同种异体骨髓间充质干细胞移植治疗大鼠脊髓损伤的抗凋亡及慢性应激机理
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摘要
一、同种异体大鼠骨髓间充质干细胞共培养及多向分化潜能鉴定
目的探讨同种异体大鼠骨髓间充质干细胞在体外的共培养,并行多向分化诱导鉴定。
方法分别通过全骨髓直接培养法及密度梯度法分离培养大鼠骨髓间充质干细胞,各传至第3代后,取等量细胞,混合后共培养,共培养之细胞再次传代后,Wright-Gemsa染色后观察其一般形态,以流式细胞仪行CD29、CD45、CD90表面标志物鉴定,并分别进行成骨细胞、成脂肪细胞诱导分化,分别以碱性磷酸酶、Von Cossa及油红染色鉴定。
结果全骨髓直接培养法原代细胞形态不均一,但是增殖能力好;密度梯度法分离得到的细胞较为均一,但增殖力不如全骨髓直接培养法;经过3代传代后,细胞均可获得相对纯化。共培养细胞增殖力较好,细胞形态较为均一,且未出现因免疫排斥导致的细胞死亡。Wright-Gemsa染色结果显示共培养细胞呈典型梭形。流式细胞仪检测显示共培养细胞阳性表达CD29、CD90,阴性表达CD45。共培养之大鼠骨髓间充质干细胞经诱导分化后分别出现成骨细胞和脂肪细胞形态,经碱性磷酸酶、VonCossa染色证实为成骨细胞,经油红染色证实为脂肪细胞。
结论采用全骨髓直接培养及密度梯度离心法,经3～5代培养后均可获得相对纯化之骨髓间充质干细胞。同种异体大鼠骨髓间充质干细胞共培养生长良好,不发生免疫排斥,证实该类细胞的极低免疫原性。在体外,共培养之大鼠骨髓间充质干细胞可诱导分化为成骨细胞、脂肪细胞,表明其具有多向分化潜能。
二、同种异体大鼠骨髓间充质干细胞的神经分化诱导鉴定
目的探讨同种异体大鼠骨髓间充质干细胞在体外的共培养,并行神经分化诱导鉴定。
方法分别通过全骨髓直接培养法及密度梯度法分离培养大鼠骨髓间充质干细胞,各传至第3代后,取等量细胞,混合后共培养,共培养之细胞再次传代后,进行神经方向诱导,并行Nestin,NSE,GFAP免疫细胞化学染色鉴定。
结果共培养之大鼠骨髓间充质干细胞经诱导分化后出现神经元和胶质细胞形态,经Nestin,NSE,GFAP免疫细胞化学染色证实为神经细胞。
结论同种异体共培养之骨髓间充质干细胞可在体外定向诱导为神经元和胶质细胞,提示移植入体内后,在适当环境下可分化为神经细胞。
三、脊髓损伤大鼠中同种异体骨髓间充质干细胞经脑脊液向损伤区的迁移
目的研究自脊髓损伤大鼠腰椎水平注射入蛛网膜下腔之同种异体骨髓间充质干细胞经脑脊液向损伤区的迁移。
方法分别通过全骨髓直接培养法及密度梯度法分离培养大鼠骨髓间充质干细胞,各传至第3代后,取等量细胞,混合后共培养,共培养之细胞再次传代后,以腺病毒转染EGFP基因。取成年SD大鼠20只,随机分为3组:对照组4只;模型组8只,治疗组8只。模型组与治疗组以改良Allen法复制大鼠下胸段脊髓损伤模型,对照组仅咬除相应节段棘突及椎板,不干预脊髓。造模后7天,于腰椎水平(L4-L5间隙)蛛网膜下腔向对照组及治疗组注射经EGFP标记之BMSCs,模型组注射同体积的Hank's缓冲液。观察各组动物造模前后及移植前后后肢运动功能,以BBB评分对其进行评估,各组分别于移植后7天、14天随机选取一半动物处死取损伤节段为中心脊髓,纵向切开脊髓后,一半行冰冻切片,于荧光显微镜下观察移植细胞向脊髓损伤区的迁移情况;另一半石蜡包埋后行HE染色,于光学显微镜下观察脊髓损伤区的细胞聚集情况。
结果三组动物造模术前BBB评分均为21分(满分)。术后第一天,对照组动物BBB评分恢复至21分。模型组和治疗组动物造模术后第3、7天BBB评分均低于对照组,有极显著性差异(P＜0.01),但两组间比较无显著性差异(P＞0.05)。两组动物自主排尿功能均在造模术后3天内恢复。BMSCs以Ad5F35-EGFP腺病毒转染后24h,荧光显微镜下即可见少量细胞表达绿色荧光。48h后,荧光显微镜下见大量细胞表达绿色荧光。经蛛网膜下腔注射EGFP标记之BMSCs后,治疗组动物从移植术后第1天开始,BBB评分持续增加。模型组动物BBB评分变化不大。移植术后第7天,模型组与治疗组动物BBB评分分别恢复至6.00±1.60和8.13±1.96分,与对照组相比均有极显著性差异(P＜0.01),且两组间比较亦有显著性差异(P＜0.05)。荧光显微镜下观察,EGFP标记之BMSCs移植后7天、14天,对照组与治疗组切片均见绿色荧光表达,模型组未见荧光表达。脊髓组织HE染色结果显示:移植后7天、14天,对照组切片脊髓结构正常;治疗组切片脊髓损伤区可见空洞,在脊髓面向蛛网膜下腔一面见大量细胞聚集;模型组切片脊髓损伤区见较大空洞,未见明显细胞聚集。
