, NSCs group followed, no control group, and the difference between the groups was statistically significant (P<0.05). After the 4th week, SEP and MEP latency period:bcl-2-NSCs group P<0.05); Volatility:bcl-2-NSCs group> NSCs group> control group, and between the groups was significant difference (P<0.05). Conclusions By Ad-EGFP as vector-mediated side B-cell lymphoma 2 gene (bcl-2) gene transfection make neural stem cells can promote cultured rat neural stem cells. bcl-2 gene-modified neural stem cell transplantation can promote the regeneration of spinal cord injury synaptic elevated HSP27 expression after spinal cord injury, reduced expression and neural cell apoptosis after spinal cord injury bcl-2 gene and improve limb movement in rats function and electrophysiological function.

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张梅,王跃新,侯晓华,洪军,殷胜春,李岩,刘庆阳. bcl-2基因修饰神经干细胞移植修复大鼠脊髓损伤[J].中国比较医学杂志,2016,26(7):35~41.

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  • 最后修改日期:2016-01-12
  • 在线发布日期: 2016-07-28
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