【作者】 王伟；

【导师】 彭昊；

【作者基本信息】 武汉大学 ， 外科学、骨外科学， 2018， 博士

【摘要】 脊髓损伤(spinal cord injury,SCI)可导致损伤节段以下不可逆的感觉、运动和括约肌功能障碍,给患者、家庭及社会带来巨大的压力与沉重的负担,随着现代工业的发展、交通运输的繁荣以及老龄社会带来的高龄人群的快速增加,脊髓损伤病例逐年增多。虽然现代医学以及外科技术有了巨大的进步,但目前认为脊髓损伤仍然是不能治愈的损伤。神经保护以及神经再生修复是治疗脊髓损伤两个方面。神经保护是通过调节脊髓损伤后的病理生理改变,减少继发性损伤;神经再生是指通过移植或者刺激自身细胞参与神经再生及修复。干细胞移植是神经再生修复的方法之一,但移植干细胞在移植早期死亡以及凋亡,影响治疗效果。如何提高干细胞移植治疗脊髓损伤的疗效是科学家及医师面临难题之一。自由基是影响移植细胞成活的重要因素之一,因此降低自由基水平可能促进移植干细胞的成活,改善干细胞移植治疗脊髓损伤的效果。自由基学说是急性脊髓损伤后二次损伤的重要机制之一。脊髓损伤后,自由基(Free Radicals)生成增多,氧化应激造成的一系列级联反应,使原始打击损伤组织相邻的正常神经组织发生继发性病理损伤,在空间上扩大损伤的范围,并进一步加深原始损伤的损伤程度;脊髓损伤后早期抗氧化治疗可以有效的阻止二次损伤在时间及空间上的蔓延,最大程度的保护损伤神经组织及相邻正常神经组织,控制炎症反应,具有神经保护作用。超氧化物歧化酶(superoxidedismutase,SOD)是人体内天然的氧自由基清除剂,有望用于SCI的治疗,但由于细胞膜结构的生物屏障作用不允许此类大分子物质进入细胞发挥作用,限制了其潜在的治疗价值,影响了其临床应用。PEP-1是一种新型转导肽,它能够将多种蛋白质和肽段转导入细胞内。国外研究发现利用基因工程方法合成的PEP-1-sod1可以通过血脑屏障并且可以转导进入脊髓神经细胞,降低脊髓损伤后的超氧自由基及一氧化氮自由基水平,减少神经细胞凋亡,促进神经功能恢复。目的探讨脂肪干细胞(Adipose-derived stem cells,ADSCs)移植联合腹腔注射PEP-1-sod1修复脊髓损伤,探讨PEP-1-sod1在脊髓损伤早期的抗氧化保护作用,对移植脂肪干细胞的保护作用,及对脂肪干细胞移植修复脊髓损伤的促进作用。方法以贴壁培养法体外分离培养S-D大鼠脂肪干细胞(adipose-derived mesenchymal stem cellsADSCs),倒置相差显微镜观察脂肪干细胞形态,流式细胞仪鉴定分离细胞的表面标志,对脂肪干细胞进行成脂肪、成骨及成软骨诱导并鉴定,观察脂肪干细胞的多向分化潜能;以携带GFP基因的腺病毒载体转染脂肪干细胞,绘制脂肪干细胞及转染后的脂肪干细胞生长曲线,测定其细胞活力。取SD大鼠并随机分为四组,A组,空白对照组(sham group),手术切开暴露脊髓后即缝合,脊髓损伤模型组(B组、C组、D组)各组动物24只;B组,实验对照组(saline group)进行无菌生理盐水注射;C组,细胞治疗组(ADSCs group),脊髓损伤模型建立后进行ADSCs注射移植;D组,PEP-1-sod1联合ADSCs治疗组(联合治疗组,combined group),在脊髓损伤模型建立后即给予PEP-1-sod1腹腔注射并进行ADSCs移植,并连续3天注射PEP-1-sod1。B组、C组、D组为脊髓损伤模型组,各组动物24只。术后1、3、7d对脊髓损伤模型组各组行SOD活性检测及MDA含量检测,术后7d行细胞凋亡检测;术后1、2、3、4w行BBB运动评分观察,术后4w BBB评分完成后处死大鼠行NeuN表达阳性细胞检测,ADSCs治疗组及联合治疗组行GFP阳性细胞检测。结果1.贴壁法筛选培养得到的脂肪干细胞呈纤维细胞样,增值能力强。流式细胞检测脂肪干细胞高表达间充质干细胞表面标记,几乎不表达造血干细胞标记。分离的脂肪干细胞具有成脂肪、成骨及成软骨分化潜能。2.荧光显微镜下观察Adv-GFP转染成功后脂肪干细胞发出绿色荧光。Adv-GFP转染不影响脂肪干细胞的增值能力。3.术后1d盐水治疗组与ADSCs治疗组SOD活性差异无显著性;术后3d、7d ADSCs治疗组SOD活性大于盐水治疗组(p<0.05);术后1d、3d、7d联合治疗组SOD活性大于ADSCs治疗组(p<0.05),MDA含量小于ADSCs治疗组(p<0.05),ADSCs治疗组MDA含量小于盐水治疗组(p<0.05)。4.术后7d脊髓损伤组均可见大量细胞凋亡,联合治疗组细胞凋亡少于ADSCs治疗组(p<0.05),ADSCs治疗组细胞凋亡少于盐水治疗组(p<0.05);联合治疗组caspase-3蛋白及Bax蛋白表达较ADSCs治疗组减少(p<0.05),ADSCs治疗组较盐水治疗组减少(p<0.05)。5.术后1周各组BBB评分无差异(P>0.05),术后2周、3周、4周各组BBB评比较为联合治疗组>ADSCs治疗组>盐水治疗组(P<0.05)。6.术后4周各组NeuN阳性细胞数比较为联合治疗组>ADSCs治疗组>盐水治疗组(P<0.05);联合治疗组GFP阳性细胞数大于ADSCs治疗组(P<0.05)。结论1.大鼠腹股沟区脂肪组织贴壁法分离的脂肪干细胞,增值能力强,高表达间充质干细胞表面标记,具有多向分化潜能。Adv-GFP转染不影响脂肪干细胞的增值能力。2.与单纯脂肪干细胞移植相比,联合应用PEP-1-sod1进一步增加损伤局部SOD活性,降低MDA含量。3.PEP-1-sod1可以减少损伤早期细胞凋亡。4.PEP-1-sod1可以促进脂肪干细胞移植治疗脊髓损伤的疗效。5.PEP-1-sod1与脂肪干细胞移植治疗脊髓损伤的协同作用,可能与其抗氧化作用及减少移植细胞凋亡有关。更多还原