结论由腰椎水平蛛网膜下腔注入之EGFP标记BMSCs可经脑脊液向大鼠下胸段脊髓损伤区迁移,并改善脊髓损伤大鼠后肢运动功能。从临床角度看,如采用BMSCs治疗脊髓损伤,经腰椎穿刺注射是一种更简单、微创的术式。
四、同种异体骨髓间充质干细胞移植对脊髓损伤大鼠脊髓神经细胞凋亡的影响
目的探讨骨髓间充质干细胞移植治疗大鼠脊髓损伤的潜在抗凋亡机制。
方法6周龄雄性SD大鼠8只,用于BMSCs分离培养。成年雄性SD大鼠84只,根据移植术后取材时间点随机分为6组,每组14只:A1.对照组,14天:A2.对照组,28天;B1.模型组,14天;B2.模型组,28天;C1.治疗组,14天;C2.治疗组,28天。模型组与治疗组以改良Allen法复制大鼠下胸段脊髓损伤模型,对照组仅咬除相应节段棘突及椎板,不干预脊髓。造模后7天,于腰椎水平(L4-L5间隙)蛛网膜下腔向对照组及治疗组注射BMSCs,模型组注射同体积的Hank's缓冲液。观察各组动物造模前后及移植前后后肢运动功能,以BBB评分对其进行评估。按组别分别于移植后第14天、第28天处死动物,其中每组8只以损伤节段或相应节段为中心灌注后取脊髓组织,石蜡包埋后行HE染色,原位末端脱氧核苷酸转移酶(TdT)介导的脱氧尿苷三磷酸(d-UTP)缺口末端标记技术(TUNEL)染色,Bcl-2、Bax免疫组织化学染色;5只以损伤节段或相应节段为中心直接取脊髓组织,提取RNA后,行RT-PCR,检测Bcl-2、Bax基因的表达:1只于损伤中心或相应区域直接取1mm×1mm×1mm脊髓组织块,行透射电子显微镜观察。
结果三组动物造模术前BBB评分均为21分(满分)。术后第一天,对照组动物BBB评分恢复至21分。模型组和治疗组动物造模术后第3、7天BBB评分均低于对照组,有极显著性差异(P＜0.01),但两组间比较无显著性差异(P＞0.05)。两组动物自主排尿功能均在造模术后3天内恢复。BMSCs移植后,治疗组动物BBB评分持续增加,但仍低于对照组,有极显著性差异(P＜0.01),模型组动物BBB评分变化不大。从移植术后第7天开始,治疗组动物BBB评分均高于模型组,有极显著性差异(P＜0.01)。脊髓组织HE染色结果表明:移植后14天、28天,对照组切片脊髓结构正常。模型组和治疗组切片均可见脊髓损伤空洞,模型组更为明显。透射电镜观察显示,移植术后14天、28天,对照组凋亡细胞均较少,未见明显坏死细胞。模型组可见大量凋亡细胞及坏死细胞。治疗组凋亡坏死细胞均较模型组少。移植后14天,模型组TUNEL阳性细胞数量多于对照组和治疗组,并有显著性差异(P＜0.01),阳性细胞主要分布于白质中。治疗组亦高于对照组,有显著性差异(P＜0.01)。移植后28天,各组TUNEL阳性细胞数量均减少,模型组仍然高于对照组和治疗组,但无显著性差异。移植后14天,模型组Bax阳性细胞表达多于对照组和治疗组,且有显著性差异(P＜0.01),治疗组Bax阳性细胞表达多于对照组,亦有显著性差异(P＜0.01)。而模型组Bcl-2阳性细胞表达少于对照组和治疗组,但无显著性差异。移植后28天,Bax阳性细胞表达减少,模型组仍然高于对照组和治疗组,但无显著性差异。Bcl-2阳性细胞表达变化不大。RT-PCR结果表明,移植后14天、28天,模型组Bax基因表达均强于对照组和治疗组,而Bcl-2基因表达弱于对照组和治疗组,但其光密度比较均无显著性差异。
