bcl-2基因修饰雪旺细胞蛛网膜下腔移植修复大鼠脊髓损伤

发布时间：2018-04-15 14:42

本文选题：蛛网膜下腔 + 移植　；参考：《山东大学》2017年博士论文

【摘要】：目的:探讨bcl-2基因转染雪旺细胞蛛网膜下腔移植对脊髓损伤大鼠损伤神经功能恢复的影响。方法:体外培养大鼠雪旺细胞,经Ad-EGFP为载体介导端B淋巴细胞瘤-2基因(bcl-2)基因转染雪旺细胞,分为3组:对照组、阴性转染组及bcl-2转染组。Western blot检测雪旺细胞被转染后第3天和第14天的bcl-2蛋白的表达。选取成年雌性SD大鼠83只,成功造模72只,随机分为对照组,SCs组及bcl-2-SCs组,每组24只。依据改良Allen打击法建造大鼠急性脊髓损伤模型,分别于造模前、造模后1天、3天、1周、2周、3周、4周进行BBB、斜板试验及改良Tarlov方法对各组大鼠进行运动功能评定。造模后7天通过RT-PCR及Western blot检测检测脊髓损伤区周围胶质原纤维酸性蛋白(GFAP)、神经丝蛋白(NF-200)的表达,采用TUNEL法检测神经细胞凋亡情况,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SCs存活及分布情况,HRP逆行示踪检测神经纤维修复情况,通过SEP和MEP观察大鼠神经电生理恢复情况。结果:Western blot结果显示Ad-EGFP介导的bcl-2基因转染大鼠雪旺细胞体外能稳定表达bcl-2;大鼠下肢运动功能评价bcl-2-SCs组优于SCs组,SCs组优于对照组。造模完成72h,bcl-2-SCs组细胞凋亡数均明显低于其他两组(P0.05)。造模后7天,与对照组和SCs组相比,bcl-2-SCs组GFAP、NF-200基因和蛋白的表达均较显著升高P0.05)。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。SCs组损伤区可见少量神经轴索样结构,脊髓空洞较小,bcl-2-SCs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-SCs组多于SCs组,对照组数量最少,各组数据有统计学差异(P0.05)。HRP阳性神经纤维数:bcl-2-SCs组SCs组对照组,各组之间有显著性(P0.05)。造模后4周,SEP和MEP的潜伏期:bcl-2-SCs组SCs组对照组,且各组之间差异有显著性意义(P0.05);波幅:bcl-2-SCs组SCs组对照组,且各组之间差异有显著性意义(P0.05)。结论:bcl-2基因修饰雪旺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GFAP、NF-200基因和蛋白的表达,减少脊髓损伤区神经细胞凋亡,提升大鼠的肢体运动功能和电生理功能。
[Abstract]:Aim: to investigate the effect of subarachnoid transplantation of Schwann cells transfected with bcl-2 gene on the recovery of nerve function in spinal cord injury rats.Methods: Schwann cells were cultured in vitro. Schwann cells were transfected with Ad-EGFP mediated terminal B lymphocytoma 2 gene (BCL 2) gene. The cells were divided into 3 groups: control group.The expression of bcl-2 protein in Schwann cells on the 3rd and 14th day after transfection was detected by Western blot in negative transfection group and bcl-2 transfection group.Seventy-two adult female Sprague-Dawley rats were selected and randomly divided into two groups: control group (n = 24) and bcl-2-SCs group (n = 24).The model of acute spinal cord injury (sci) in rats was established according to the modified Allen attack method. The motor function of each group was evaluated by slanting plate test and modified Tarlov method before the model was made and 1 day, 2 weeks, 3 weeks and 4 weeks after the establishment of the model.RT-PCR and Western blot were used to detect the expression of glial fibrillary acidic protein (GFAP) and neurofilament protein (NF-200) around the injured area of spinal cord. The apoptosis of neurons was detected by TUNEL method.The survival and distribution of EGFP labeled SCs were observed by HE staining and fluorescence microscope 4 weeks after the model. The nerve fiber repair was detected by retrograde tracing. The electrophysiological recovery of rat nerve was observed by SEP and MEP.Results the expression of bcl-2in Schwann cells transfected with bcl-2 gene mediated by Ad-EGFP was stable in vitro, and the evaluation of motor function of lower extremity in bcl-2-SCs group was superior to that in SCs group.The number of apoptotic cells in 72h Bcl-2-SCs group was significantly lower than that in the other two groups (P 0.05).After 7 days, compared with control group and SCs group, the expression of GFAP-NF-200 gene and protein in Bcl-2-SCs group was significantly higher than that in control group and SCs group.At 4 weeks after modeling, the absence of spinal cord tissue and the formation of syringomyelia were observed in the control group, and a few axonal structures were observed in the injured area without axons passing through .SCs, and more axonal structures were observed in the smaller syringomyelia bcl-2-SCs group.The number of positive cells labeled in the syringomyelia group was higher than that in the SCs group, and the number of the control group was the least. There was significant difference in the number of positive nerve fibers between the two groups (P 0.05). The number of HRP-positive nerve fibers in the SCs group was 10% bcl-2-SCs, and there was a significant difference between the two groups (P 0.05).Four weeks after modeling, the latent period of SCs and MEP in the SCs group was significantly higher than that in the control group (P 0.05), and the amplitude in the SCs group was significantly higher than that in the control group (P 0.05).Conclusion Schwann cell transplantation with the BCL 2 gene can promote the regeneration of nerve synapses in spinal cord injury rats, increase the expression of GFAPN NF-200 gene and protein in spinal cord injury area, and reduce neuronal apoptosis in spinal cord injury area.Enhance the limb motor function and electrophysiological function of rats.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R651.2

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