【作者】 刘佳；

【导师】 王廷华； 李力燕；

【作者基本信息】 昆明医学院 ， 神经外科学， 2010， 博士

【摘要】 【目的】建立三种干细胞即神经干细胞(NSC)、骨髓问充质细胞(BMSC)和造血干细胞(HSC),及两种支持细胞即嗅鞘细胞(OEC)和雪旺细胞(SC)的体外培养技术,并将两种支持细胞分别和三种干细胞分别联合移植入SD大鼠和猴损伤的脊髓,筛选出细胞联合移植治疗SCI的优化方案,并探讨与其相关的分子表达,找出相对重要的调节分子,研究该分子在SCI中的作用。为寻找细胞移植治疗SCI的有效方法和相关的分子机制提供实验依据,为指导临床应用干细胞移植治疗SCI提供新思路。【方法】1.建立嗅鞘细胞、雪旺细胞、神经干细胞、骨髓间充质细胞和造血干细胞的体外培养技术,并对培养细胞进行鉴定。2.建立SD大鼠脊髓T10全横断损伤模型。将动物分假手术组、单纯脊髓全横断手术组、脊髓全横断手术注射免疫抑制剂组,NSC移植组、BMSC移植组、HSC移植组、SC细胞移植组,OEC移植组,以及NSC+OEC,BMSC+OEC,HSC+SC联合移植组。术后14d沿原切口暴露损伤脊髓,选取距离瘢痕5mm的脊髓上下端和瘢痕各取左右两个位点(脊髓上下端旁开脊髓后正中沟0.5mm,瘢痕处的左右侧位点参照脊髓上下端的左右位点定位),共6个位点用立体定向仪进行细胞移植(1.0×10~5/5uL,每个位点)。用5mg/kg/天腹腔注射环孢素(CsA)以减轻细胞移植后的免疫排斥反应。3.后肢运动功能评分:移植术后1～24周,采用双盲法由三名观察人员每周末进行大鼠后肢BBB运动功能评分。最后将三人所得分值求平均分后进行统计学处理。筛选出促进脊髓损伤功能恢复最好的一组为NSC+OEC联合移植组。4.神经电生理检测:对NSC+OEC联合移植组,及其相应对照组进行神经电生理检测,进一步探讨NSC+OEC联合移植组促进SCI的实验依据。(1)运动诱发电位(MEP)的测定采用了神经电生理经颅磁刺激器对NSC+OEC联合移植组14d组,及其对照组假手术组、脊髓全横断手术注射免疫抑制剂组、NSC移植组和OEC移植组的MEP进行检测。3.6%水合三氯乙醛麻醉固定动物后,用盘状电极通过导电膏粘附于大鼠耳朵作为地线,记录电极置于大腿股四头肌,正负电极间距1cm,磁力板中心置于大脑皮质运动区之上刺激大脑皮质,以刺激强度40%刺激大脑皮质,记录股四头肌收集到得电位变化,待波形稳定且重复3次时中止并描记结果。(2)皮层体感诱发电位(CSEP)的测定本实验采用了皮层体感诱发仪(MEDTRONIC公司产品-KEYPOINY)对移植14d的NSC+OEC联合移植组,及其对照组假手术组、脊髓全横断手术注射免疫抑制剂组、NSC移植组和OEC移植组进行了检测。电流为方波脉冲型,刺激电极置于大鼠左下肢胫后神经处,正负电极间距为1cm,参考电极置于大鼠鼻正中皮下,记录电极置于大鼠右侧大脑皮层处皮下,接地电极置于大鼠耳朵。参数设置:扫描速度10ms/D,灵敏度10uv/D,滤波低频10Hz,滤波高频2KHz,刺激强度以引起大鼠后爪抖动为止。每次均记录300次波形后将其叠加平均得到最终的检测结果。5.植入细胞存活检测:在第12和24W末取部分动物用3.6%的水合氯醛进行腹腔注射麻醉,4%的多聚甲醛灌注固定后取材、冰冻切片,荧光显微镜下观察植入细胞存活,迁移。6.PCR扩增疤痕组织的神经营养因子及其受体,和凋亡相关因子:将14d、21d、28d的各组全横断脊髓(手术注射免疫抑制剂组、NSC移植组、OEC移植组和NSC+OEC联合移植组)活体取出疤痕部位及临近组织,匀浆、裂解、提取总RNA、逆转录合成cDNA,进行神经营养因子NGF、BDNF、NT-3、CNTF、PDGF-B、IGF-1、bFGF、TGF-β1,及其受体TrkA、TrkB和TrkC,和凋亡相关因子Bax、Bcl-2、Caspase-3及内参照β-actin的PCR扩增,琼脂糖凝胶电泳检测扩增产物。测光密度值,进行统计学分析,筛选出有变化的因子。推测其中与SCI有关的重要分子。7.用高等动物灵长类猴验证NSC+OEC联合移植治疗SCI的效果:建立恒河猴半横断SCI模型。动物分半横断手术注射免疫抑制剂组、NSC移植组、OEC移植组和NSC+OEC联合移植组。通过运动功能评分(1-9周),神经电生理和核磁共振检测(1月),和形态学观察来阐明NSC+OEC联合移植对SCI的促进作用。8.选择通过PCR扩增后确定的疤痕组织内有变化的PDGF-B作为切入点:通过免疫组化,RT-PCR,Western Blot,细胞培养,RNA干扰,抗体封闭等技术来研究PDGF-BB在SCI中的作用和可能的作用机制。【结果】1.本实验成功建立了OEC、SC、NSC、BMSC和HSC的体外培养方法,并进行了鉴定。2.运动功能评分:所有大鼠脊髓全横断后,双侧后肢完全瘫痪,不能排尿。大鼠靠前肢拖动身体前行。