Lentiviral-mediated transplantation of olfactory ensheathing cells modified by ciliary neurotrophic factor for treatment of spinal cord injury

doi:10.3969/j.issn.2095-4344.1723

Abstract

Abstract:

BACKGROUND: Previous studies have confirmed that both ciliary neurotrophic factor and olfactory ensheathing cells have a certain therapeutic effect on spinal cord injury, but there is no report on whether their combination treatment has a better therapeutic effect.
OBJECTIVE: To explore the effect of lentiviral-mediated transplantation of olfactory ensheathing cells modified with ciliary neurotrophic factor gene in rats with spinal cord injury and its related mechanisms.
METHODS: (1) In vitro experiment: There were four groups in the in vitro experiment: spinal cord neurons in control group and model group were cultured alone, while in overexpression co-culture group and normal co-culture group, Transwell chamber was used to establish the co-culture model of spinal cord neurons and olfactory ensheathing cells modified by ciliary neurotrophic factor gene or not. Alginate was given to damage the cells in the latter three groups. After 12 hours of co-culture, cell counting kit-8 assay was used to detect cell viability; qRT-PCR and western blot were used to detect the expression of BAX, BCL-2, Caspase 3 and ciliary neurotrophic factor in spinal neurons. (2) In vivo experiment: There were four groups in the in vivo experiment: sham operation group, model group, treatment group, and combination treatment group. Modified Allen’s method was used to make the rat model of spinal cord injury in the latter three groups. At 1 week after modeling, 5 µL of olfactory ensheathing cell suspension or ciliary neurotrophic factor overexpressing olfactory ensheathing cell suspension was injected 1 mm above and below the injured site in the treatment and combination treatment groups, respectively; similarly, the same volume of culture medium was given in the sham operation and model groups. At 4 weeks after treatment, Basso, Beattie and Bresnahan score and grid-climbing test were conducted to evaluate the motor ability of rats; hematoxylin-eosin staining was used to detect the pathological progression of spinal cord injury; and qRT-PCR and western blot were used to detect the expression of BAX, BCL-2, Caspase 3 and ciliary neurotrophic factor in spinal cord tissues.
RESULTS AND CONCLUSION: Olfactory ensheathing cells modified with ciliary neurotrophic factor gene could significantly alleviate cell injury and spinal cord injury (P < 0.001), increase the expression of ciliary neurotrophic factor (P < 0.01) and decrease the expression of apoptosis-related molecules BAX, BCL-2 and Caspase 3 (P < 0.01). To conclude, transplantation of olfactory ensheathing cells modified by ciliary ganglion neurotrophic factor gene can improve the motor function after spinal cord injury, which may be related to the down-regulation of BAX, BCL-2 and Caspase-3 expression and reduction in the apoptosis of spinal cord neurons.

Key words: spinal cord injury, olfactory ensheathing cells, spinal cord neurons, ciliary neurotrophic factor, neuronal apoptosis

CLC Number:

Ma Jiehua, Zhang Dan, Huo Yanli, Wei Yawei, Sun Lin, Zhao Yu. Lentiviral-mediated transplantation of olfactory ensheathing cells modified by ciliary neurotrophic factor for treatment of spinal cord injury[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(17): 2709-2715.