结论经蛛网膜下腔注射BMSCs可改善大鼠下胸段脊髓损伤模型的后肢运动功能,同时减少神经细胞的凋亡,降低Bax基因及其蛋白的表达,增加Bcl-2基因及其蛋白的表达。提示BMSCs移植治疗大鼠脊髓损伤具有潜在抗凋亡作用,这一作用很有可能是通过对凋亡调控基因Bax和Bcl-2的影响而实现的。
五、同种异体骨髓间充质干细胞移植对脊髓损伤大鼠应激状态及中枢AMPA受体蛋白表达的影响
目的探讨同种异体骨髓间充质干细胞移植对脊髓损伤大鼠应激状态及海马和杏仁核AMPA受体蛋白表达的影响。
方法成年雄性SD大鼠48只,根据移植术后取材时间点随机分为6组,每组8只:A1.对照组,14天;A2.对照组,28天;B1.模型组,14天;B2.模型组,28天;C1.治疗组,14天;C2.治疗组,28天。模型组与治疗组以改良Allen法复制大鼠下胸段脊髓损伤模型,对照组仅咬除相应节段棘突及椎板,不干预脊髓。造模后7天,于腰椎水平(L4-L5间隙)蛛网膜下腔向对照组及治疗组注射BMSCs,模型组注射同体积的Hank's缓冲液。观察各组动物造模前后及移植前后后肢运动功能,以BBB评分对其进行评估,并记录同时段动物体重。按组别分别于移植后第14天、第28天处死动物,取血,以酶联免疫吸附测定法(ELISA)测定血浆ACTH和血清皮质酮含量;灌注后取脑,冰冻切片后行GluR1、GluR2免疫组织化学染色。
结果三组动物造模术前BBB评分均为21分(满分)。术后第一天,对照组动物BBB评分恢复至21分。模型组和治疗组动物造模术后第3、7天BBB评分均低于对照组,有极显著性差异(P＜0.01),但两组间比较无显著性差异(P＞0.05)。两组动物自主排尿功能均在造模术后3天内恢复。BMSCs移植后,治疗组动物BBB评分持续增加,但仍低于对照组,有极显著性差异(P＜0.01),模型组动物BBB评分变化不大。从移植术后第7天开始,治疗组动物BBB评分均高于模型组,有极显著性差异(P＜0.01)。由于手术的干扰,三组动物在造模手术后体重均下降,术后逐渐恢复,至移植前,对照组体重高于模型组和治疗组,但无显著性差异(P＞0.05)。同样由于手术的干扰,各组动物体重在移植手术后均有明显降低。之后各组动物体重呈增加趋势,对照组体重增加快于同时段模型组和治疗组,从移植术后3天开始,差异有显著性意义(P＜0.05或P＜0.01)。从移植术后7天开始,各时段治疗组体重均高于模型组,但无显著性差异(P＞0.05)。移植后14天(即脊髓损伤后21天),模型组血浆ACTH较对照组和治疗组高,皮质酮比正常组要低。移植后28天(即脊髓损伤后35天),模型组ACTH下降,接近正常值,皮质酮持续升高。移植术后14天,GluR1阳性细胞数,模型组和治疗组在CA1区均高于对照组,均有显著性差异(P＜0.05或P＜0.01),在其他区无显著性差异;GluR2阳性细胞数有类似趋势,但均无显著性差异。移植术后28天,GluR1阳性细胞数,模型组在CA1、CA3、DG区均高于对照组,在CA1、CA3区亦高于治疗组,且均有显著性差异(P＜0.05或P＜0.01),模型组和治疗组在DG区均高于各自移植后14天阳性细胞数,均有显著性差异(P＜0.05或P＜0.01);GluR2阳性细胞数,治疗组在BLA区高于对照组,且有显著性差异(P＜0.05),在其他区域各组虽表现出与GluR1表达类似的趋势,但均无显著性差异。
结论BMSCs移植可改善大鼠下胸段脊髓损伤模型的后肢运动功能,同时可缓解脊髓损伤大鼠的慢性应激状态,其可能的机制为通过对AMPA受体蛋白GluR1及GluR2表达的影响而实现。
Part I : Co-culture and multilineage differentiation potentialcharacterization of allogenic rat bone mesenchymal stem cells
Objective To investigate the Co-culture of allogenic SD rat bone mesenchymal stem cells (BMSCs) in vitro, and to perform multilineage differentiation potential characterization.