1周末时,手术损伤组大鼠双后肢开始出现1个后肢关节(踝关节)的轻微活动。细胞移植组则开始出现1或2个后肢关节轻微活动(多为膝关节或/和踝关节)。在第2周末时,手术损伤组和细胞移植组大鼠在细胞移植后排尿障碍均较第1周末时稍有好转,膀胱残余尿仍较多,手术损伤组部分大鼠开始出现2个后肢关节的轻微活动,而细胞移植组部分大鼠开始出现1或2个后肢关节大幅运动。各细胞移植组的大鼠BBB评分在整个恢复的过程中均有明显的提高,但变化最明显的的就是OECs+NSCs组。说明OEC+NSC移植对运动功能恢复明显改善。3.神经电生理感觉和运动诱发电位检测3.1 CSEP检测N1波变化:脊髓全横断组没有测到波形,潜伏期无限延长,与假手术组比较差异显著;说明手术切断脊髓后上行传导通路明显受损。细胞移植后,OEC、NSC移植组均分别能测到N1波,其与SCI组相比较,有显著性统计学差异(P=0.001,≤0.001、P=0.045,＜0.05)。但OEC+NSC移植组分别与OEC组和NSC组比较,均无统计学差异(P=0.720、0.323,＞0.05)。说明OEC+NSC移植对感觉神经生理功能恢复改善不明显。P1波变化:脊髓全横断组亦没有测到波形,说明潜伏期无限延长,与假手术组比较差异显著;然而OEC、NSC移植组能测到P1波,其与SCI组完全消失的情况相比较,差异显著,P=0.001,≤0.001;然而,需注意的是,仅OEC移植组P1波的潜伏期较与假手术组比较明显延长,有统计学差异;而NSC移植组P1波的潜伏期与SCI组相比较,P=0.069,＞0.05,无统计学差异;OEC+NSC移植组P1波的潜伏期与OEC组和NSC组的相比较,P值分别为0.688、0.247,＞0.05,无统计学差异。进一步说明OEC+NSC移植对感觉神经生理功能恢复改善不明显。3.2 MEP检测:本实验中,假手术大鼠均能测到明显的诱发电位,这种诱发电位的波幅为均值在6mv左右,潜伏期5ms左右。而SCI后,波幅和潜伏期均测不到,MEP消失。但给予NSC或者OEC移植后,均能测到MEP。只是其潜伏期有些延长(说明传导速度减慢),且恢复波幅明显降低(说明突触功能受损)。细胞联合治疗对MEP没有明显影响。4.植入细胞的存活:脊髓切片上可见到GFP标记的绿色荧光细胞。而联合移植组脊髓既可见绿色荧光细胞,又可见Hochest33342标记的细胞核。标记细胞在损伤脊髓瘢痕,及其上段、下端均可见到。说明移植细胞向头、尾侧迁移,并在宿主脊髓存活。5.PCR扩增疤痕组织的神经营养因子及其受体,和凋亡相关因子的表达变化发现:①在疤痕组织中均未检测到BDNF、NT-3、bFGF、TrkA、TrkB表达;②NGF各时间点均没有变化。③凋亡基因变化:Bcl-2:在联合移植21d时与NSC移植组比较明显降低,P=0.011,＜0.05,差异有统计学意义;Caspas-3:联合移植组在14d与NSC移植组、OEC移植组相比较,明显增加(P=0.039、0.032,＜0.05),差异有显著统计学意义;但至在21d时,联合移植组与NSC移植组比较Caspas-3表达明显减P=0.006,＜0.01,差异有显著统计学意义;在28d时,各处理方式之间比较,差异无统计学意义。Bax在联合移植组14d时与NSC移植比较,明显增加,P=0.031,＜0.05,差异有统计学意义;但至21d时,联合移植组与手术组比较明显减少,P=0.040,＜0.05,差异有统计学意义;在28d时,各处理方式之间比较,差异无统计学意义。④NTF变化:CNTF在联合移植14d时,与OEC移植组及NSC移植比较表达明显增加,P值分别为0.007、0.001、0.023,均＜0.05,差异有显著统计学意义;在21d,联合移植组与NSC移植组比较明显减少,P值为0.000,＜0.01,差异有显著统计学意义;在28d,联合移植组与OEC移植组、NSC移植组比较均明显减少,P值分别为0.028、0.028,均＜0.05,差异有显著统计学意义。IGF-1:在14d时,联合移植组与OEC移植组及NSC移植比较,明显增加,尸值分别为0.003、0.001,均＜0.05,差异有显著性统计学意义;而在21d和28d时,各处理方式之间比较,差异均无统计学意义;PDGF-B:在14d时,联合移植组、OEC、NSC移植与手术组比较,明显减少,P=0.044,＜0.05,差异有统计学意义;联合移植组与NSC移植比较,明显减少,P=0.047,＜0.05,差异有统计学意义。在21d时,联合移植组与NSC移植组、OEC移植组及手术组比较明显减少,P=0.039、0.039、0.031,＜0.05,差异有统计学意义。在28d时,联合移植组与OEC移植组之间比较显著减少,P=0.011,＜0.05,差异有统计学意义。TGF-betal:在14d时,联合移植组与NSC移植组、OEC移植组和手术组比较明显增加,p=0.001、0.019、0.001,＜0.05,差异有统计学意义;而在21d和28d时,各处理方式之间比较,差异无统计学意义。