慢病毒介导睫状神经营养因子基因修饰嗅鞘细胞移植治疗脊髓损伤
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摘要
背景:以往研究证实,睫状神经营养因子与嗅鞘细胞单独应用均对脊髓损伤具有一定的治疗作用,但两者联合应用是否有更好的治疗效果尚未见报道。目的:慢病毒介导睫状神经营养因子基因修饰嗅鞘细胞移植对大鼠脊髓损伤的作用及其相关机制。方法:①体外实验:实验分为4组,对照组进行脊髓神经元细胞单独培养,模型组进行脊髓神经元细胞单独培养,正常嗅鞘细胞共培养组采用Transwell小室进行脊髓神经元细胞与正常嗅鞘细胞共培养,过表达嗅鞘细胞共培养组采用Transwell小室进行脊髓神经元细胞与睫状神经营养因子过表达嗅鞘细胞共培养,后3组给予海仁藻酸建立神经元细胞损伤模型。共培养12 h后,采用CCK8法检测细胞活力,qRT-PCR与Western blot检测脊髓神经元细胞中BAX、BCL-2、Caspase3、CNTF的表达;②体内实验:实验分为4组,假手术组、模型组、治疗组、联合治疗组,后3组采用改良Allen’s重物打击法制作脊髓损伤大鼠模型,造模1周后,治疗组、联合治疗组分别在损伤区上、下1 mm处各注射5μL嗅鞘细胞悬液或睫状神经营养因子过表达嗅鞘细胞悬液,假手术组与模型组大鼠依照同样的方法注射等量培养基;③治疗后4周,采用BBB评分与爬网格实验评估大鼠运动能力、苏木精-伊红染色检测脊髓损伤的病理进展、qRT-PCR与Western blot检测脊髓组织中BAX、BCL-2、Caspase3、CNTF的表达。结果与结论:①睫状神经营养因子基因修饰的嗅鞘细胞能显著抑制细胞损伤以及脊髓损伤(P<0.001);显著提高睫状神经营养因子的表达水平(P <0.01)以及显著降低凋亡相关分子BAX、BCL-2、Caspase3的表达水平(P <0.01);②睫状神经营养因子基因修饰的嗅鞘细胞移植能够改善脊髓损伤引起的运动功能下降,其机制可能与下调BAX、BCL-2、Caspase3表达,降低脊髓神经元细胞凋亡相关。
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
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
引文
[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 InjuryRepair 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.JNeurotrauma.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 RhoAshRNA 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 receptorαtogether 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 glutamateinduced 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 CNTFExpression.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.JNeurotrauma.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.JNeurosurg 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.
[2]Ahuja CS,Nori S,Tetreault L,et al.Traumatic Spinal Cord InjuryRepair 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.JNeurotrauma.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 RhoAshRNA 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 receptorαtogether 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 glutamateinduced 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 CNTFExpression.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.JNeurotrauma.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.JNeurosurg 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.