【Abstract】 Spinal cord injury is a devastating condition,which can cause irreversible sensory-motor deficits and sphincter dysfunction below the injured segment.Those injuries not only affect the physical and psychological well-being of those patients,but also bring huge socioeconomic burden to their families and society.With the development of modern industry and transportation and the coming of the aging society,the cases of spinal cord injury have increased year by year.Although modern medicine and surgical techniques have made great progress,it is still believed that spinal cord injury is still an unhealed injury.Neuroprotection and nerve regeneration are two important aspects in the treatment of spinal cord injury.Neuroprotection is a way to reduce secondary injury after primary spinal cord injury by regulating the secondary pathophysiological changes.Neuro-regeneration refers to transplantation or stimulating its own cells to repair injured spinal cord.Stem cell transplantation therapy holds the potential to promote repair and functional plasticity following spinal cord injury,but the death and apoptosis of transplanted stem cells in the early stage of transplantation affect it’s therapeutic effect.How to improve it’s therapeutic effect is one of the problems.Free radicals are one of the crucial factors that affect the survival of transplanted cells.Therefore,reducing the level of free radicals may promote the survival of transplanted stem cells and improve the effect of stem cell transplantation for spinal cord injury.One of the best validated secondary injury mechanisms in acute spinal cord injury concerns the post-traumatic generation of free radicals and their oxidative damage.Free radicals induce a series of cascade reactions,so the injury expands.Early antioxidant therapy can effectively prevent the spread of the damage and regulate the inflammation.Superoxide dismutase(SOD)is a natural free radical scavenger in human body.So it was expected to be used in the treatment of SCI.However,because of the biological barrier,it is not allowed to enter into cells,which affects its clinical application.Recently,a 21-residue peptide carrier,PEP-1,was developed as a tool for delivering biologically active molecules into cells.This peptide carrier allows the delivery of a variety of peptides and proteins into cells in a biologically active form.A recent report demonstrated that the PEP-1-SOD1 fusion protein can be delivered both efficiently and in a biologically active form into injured spinal cords in vivo.Systemically injected PEP-1-SOD1 can decrease the levels of free radicals,and thereby can improve functional recovery after SCI.Objective To investigate the effects of PEP-1-SOD1 on adipose-derived stem cells(ADSCs)transplantation in the therapy of spinal cord injury(SCI).To discuss the antioxidant effects of PEP-1-sodl on early spinal cord injury and transplanted adipose derived stem cells.Methods ADSCs were isolated from the adipose tissues of rats,cultured in vitro,fluorescence-activated cell sorting(FACS)analysis was done to access the phenotype of ADSCs.of ADSCs were performed to identify the Multidirectional differentiation potential of ADSCs were ADSCs were identified by Osteogenic,adipogenic and chondrogenic differentiation induction in vitro,than it was