Tumor necrosis factor-alpha antagonist combined with tyrosine kinase C gene- modified neural stem cell transplantation for spinal cord injury

doi:10.3969/j.issn.2095-4344.2017.21.010

Abstract

Abstract:

BACKGROUND: Whether controlling of post-injury inflammatory response combined with neural stem cell (NSC) transplantation can improve the curative efficacy for spinal cord injury still remains unclear.
OBJECTIVE: To investigate the repair of spinal cord tissue, myelin regeneration, axon regeneration, motor function recovery and the possible mechanism after early application of tumor necrosis factor α antagonist (Etanercept) combined with tyrosine kinase C (TrkC) gene-modified NSC transplantation.
METHODS: TrkC-overexpressed NSCs (TrkC-NSCs) were constructed by lentiviral transfection technique. The rat models of spinal cord transection injury were prepared, and then subjected to Etanercept combined with TrkC-NSCs transplantation. The number of neurons and neuroregeneration after injury were measured by Nissl’s staining, immunofluorescence and western blot. The rat motor function was detected by Basso Beattie Bresnahan score and evoked potential. The myelin regeneration was detected by electron microscopy and toluidine blue staining.
RESULTS AND CONCLUSION: Compared with the other groups, the Etanercept combined with TrkC-NSCs transplantation group had more survived anterior horn motor neurons at 28 days after injury, more myelin-encapsulated axons, higher Basso Beattie Bresnahan score, greater amplitude of the evoked potentials, and relatively shorter latency (all P < 0.05). These findings indicate that early application of tumor necrosis factor-α antagonist combined with TrkC-NSCs transplantation after spinal cord injury in rats can effectively promote myelin regeneration, axon regeneration, and further promote motor function recovery.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Tissue Engineering, Spinal Cord Injuries, Neural Stem Cells, Apoptosis

CLC Number:

R394.2

Wang Le, Chen Ning-ning, Li Ting-ting, Zhao Xiao-yang, Wei Fu-xin, Cui Shang-bin, Wan Yong,Liu Shao-yu. Tumor necrosis factor-alpha antagonist combined with tyrosine kinase C gene- modified neural stem cell transplantation for spinal cord injury[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(21): 3338-3345.