Methods Rat BMSCs were isolated and cultured by complete marrow direct culture method and density gradient method respectively, and the 3rd passage cells were harvested and co-cultured with same concentration. The passage cells of the co-cultured cells were obtained and Wright-Gemsa staining were performed for morphological observation; and to identify the surface marker of CD29, CD45, CD90 by flow cytometry; moreover, the passage cells were induced to differentiate into osteoblast and adipocyte respectively, then performed alkaline phosphatase, Von Cossa and oil red staining characterization.
Results The primary cells of complete marrow direct culture method are uneven in shape, but with excellent reproductive activity; the cells obtained by density gradient method are fairly uniform in shape, but with lower reproductive activity; after 3 passages, the cells obtained by these two methods both got relative purification. The co-cultured cells were good in reproductive activity and relatively uniform in shape, without cell death because of immunologic rejection. After Wright-Gemsa staining, the cells displayed typical spindle-shaped cells; and the co-cultured cells were positive for CD29 and CD90, but negative for CD45 by flow cytometry. After induction, the co-cultured cells appeared morphological changes of osteoblast and adipocyte, and were confirmed be osteoblast by alkaline phosphatase staining and Von Cossa staining; be adipocyte by oil red staining.
Conclusion By use complete marrow direct culture method and density gradient method, after 3～5 passages, can obtain relative purified BMSCs. The co-cultured allogenic BMSCs were good in growth, without immunologic rejection, imply the weak immunogenicity of BMSCs. In vitro, the co-cultured rat BMSCs can be induced osteoblast and adipocyte, indicate the multilineage differentiation potential of BMSCs.
Part II: Neural differentiation potential characterization of allogenicrat bone mesenchymal stem cells
Objective To investigate the Co-culture of allogenic SD rat bone mesenchemal stem cells (BMSCs) in vitro, and to perform neural differentiation potential characterization.
Methods Rat BMSCs were isolated and cultured by complete marrow direct culture method and density gradient method respectively, and the 3rd passage cells were harvested and co-cultured with same concentration. The passage cells of the co-cultured cells were obtained and induced to differentiate into neurocyte, then performed immunocytochemistry of Nestin, NSE, GFAP.
Results After induction, the co-cultured cells appeared morphological changes of neuron and glial cell, and were confirmed by Nestin, NSE, GFAP immunostaining.
Conclusion The co-cultured allogenic BMSCs can be induced into neuron and glial cell in vitro, indicate that in appropriate circumstance, BMSCs can be induced into neurocyte in vivo.
Part III: Migration of allogenic rat bone mesenchymal stem cells toinjured sites through cerebrospinal fluid in rat with spinal cord injury
Objective To investigate the migration of allogenic rat bone mesenchymal stem cells to injured sites through cerebrospinal fluid in rat with spinal cord injury.
Methods Rat BMSCs were isolated and cultured by complete marrow direct culture method and density gradient method respectively, and the 3rd passage cells were harvested and co-cultured with same concentration. The passage cells of the co-cultured cells were labeled by transfection of recombinant adenovirus containing enhanced green fluorescence protein (EGFP) gene (Ad5F35-EGFP). A total of 20 adult male Sprague-Dawley (SD) rats weight 222-289 g were divided into 3 groups: control group, n=4; model group, n=8; treatment group, n=8. The rats in model and treatment group were performed a partial low thoracic spinal cord injury (SCI) by modified Allen's method (weight drop method) at T10 under chloral hydrate anesthesia. Rats in ontrol group received only laminectomy, without spinal cord interference. At day 7 After thoracic SCI, the dura at L4-L5 intervertebral space was exposed with partial removal of the L5 spinous process and L4-L5 ligamentum flavum under chloral hydrate anesthesia; 100μl of Hank's buffered saline solution contained BMSCs (EGFP transfected) or the same amount of Hank's buffered saline solution was injected into the subarachnoid space by single shot with a tip-bent 25 G needle. Then observed the hind limb motor function of all animals, and evaluated by Basso-Beatie-Bresnahan (BBB) scale. Half of the rats in each group had spinal cord tissue harvested at day 7 after cell transplantation. The other were harvested at day 14 after cell transplantation. The spinal cord segments containing the injured sites was removed from the spinal column after perfusion. Spinal cords were postfixed in 4% paraformaldehyde overnight and half-cut longitudinally in the sagittal direction. One half of the spinal cord was transferred to 30% sucrose solution, frozen, and cut in a cryostat at 10μm thickness for the examination of migration of transplanted cells to injured sites under fluorescent microscopy. The other half of the spinal cord was paraffin-embedded and sectioned in the same size as above for hematoxylin and eosin staining for the examination of cell aggregation in injury sites under optical microscope.