infected with adenovirus carrying GFP gene and its biological characteristic was accessed.Rats were randomly divided into four groups sham group saline group,ADSCs groupPEP-1-SOD 1-ADSCs group.The saline group underwent sterile saline injection;The ADSCs group transplanted ADSCs after SCI.The PEP-1-SOD1-ADSCs group treated with PEP-1-sodl combined ADSCs transplantion.After 1,3 and 7 days,the SOD activity in spinal cord was assessed by pyrogallol autoxidation assay;MDA content was determined using thiobarbituric acid colorimetric method and apoptosis were detected by TUNNEL staining.At 1,2,3 and 4 weeks after operation,the BBB exercise were scored,NeuN expression was assessed by immunohistochemistry and GFP positive cells were determined by fluorescence microscopy.Results 1.ADSCs obtained by the Adherent culture had strong proliferation ability The FACS analysis showed that ADSCs exhibited some parallel characteristics of typical MSCs and null expression of hemopoietic stem cell marker.ADSCs had osteogenic,adipogenic and chondrogenic differentiation potential.2.Adv-GFP infected ADSCs showed bright circular,the proliferation ability was not impaired.3.1,3and 7days post surgery,the SOD activities of PEP-1-sod 1-ADSCs group was stronger than ADSCs group(P<0.05),the MDA levels of PEP-1-sod 1-ADSCs group was less than ADSCs group(P<0.05).1 days post surgery,the SOD activities of Saline group and ADSCs group was of no statistic differences.3and 7days post surgery,the SOD activities of Saline group was less than ADSCs group.4.7 days post surgery,there was less apoptotic cells in PEP-1-sodl-ADSCs group than ADSCs group(p<0.05),apoptotic cell numbers in ADSCs group was less than saline group(p<0.05).5.The BBB scores of SCI group were not significantly different 1 week after surgery,2,3,4weeks after surgery the BBB scores were descendingly ordered as PEP-1-sodl-ADSCs group<ADSCs group<saline group(P<0.05).6.4weeks after surgery Groups comparison of NeuN positive cells were for PEP-1-sodl-ADSCs group>ADSCs group>saline group(P<0.05),the GFP positive cells of PEP-1-sodl-ADSCs group was higher than that of ADSCs group(P<0.05).Conclusions 1.high purified ADSCs can be obtained by adherent method.The obtained cells showed MSCs surface markes and had multi-directional differentiation capbilities.Adv-GFP infected ADSCs showed bright circular,its proliferation ability was not impaired.2.compared with purely ADSCs transplantation,PEP-1-SOD1 further increased the activity of local SOD,decreased the content of MDA.3.PEP-1-SOD1 reduced the early Apoptosis of local and transplanted cells.4.PEP-1-SOD1 improved the effects of ADSCs transplantation to repair SCI.5.It demonstrated synergistic effect of PEP-1-sod1 combined adipose stem cell transplantation in the treatment of spinal cord injury;it might relate to the antioxidant and anti-apoptosis effect of PEP-1-sod1.更多还原