Figures/Tables 2

2.1 慢病毒LV/TrkC转染神经干细胞后筛选结果原代提取的神经干细胞在培养24 h后，50%-75%的细胞呈贴壁状态，48 h后出现细胞集落生长，并开始悬浮；72 h后细胞逐渐悬浮并细胞增殖速度增加；约4 d出现小的神经球(neurosphere)，待生长六七天时，小神经球生长至150- 200 μm则可进行传代。传至2代时，细胞形态基本均一，增殖旺盛，折光性好，Nestin染色阳性比例高(图1)。将细胞种植到24-well中培养，测试不同滴度LV-GFP感染神经干细胞后绿色荧光强度。发现MOI值=10时，镜下所见的神经球绿色荧光表达最为强烈，细胞质和细胞核内均有GFP-TrkC表达。培养到第7天仍有强GFP绿色荧光表达。通过转染成功后加入嘌呤霉素，筛选出了高表达TrkC基因的神经干细胞(图2)。 2.2 大鼠后肢运动功能变化实验用BBB评分对各组大鼠的后肢运动功能情况进行评估，尽管OP组的运动恢复十分有限，单纯ET组的BBB评分在损伤2周后开始较OP组有明显的恢复而且这种恢复逐渐明显一直到末次观察(损伤后4周)(P < 0.05)(图3)，这表明肿瘤坏死因子α在脊髓横断损伤模型的继发性损伤中也扮演重要角色。另外从损伤后2周开始，NSC+ET组相比OP组明显促进了大鼠运动功能恢复(P < 0.05)，这种恢复在3周和4周更为明显(P < 0.01)；而联合治疗在4周后功能恢复好于两个单纯治疗组(P < 0.05)。在NSC+ET组，损伤后4周10只大鼠里有3只可以做到负重步行(BBB评分10分)，结果表明Etanercept早期应用可以有利于脊髓运动功能恢复，联合TrkC-NSC治疗恢复更好。 2.3 大鼠运动诱发电位变化测量各组大鼠的运动诱发电位(MEP)，运动诱发电位作为临床常用的脊髓检测制备，主要用于评估运动神经系统。通过运动诱发电位的波幅和潜伏期可初步估计出脊髓内兴奋的轴突数目和神经的信号传导能力。运动诱发电位的信号在OP组非常差，ET组，NSC组和NSC+ET组的运动诱发电位波幅较OP组明显升高，各组运动诱发电位波幅最大的是NSC+ET组(P < 0.05)。但相比假手术组仍有较大差距。这些结果表明早期应用Etanercept联合TrkC-NSC移植可促进神经再生；另外，Etanercept的抗炎和抗凋亡作用促进了神经细胞存活以及运动神经功能恢复。相比BBB评分，运动诱发电位的波幅在ET组和NSC组是差异无显著性意义(P > 0.05)。运动诱发电位仅在NSC+ET组的潜伏期较OP组缩短，差异有显著性意义(P < 0.05；图4)。 2.4 大鼠脊髓前角神经元计数变化实验利用尼氏染色来评估脊髓损伤后早期应用Etanercept联合TrkC-NSC移植对脊髓前角运动神经元的数目的影响，计数位置为损伤处尾端。统计可知NSC组，ET组和NSC+ET组的前角运动神经元个数相比OP组都有显著增加(P < 0.05)，NSC+ET组的前角运动神经元个数相比其他组均有增加(P < 0.05)，说明脊髓损伤后早期应用Etanercept联合TrkC-NSC移植28 d后相比单纯TrkC-NSC移植或单纯Etanercept治疗可得到更多存活的前角运动神经元。因评估的位置是脊髓损伤横断处的尾端，故存活神经元可能与ET对损伤后残存神经元的保护，或移植后神经干细胞迁移分化有关(图5)。 2.5 大鼠脊髓神经元相关标记物情况为评估脊髓损伤后早期应用Etanercept联合TrkC-NSC移植治疗的神经再生情况，以及Etanercept的抗炎抗凋亡作用影响下NSC+ET组相比NSC或ET组的恢复情况，实验利用几种神经元再生的相关标记物。NF200的表达在ET，NSC组和NSC+ET组相比OP组均有升高(P < 0.05)，而NF200表达最高的是NSC+ET联合治疗组(P < 0.05)。与突触功能密切相关的蛋白synapsin Ⅰ，在ET，NSC组和NSC+ET组相比OP组均有升高(P < 0.05)，表达最高的是NSC+ET联合治疗组(P < 0.05)。另外结果还显示，NSC组的NF200及synapsinⅠ表达要高于ET组(P < 0.05)(图6)。实验还利用免疫荧光检测NF200在脊髓损伤处的生成情况，为评估各组轴突再生的程度。ET组，NSC组和NSC+ET组在损伤后4周的神经丝数目相比对照组均明显增加(P < 0.05)。在NSC组中NF200阳性的神经丝数目要高于ET组(P < 0.05)，NSC+ET组的神经丝数目最多(P < 0.05)，表明联合治疗效果优于单独ET或TrkC-NSC移植，这其中的效果大部分归功于早期应用Etanercept对肿瘤坏死因子α的拮抗作用(图6)。 2.6 大鼠脊髓损伤区域的髓鞘再生为检测脊髓损伤后早期应用Etanercept联合TrkC-NSC移植治疗能否促进脊髓髓鞘再生，实验使用甲苯胺蓝轴突染色后光镜计数。结果显示，OP组的脱髓鞘情况非常严重，而在ET组，NSC组和NSC+ET组中实验发现有更多的具有髓鞘包绕的轴突(P < 0.05)。而NSC组中轴突被髓鞘包绕的个数和比例都高于ET组，差异有显著性意义(P < 0.05)。NSC+ET组得到的具备髓鞘包绕的轴突数量和比率均高于其他组(P < 0.05)。电镜观察可见到脱髓鞘情况和甲苯胺蓝染色计数结果一致，在对照组中实验可见包绕轴突的髓鞘的薄层分离，髓鞘结构疏松(图7)。"

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