Trkc:在14d时,联合移植组与NSC移植组比较,明显降低,P值分别为0.031,＜0.05,差异有统计学意义;在21时,联合移植组与手术组比较(P=0.015,＜0.05),和NSC移植组比较(P=0.010,＜0.05)。差异有统计学意义,而在28d时,各处理方式之间比较,差异无统计学意义。可以看出,PDGF-B是细胞移植促进损伤脊髓修复中变化最明显的分子,提示其可能是细胞移植改进功能可塑性相关的重要分子。6.OEC+NSC移植对恒河猴SCI功能恢复的影响6.1后肢运动功能评分:改良的Tarlov’s Method评分结果显示损伤侧、细胞联合移植侧评分均较假手术组明显降低,有统计学差异(P＜0.05)。第1周、第2周、第3周、第4周、第7周,OEE+NSE移植细胞联合移植侧Tarlov’s Method评分均明显高于损伤侧评分,均有统计学意义(P＜0.05)。6.2移植细胞在宿主脊髓存活和迁移情况:在宿主脊髓可观察到大量荧光细胞,向头端或尾端迁徙;说明移植细胞在宿主脊髓存活,迁移。6.3神经电生理:CSEP:SCI后各组动物的CSEP信号均消失。损伤侧N1波波幅明显低于损伤对侧,有统计学差异(P＜0.05);而NSC联合OEC细胞移植1个月,细胞联合移植侧N1波幅明显高于损伤侧,有统计学意义(P＜0.05);MEP:SCI后各动物MEP信号均消失。而细胞移植1个月能检测到MEP,只是损伤侧损伤平面以下节段MEP的波幅明显低于损伤侧损伤平面以上节段的MEP波幅,有统计学差异(P＜0.05);而损伤侧与细胞联合移植侧测得的波幅相比较没有明显差异,没有统计学意义(P＞0.05)。比较之,损伤侧损伤平面以上节段测得的潜伏期较损伤侧损伤平面以下节段测得波幅明显延长,有统计学差异(P＜0.05);而细胞联合移植测得的潜伏期较损伤侧测得的潜伏期明显缩短,有统计学差异(P＜0.05)。6.4 MRI:脊髓左侧半横断+细胞移植瘢痕横切面积明显小于单纯脊髓左侧半横断组脊髓损伤对照节段面积,有统计学差异(P＜0.05);而脊髓损伤上1节段和损伤下1阶段脊髓横切面面积无明显差异,均没有统计学差异(P＞0.05)。7.PDGF-B在大鼠脊髓全横断损伤后的变化和作用研究:SCT后1天直至损伤后28天,PDGF-B蛋白质和mRNA在损伤脊髓中明显上调。PDGF-B免疫反应的产物分布在疤痕星型胶质细胞中。同时星型胶质细胞明显增值,疤痕组织内的PDGF-B信号分子转录激活因子3(STAT-3)的上调相一致。通过PDGB-B抗体封闭和iRNA对PDGF-B活性分别在蛋白水平和基因水平进行阻断和干扰,导致以下几方面的显著改变:(1)显著下调了PDGF-B下游信号分子的水平;(2)显著减少了星形细胞胶质增生和疤痕的形成;(3)增加轴突再生和出芽;(4)后肢运动功能和感觉功能的显著改善。【结论】1本实验通过不同的细胞组合治疗方法,在低等啮齿类动物SD大鼠和高等灵长类动物恒河猴SCI模型上证明了NSC联合OEC移植能够最有效促进损伤脊髓的运动功能恢复。是目前较为乐观的有效策略。移植细胞能够在大鼠和猴损伤脊髓长时间存活、迁徙。NSC联合OEC移植作为一种治疗SCI的有效方法,具有潜在的临床应用价值。2 NSC联合OEC移植有效促进损伤脊髓的运动功能恢复与多种细胞因子的表达调节有关。其中PDGF-B是较重要的分子。3 PDGF-B蛋白和基因干扰,能显著减少了星形胶质增生,抑制疤痕形成,促进轴突再生,并改善后肢运动功能。从而证明PDGF在SCI修复中是一个关键的调节分子。更多还原

【Abstract】 [Objective]In present study, based on a great deal of reports and effective strategies of clinical therapy of SCI, we used different supporting cells (SC and OEC) to combined with different stem cells like neural stem cells(NSC), mensenchymal stem cells(BMSC) and heomopoietic stem cells(HSC) to seek for the optimal method which promotes SCI restoration, by means of co-transplantation of different supporting cells and different stem cells based on the theory that supporting cells can play a role in bridge grafting and NTF secretion and stem cells can differentiate into target cells so as to replace of host lost neurons. Our objective is to get the most effective methods for the SCI therapy by cellular coordination. Then we ask the possible mechnism.