Results Before surgery, all animals' BBB scale score was 21 (the maximum value). At day 1 after surgery, the animals of control group reached 21 BBB scale score. Three, seven days after surgery, BBB scale score of the animals in model group and treatment group was lower than control, with significant difference (P<0.01), but without significant difference (P>0.05) between this two groups. All animals' urinary function recovered within 3 days after surgery. 24h after transfection through Ad5F35-EGFP adenovirus, some BMSCs expressed GFP under fluorescent microscope, 48h after transfection, a great many BMSCs expressed GFP. After transplantation of EGFP-BMSCs following a subarachnoid injection, BBB scale score of the animals in treatment group increased progressively. 7 days after grafting procedure, the animals of model group and treatment group reached a mean value of 6.00±1.60 and 8.13±1.96 in the BBB scale respectively, still lower than control group, with significant difference (P<0.01), and treatment group's BBB score higher than those in model group, with significant difference (P<0.05). Under fluorescent microscope, both of control group and treatment group animals' sections expressed GFP 7 and 14 days after grafting procedure, but the GFP expression was negative in model group. HE staining showed that 7 and 14 days after grafting procedure, sections of control group exhibited normal neural morphological change, while huge cell aggregations were observed on the surface of the spinal cord facing the subarachnoid space in treatment group, cell aggregations could not be seen in the model group.
Conclusion EGFP-BMSCs following a subarachnoid injection could migrate from a lumbar site into the injured low thoracic spinal cord through the cerebrospinal fluid, and could improve the hind limb motor function of SCI rats. This would potentially establish a simpler, less invasive procedure for BMSCs transplantation to spinal cord injury by lumbar puncture in clinical application.
Part IV: The influence of allogenic bone mesenchymal stem cellstransplantation to the neurocyte apoptosis of injured rat spinal cord.
Objective To investigate a potential anti-apoptotic mechanism of transplanted BMSCs for rat SCI.
Methods A total of & male SD rats weight 90-102 g were used for BMSCs harvest, and a total of 84 adult male SD rats weight 235-319 g were divided into 6 groups depending on the timing of the lumbar injection after thoracic SCI, n=14 in every group: Group A1, control group, day 14 post; Group A2, control group, day 28 post; Group B1, model group, day 14 post; Group B2, model group, day 28 post; Group C1, treatment group, day 14 post; Group C2, treatment group, day 28 post. The rats in model and treatment group were performed a partial low thoracic spinal cord injury (SCI) by modified Allen's method at T10 under chloral hydrate anesthesia. Rats in ontrol group received only laminectomy, without spinal cord interference. At day 7 After thoracic SCI, the dura at L4-L5 intervertebral space was exposed with partial removal of the L5 spinous process and L4-L5 ligamentum flavum under chloral hydrate anesthesia; 100μl of Hank's buffered saline solution contained BMSCs or the same amount of Hank's buffered saline solution was injected into the subarachnoid space by single shot with a tip-bent 25 G needle. Then observed the hind limb motor function of all animals, and evaluated by BBB scale. Rats were anesthetized at day 14, 28 postoperatively according to the timing point of group. Every 8 rat spinal cord segments containing the injured sites or counterparts in each group were harvested after perfused with normal saline followed 4% paraformaldehyde intracardially. Spinal cords were postfixed in neutral formalin for 24-48h and were paraffin-embedded and sectioned at 4 urn thickness. Hematoxylin and eosin staining were performed for histological observation. Neurocyte apoptosis was examined by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling ( TUNEL) staining, the expression of Bax and Bc1-2 protein were tested with immunohistochemistry. 5 rat spinal cord segments containing the injured sites or counterparts in each group were harvested directly, to investigate the mRNA expression of Bax and Bcl-2 by reverse transcriptase -polymerase chain reaction (RT-PCR). 1 rat spinal cord segments containing the injured sites or counterparts in each group were harvested directly, a 4 1mm×1mm×1mm spinal tissue were cut from the center of injured sites, for ultramicrostructural observation under transmission electron microscope (TEM).