Figures/Tables 2

2.1 荧光定量PCR与蛋白免疫印迹法验证睫状神经营养因子过表达稳定细胞株稳定过表达组嗅鞘细胞中睫状神经营养因子mRNA显著升高(P < 0.001)，睫状神经营养因子蛋白表达量显著增加(P < 0.001)，见图1。 2.2 CCK8检测睫状神经营养因子过表达嗅鞘细胞对脊髓神经元细胞的保护作用采用Transwell小室建立嗅鞘细胞与脊髓神经元细胞共培养体系，并于脊髓神经元细胞中加海仁藻酸制作细胞损伤模型，24 h后加入CCK8溶液，测定细胞活力。与对照组相比，海仁藻酸可以显著抑制脊髓神经元细胞的活性(P < 0.001)；嗅鞘细胞与脊髓神经元细胞共培养可以抑制海仁藻酸对脊髓神经元细胞的损伤(P < 0.01)；嗅鞘细胞过表达睫状神经营养因子以后对脊髓神经元细胞损伤的抑制效果更显著(P < 0.01)，见图2。 2.3 睫状神经营养因子过表达嗅鞘细胞对脊髓损伤的影响采用改良Allen’s重物打击法制作脊髓损伤模型，随后按照不同的实验分组进行嗅鞘细胞移植。BBB评分结果显示，脊髓损伤模型建立之后，大鼠的运动功能几乎丧失(P < 0.001)；嗅鞘细胞移植4周后，动物后肢运动能力得到了一定程度的恢复(P < 0.01)；与正常嗅鞘细胞组相比，睫状神经营养因子过表达嗅鞘细胞移植后动物运动能力显著改善(P < 0.05)。爬网格实验结果也证明，嗅鞘细胞移植能够显著改善因脊髓损伤丧失的运动能力(P < 0.01)；睫状神经营养因子过表达嗅鞘细胞组大鼠于网格上攀爬的时间更久(P < 0.05)，BBB评分与爬网格实验结果证实睫状神经营养因子过表达嗅鞘细胞更能有效改善因脊髓损伤引起的运动功能下降，具有较好的治疗效果，见图3。 2.4 苏木精-伊红染色检测脊髓损伤的病理进展与假手术组相比，脊髓损伤组大鼠脊髓出现明显的损伤，表现出大量的空洞；在嗅鞘细胞移植后，脊髓形态得到了一定程度的恢复；在移植过表达睫状神经营养因子的嗅鞘细胞后，脊髓损伤得到了显著性的修复，见图4。 2.5 荧光定量PCR检测脊髓神经元细胞与脊髓组织中BAX、BCL-2、Caspase3、CNTF的mRNA表达在海仁藻酸致脊髓神经元细胞损伤以及动物脊髓损伤模型中，CNTF mRNA的表达明显下降(P < 0.001)，而细胞凋亡因子BAX、BCL-2、Caspase3的mRNA表达显著升高(P < 0.001)；嗅鞘细胞共培养或移植后，CNTF mRNA的表达得到了一定程度的恢复(P < 0.01)，细胞凋亡因子BAX、BCL-2、Caspase3的mRNA表达明显被抑制(P < 0.01)；在嗅鞘细胞中稳定过表达睫状神经营养因子后，CNTF mRNA表达显著升高(P < 0.01)，细胞凋亡因子BAX、BCL-2、Caspase3的mRNA表达显著下降(P < 0.01)。因此，嗅鞘细胞可能是通过释放CNTF抑制细胞凋亡因子的表达，减轻因脊髓损伤引起的运动功能障碍，见图5。 2.6 蛋白免疫印迹法检测脊髓神经元细胞与脊髓组织中BAX、BCL-2、Caspase3、CNTF的蛋白表达在海仁藻酸致脊髓神经元细胞损伤以及动物脊髓损伤模型中，CNTF的蛋白表达明显降低(P < 0.001)，细胞凋亡相关分子BAX、BCL-2、Caspase3的蛋白表达显著升高(P < 0.001)；嗅鞘细胞共培养或移植后，CNTF的蛋白表达得到了一定程度的恢复(P < 0.01)，细胞凋亡因子BAX、BCL-2、Caspase3蛋白的表达明显被抑制(P < 0.01)；在嗅鞘细胞中稳定过表达睫状神经营养因子后，CNTF的蛋白表达显著升高(P < 0.01)，细胞凋亡因子BAX、BCL-2、Caspase3的蛋白表达显著下降(P < 0.01)。因此，嗅鞘细胞可能是通过CNTF抑制脊髓神经元的凋亡而对脊髓损伤起到了一定程度的保护作用，见图6。"

References

[1] Fan B, Wei Z, Yao X, et al. Microenvironment Imbalance of Spinal Cord Injury. Cell Transplant. 2018;27(6):853-866.

[2] Ahuja CS, Nori S, Tetreault L, et al. Traumatic Spinal Cord Injury- Repair and Regeneration. Neurosurgery. 2017;80(3S):S9-S22.

[3] Furlan JC, Fehlings MG. The impact of age on mortality, impairment, and disability among adults with acute traumatic spinal cord injury. J Neurotrauma. 2009;26(10):1707-1717.

[4] Wu Q, Li YL, Ning GZ, et al. Epidemiology of traumatic cervical spinal cord injury in Tianjin, China. Spinal Cord. 2012;50(10): 740-744.

[5] Raspa A, Pugliese R, Maleki M, et al. Recent therapeutic approaches for spinal cord injury. Biotechnol Bioeng. 2016;113(2): 253-259.

[6] Lee N, Rydyznski CE, Rasch MS, et al. Adult ciliary neurotrophic factor receptors help maintain facial motor neuron choline acetyltransferase expression in vivo following nerve crush. J Comp Neurol. 2017;525(5): 1206-1215.

[7] Cen LP, Liang JJ, Chen JH, et al. AAV-mediated transfer of RhoA shRNA and CNTF promotes retinal ganglion cell survival and axon regeneration. Neuroscience. 2017;343:472-482.

[8] Yin DP, Chen QY, Liu L.Synergetic effects of ciliary neurotrophic factor and olfactory ensheathing cells on optic nerve reparation (complete translation).Neural Regen Res. 2016;11(6):1006-1012.

[9] Lee N, Serbinski CR, Braunlin MR, et al. Muscle and motor neuron ciliary neurotrophic factor receptor α together maintain adult motor neuron axons in vivo. Eur J Neurosci. 2016;44(12):3023-3034.

[10] Gu Y, Wang J, Ding F, et al. Neurotrophic actions of bone marrow stromal cells on primary culture of dorsal root ganglion tissues and neurons. J Mol Neurosci. 2010;40(3):332-341.