[Method]Three Stem cells consisting of neural stem cells(NSC), mensenchymal stem cells(BMSC) and heomopoietic stem cells(HSC) as well as two support cells including olfactory ensheathing cell (OEC) , and Schwann cells (SC) were as seedcells to be impanted into spinal cord following injury. The the most Optimal strategyof stem cell transplantation into transected spinal cord, and the role of PDGF were investigated by using cells culture, Immunohistochemistry (IHC), Western-blot, Reverse Transcription polymerase Chain Reaction (RT-PCR), RNA intererence and antibody block method, and Behavioral test as well as electrophysiological test. [Result]1. Aafter spinal cord was transected, rats showed flaccid paralysis in hindlimbs immediately, indicating that the transection of the cord was completely transected. Moreover, cell transplantation results in spontaneous, howbeit incomplete, recovery of the hindlimb locomotor functions after spinal cord transection (SCT). The BBB score of cell transplantaion group was significant higher than operation group at all of the different time points (P<0.05), especially from 2 wekks to 6 month. Of 11 combination strategy, we found that NSC and OEC co-transplantation could provide the most significant amelioration for the recovery of rats behavior following SCT. This effect was confirmed from the test employed by monkey SCI model. The mechanism for this improvement is involved in regulation of various NTF in host spinal cord. Involved factors by RT-PCR including in brain derived neurotrophic factor (BDNF), Cilliary neurotrophic factor(CNTF), Isulin-like growth factor, (IGF), Fibroblast growth factor(FGF), Glia derived neurotrophic factor(GDNF), etc.2. The optimal strategy of stem cell transplantation into transected spinal cord was test in primate monkey so as to