Results Before surgery, all animals' BBB scale score was 21 (the maximum value). At day 1 after surgery, the animals of control group reached 21 BBB scale score. Three, seven days after surgery, BBB scale score of the animals in model group and treatment group was lower than control, with significant difference (P<0.01), but without significant difference (P>0.05) between this two groups. All animals' urinary function recovered within 3 days after surgery. After transplantation of BMSCs, BBB scale score of the animals in treatment group increased progressively, but still lower than control group, with significant difference (P<0.01), and treatment group's BBB score higher than those in model group from 7 day after grafting procedure, with significant difference (P<0.01). HE staining showed that 14 and 28 days after grafting procedure, sections of control group exhibited normal neural morphological change, while cavities of SCI were observed in those of the model and treatment group, the model group was more significant than treatment group. Under TEM, 14 and 28 days after grafting procedure, sections of control group exhibited few apoptotic neurocyte, while in the sections of model and treatment group, a lot of apoptotic and necrotic neurocytes were observed, the model group was more significant than treatment group. The TUNEL staining showed that the TUNEL positive cells in model group were higher than those in control group and treatment group at 14 day after injection, with significant difference (P < 0.01), and positive cells in treatment group were higher than those in control group as well, with significant difference (P<0.01), the TUNEL positive cells mainly distributed in white matter. The positive cells declined at 28 day after injection, model group were still higher than those in the other two groups, but without significant difference. The result of immunostaining against Bax and Bcl-2 demonstrated that model group showed higher Bax positive expression than those in the other two groups, with significant difference (P<0.01) at 14 day after injection, and Bax positive cells in treatment group were higher than those in control group as well, with significant difference (P<0.01), which on the contrary in Bcl-2 positive expression, but without significant difference. The Bax positive cells declined at 28 day after injection, model group were still higher than those in the other two groups, but without significant difference (P>0.05), the change of Bcl-2 positive expression was insignificant. The RT-PCR of Bax and Bcl-2 demonstrated that model group showed higher Bax gene positive expression than those in other two groups, but without significant difference (P>0.05) in relative optical density at 14 and 28 days after injection, which on the contrary in Bcl-2 positive expression, without significant difference (P>0.05) either.
Conclusion Transplantation of BMSCs by a subarachnoid injection could improve the hind limb motor function of low thoracic SCI rats, and could reduce spinal neurocyte apoptosis, decrease the genic and proteinic expression of Bax, increase the genic and proteinic expression of Bcl-2, which implied a potential anti-apoptotic mechanism of transplanted BMSCs for rat SCI, the anti-apoptotic effect maybe achieved by the effect to apoptotic controlling gene Bax and Bcl-2.
Part V: The influence of allogenic bone mesenchymal stem cells transplantation to the stress state and AMFA receptor protein expressionof brain in rat with spinal cord injury.
Objective To investigate the effect of allogenic bone mesenchymal stem cells transplantation to the stress state and AMPA receptors protein expression of hippocampus and basolateral amygdale (BLA) in rat with SCI.
Methods A total of 48 adult male SD rats weight 250-319 g were divided into 6 groups depending on the timing of the lumbar injection after thoracic SCI, n=8 in every group: Group A1, control group, day 14 post; Group A2, control group, day 28 post; Group B1, model group, day 14 post; Group B2, model group, day 28 post; Group C1, treatment group, day 14 post; Group C2, treatment group, day 28 post. The rats in model and treatment group were performed a partial low thoracic SCI by modified Allen's method at T10 under chloral hydrate anesthesia. Rats in ontrol group received only laminectomy, without spinal cord interference. At day 7 After thoracic SCI, the dura at L4-L5 intervertebral space was exposed with partial removal of the L5 spinous process and L4-L5 ligamentum flavum under chloral hydrate anesthesia; 100μl of Hank's buffered saline solution contained BMSCs or the same amount of Hank's buffered saline solution was injected into the subarachnoid space by single shot with a tip-bent 25 G needle. Then observed the hind limb motor function of all animals, and evaluated by BBB scale. Rats were anesthetized at day 14, 28 postoperatively according to the timing point of group, to obtain blood for detection of plasmic ACTH and serumal corticosterone concentration by enzyme linked immunosorbent assay (ELISA), and to harvest brains after perfused with normal saline followed 4% paraformaldehyde intracardially, brains were postfixed in 4% paraformaldehyde overnight and were transferred to 30% sucrose solution, frozen, and cut in a cryostat at 30μm thickness for the immunohistochemistry staining of GluR1 and GluR2.