[11] Wen SY, Li AM, Mi KQ, et al.In vitro neuroprotective effects of ciliary neurotrophic factor on dorsal root ganglion neurons with glutamate- induced neurotoxicity.Neural Regen Res. 2017;12(10):1716-1723.

[12] Feng GY, Liu J, Wang YC, et al. Effects of Alpha-Synuclein on Primary Spinal Cord Neurons Associated with Apoptosis and CNTF Expression. Cell Mol Neurobiol. 2017;37(5):817-829.

[13] Yao R, Murtaza M, Velasquez JT, et al. Olfactory Ensheathing Cells for Spinal Cord Injury: Sniffing Out the Issues. Cell Transplant. 2018;27(6): 879-889.

[14] Wright AA, Todorovic M, Tello-Velasquez J, et al. Enhancing the Therapeutic Potential of Olfactory Ensheathing Cells in Spinal Cord Repair Using Neurotrophins. Cell Transplant. 2018;27(6):867-878.

[15] Haggerty AE, Maldonado-Lasunción I, Oudega M. Biomaterials for revascularization and immunomodulation after spinal cord injury. Biomed Mater. 2018;13(4):044105.

[16] Nakhjavan-Shahraki B, Yousefifard M, Rahimi-Movaghar V, et al. Transplantation of olfactory ensheathing cells on functional recovery and neuropathic pain after spinal cord injury; systematic review and meta-analysis. Sci Rep. 2018;8(1):325.

[17] Basso DM, Beattie MS, Bresnahan JC, et al. MASCIS evaluation of open field locomotor scores: effects of experience and teamwork on reliability. Multicenter Animal Spinal Cord Injury Study. J Neurotrauma. 1996;13(7): 343-359.

[18] 岳妍,谭波涛,刘媛,等. FTY720对急性脊髓损伤大鼠神经功能及血脊髓屏障的影响[J]. 解放军医学杂志,2015,40(3): 200-205.

[19] Stevens RD, Bhardwaj A, Kirsch JR, et al. Critical care and perioperative management in traumatic spinal cord injury. J Neurosurg Anesthesiol. 2003;15(3):215-229.

[20] Lang-Lazdunski L, Heurteaux C, Mignon A, et al. Ischemic spinal cord injury induced by aortic cross-clamping: prevention by riluzole. Eur J Cardiothorac Surg. 2000;18(2):174-181.

[21] Yu WR, Liu T, Fehlings TK, et al. Involvement of mitochondrial signaling pathways in the mechanism of Fas-mediated apoptosis after spinal cord injury. Eur J Neurosci. 2009;29(1):114-131.

[22] Manthorpe M, Skaper SD, Williams LR, et al. Purification of adult rat sciatic nerve ciliary neuronotrophic factor. Brain Res.1986; 367(1-2): 282-286.

[23] 习杨彦彬,王廷华. CNTF基因在正常大鼠和猫脊髓组织中的表达[J]. 昆明医科大学学报, 2006, 27(6):34-37.

[24] 曾洪艳,李力燕,郭小兵,等. 神经营养因子、凋亡相关因子和轴突导向因子在大鼠神经管畸形发育中的表达[J]. 昆明医科大学学报, 2012, 33(11):13-18.

[25] Tripathi RB, McTigue DM. Chronically increased ciliary neurotrophic factor and fibroblast growth factor-2 expression after spinal contusion in rats. J Comp Neurol. 2008;510(2):129-144.

[26] Yetiser S, Kahraman E. An analysis of time-dependent changes of neurotrophic factors (BDNF, CNTF) in traumatic facial nerve injury of a nerve-cut and nerve-crush model in rats. Otol Neurotol. 2008; 29(3): 392-396.

[27] Santos MD, Yasuike M, Kondo H, et al. A novel type-1 cytokine receptor from fish involved in the Janus kinase/signal transducers and activators of transcription (Jak/STAT) signal pathway. Mol Immunol. 2007;44(13): 3355-3363.

[28] 陈珊珊,郭小兵,金华.动脉移植BMSCs对大鼠缺血再灌注损伤脊髓CNTF和STAT3的影响[J]. 中风与神经疾病杂志,2014, 31(6):492-496.

[29] 李越,余化霖,陈莉发,等. 嗅球成鞘细胞植入大鼠挫伤脊髓内的迁移分布特征[J]. 中华创伤杂志, 2011, 27(1):78-82.

[30] 姜泳,迟晓飞,邹喜军,等. 重复经颅磁刺激联合嗅鞘细胞移植治疗脊髓损伤[J]. 中国组织工程研究,2017, 21(1):98-102.

[31] 布林,郑鸿,姜汉国,等. 移植神经生长因子和脑衍生神经生长因子基因修饰的嗅鞘细胞对脊髓损伤后细胞凋亡的影响[J].中国组织工程研究, 2005, 9(18):124-125.