provide some evidences fo humanbeing usage.3. Based on the results from previous data that quantitative analysis demonstrated that the Pletlet derived neurotrophic factors (PDGF) was downregulated in the spinal cord following NSC and OEC transplantaion (P<0.05). This indicated that PDGF maybe a vital molecules that exert negtive effect in improving neuroplasticity. This push us to determine the role of PDGF in the traumatic spinal cord. Despite PDGF-BB plays a crucial role in regulating neuronal survival and cell differentiation during embryonic development and in the adulthood. The roles it plays in astrogliosis and the formation of scar tissue after spinal cord transection (SCT), however, has not been studied. Thereforem This part we explore the roles of PDGF-BB in regulating astrogliosis at molecular level and to correlate them with axonal regeneration and sprouting and functional recovery. As early as 1 day after SCT and continuing through 28days, PDGF-BB protein and mRNA were up-regulated in the injured spinal cord. PDGF-BB immunoreactive products were detected in both neurons and astrocytes, at 1 day after SCT and these gradually increased in astrocytes till 28days. This was paralleled by scar tissue formation, and upregulation of signal transducer and activator of transcription 3 (STAT-3), all of which are the downstream signaling molecules of PDGF-BB. Blocking of and interfering with the activity of PDGF-BB with PDGB-BB-antibody and siRNA (to spell out in full) respectively resulted in significant (i) down-regulation in the downstream signaling molecules of PDGF-BB; (ii) reduction in astrogliosis and scar tissue formation; (iii) reduction of STAT-3; and (iii) improvements in hindlimb locomotor and sensory functions.Taken together, our data suggest that OEC and NSC transplantaion could be as a optimal strategy into damaged spinal cord for future clinic usage, and the possible mechnism involved in several cytokines regulation. Of them, PDGF play a crucial role in neuroplasticity following spinal cord injury and cell transplantion.更多还原