Results Before surgery, all animals' BBB scale score was 21 (the maximum value). At day 1 after surgery, the animals of control group reached 21 BBB scale score. Three, seven days after surgery, BBB scale score of the animals in model group and treatment group was lower than control, with significant difference (P<0.01), but without significant difference (P>0.05) between this two groups. All animals' urinary function recovered within 3 days after surgery. After transplantation of BMSCs, BBB scale score of the animals in treatment group increased progressively, but still lower than control group, with significant difference (P<0.01), and treatment group's BBB score higher than those in model group from 7 day after grafting procedure, with significant difference (P<0.01). After low thoracic SCI and sham operation, all animals' body weight decreased because of operation interference, but increased gradually from then on. At timing point of day 7 (pre-grafting procedure), animals' body weight in the control group were higher than those in the model and treatment group, but without significant difference (P>0.05). After grafting procedure, all animals' body weight decreased again due to operation interference as before. Then all animals' body weight increased gradually. Animals' body weight in the control group were higher than those in the model and treatment group, and with significant difference (P<0.05或P<0.01) from 3 days after grafting procedure. From 7 days after grafting procedure, animals' body weight in the treatment group were higher than those in the model group, but without significant difference (P>0.05). At the timing point of 14 days after grafting procedure (21 days after SCI), the plasmic ACTH concentrations in the model group were higher than those in the other groups, which were on the contrary in the concentrations of serumal corticosterone. At the timing point of 28 days after grafting procedure (35 days after SCI), the plasmic ACTH concentrations in the model group decreased and near to normal level, but at the same time, the concentrations of serumal corticosterone increased. At the timing point of 14 days after grafting procedure, GluR1 positive cells of the model group increased in CA1, CA3, DG regions of hippocampus and BLA, with significant difference in CA1 (P<0.01); GluR2 positive cells had similar tendency, but without significant difference. At the timing point of 28 days after grafting procedure, GluR1 positive cells of the model group were higher than those of the control group in CA1, CA3, DG regions and of the treatment group in CA1, CA3 regions of hippocampus, with significant difference (P<0.05 or P<0.01, respectively); GluR1 positive cells of the model and treatment group were higher than their counterpart at the timing point of 14 days after grafting procedure, with significant difference (P<0.05 or P<0.01, respectively); GluR2 positive cells of the treatment group were higher than those of the control group in BLA, with significant difference (P<0.05); GluR2 positive cells had similar tendency in the other regions, but without significant difference.
Conclusions Transplantation of BMSCs could improve the hind limb motor function of low thoracic SCI rats, and could relieve the stress state of SCI rats, which implied a potential anti-chronic stress mechanism of transplanted BMSCs for rat SCI, the anti-chronic stress effect maybe achieved by influence to the expression of AMPA receptor protein of GluR1 and GluR2.
目的探讨同种异体大鼠骨髓间充质干细胞在体外的共培养,并行多向分化诱导鉴定。
方法分别通过全骨髓直接培养法及密度梯度法分离培养大鼠骨髓间充质干细胞,各传至第3代后,取等量细胞,混合后共培养,共培养之细胞再次传代后,Wright-Gemsa染色后观察其一般形态,以流式细胞仪行CD29、CD45、CD90表面标志物鉴定,并分别进行成骨细胞、成脂肪细胞诱导分化,分别以碱性磷酸酶、Von Cossa及油红染色鉴定。
结果全骨髓直接培养法原代细胞形态不均一,但是增殖能力好;密度梯度法分离得到的细胞较为均一,但增殖力不如全骨髓直接培养法;经过3代传代后,细胞均可获得相对纯化。共培养细胞增殖力较好,细胞形态较为均一,且未出现因免疫排斥导致的细胞死亡。Wright-Gemsa染色结果显示共培养细胞呈典型梭形。流式细胞仪检测显示共培养细胞阳性表达CD29、CD90,阴性表达CD45。共培养之大鼠骨髓间充质干细胞经诱导分化后分别出现成骨细胞和脂肪细胞形态,经碱性磷酸酶、VonCossa染色证实为成骨细胞,经油红染色证实为脂肪细胞。
结论采用全骨髓直接培养及密度梯度离心法,经3～5代培养后均可获得相对纯化之骨髓间充质干细胞。同种异体大鼠骨髓间充质干细胞共培养生长良好,不发生免疫排斥,证实该类细胞的极低免疫原性。在体外,共培养之大鼠骨髓间充质干细胞可诱导分化为成骨细胞、脂肪细胞,表明其具有多向分化潜能。
二、同种异体大鼠骨髓间充质干细胞的神经分化诱导鉴定
目的探讨同种异体大鼠骨髓间充质干细胞在体外的共培养,并行神经分化诱导鉴定。
方法分别通过全骨髓直接培养法及密度梯度法分离培养大鼠骨髓间充质干细胞,各传至第3代后,取等量细胞,混合后共培养,共培养之细胞再次传代后,进行神经方向诱导,并行Nestin,NSE,GFAP免疫细胞化学染色鉴定。
结果共培养之大鼠骨髓间充质干细胞经诱导分化后出现神经元和胶质细胞形态,经Nestin,NSE,GFAP免疫细胞化学染色证实为神经细胞。
结论同种异体共培养之骨髓间充质干细胞可在体外定向诱导为神经元和胶质细胞,提示移植入体内后,在适当环境下可分化为神经细胞。
三、脊髓损伤大鼠中同种异体骨髓间充质干细胞经脑脊液向损伤区的迁移
目的研究自脊髓损伤大鼠腰椎水平注射入蛛网膜下腔之同种异体骨髓间充质干细胞经脑脊液向损伤区的迁移。
方法分别通过全骨髓直接培养法及密度梯度法分离培养大鼠骨髓间充质干细胞,各传至第3代后,取等量细胞,混合后共培养,共培养之细胞再次传代后,以腺病毒转染EGFP基因。取成年SD大鼠20只,随机分为3组:对照组4只;模型组8只,治疗组8只。模型组与治疗组以改良Allen法复制大鼠下胸段脊髓损伤模型,对照组仅咬除相应节段棘突及椎板,不干预脊髓。造模后7天,于腰椎水平(L4-L5间隙)蛛网膜下腔向对照组及治疗组注射经EGFP标记之BMSCs,模型组注射同体积的Hank's缓冲液。观察各组动物造模前后及移植前后后肢运动功能,以BBB评分对其进行评估,各组分别于移植后7天、14天随机选取一半动物处死取损伤节段为中心脊髓,纵向切开脊髓后,一半行冰冻切片,于荧光显微镜下观察移植细胞向脊髓损伤区的迁移情况;另一半石蜡包埋后行HE染色,于光学显微镜下观察脊髓损伤区的细胞聚集情况。
结果三组动物造模术前BBB评分均为21分(满分)。术后第一天,对照组动物BBB评分恢复至21分。模型组和治疗组动物造模术后第3、7天BBB评分均低于对照组,有极显著性差异(P＜0.01),但两组间比较无显著性差异(P＞0.05)。两组动物自主排尿功能均在造模术后3天内恢复。BMSCs以Ad5F35-EGFP腺病毒转染后24h,荧光显微镜下即可见少量细胞表达绿色荧光。48h后,荧光显微镜下见大量细胞表达绿色荧光。经蛛网膜下腔注射EGFP标记之BMSCs后,治疗组动物从移植术后第1天开始,BBB评分持续增加。模型组动物BBB评分变化不大。移植术后第7天,模型组与治疗组动物BBB评分分别恢复至6.00±1.60和8.13±1.96分,与对照组相比均有极显著性差异(P＜0.01),且两组间比较亦有显著性差异(P＜0.05)。荧光显微镜下观察,EGFP标记之BMSCs移植后7天、14天,对照组与治疗组切片均见绿色荧光表达,模型组未见荧光表达。脊髓组织HE染色结果显示:移植后7天、14天,对照组切片脊髓结构正常;治疗组切片脊髓损伤区可见空洞,在脊髓面向蛛网膜下腔一面见大量细胞聚集;模型组切片脊髓损伤区见较大空洞,未见明显细胞聚集。
结论由腰椎水平蛛网膜下腔注入之EGFP标记BMSCs可经脑脊液向大鼠下胸段脊髓损伤区迁移,并改善脊髓损伤大鼠后肢运动功能。从临床角度看,如采用BMSCs治疗脊髓损伤,经腰椎穿刺注射是一种更简单、微创的术式。
四、同种异体骨髓间充质干细胞移植对脊髓损伤大鼠脊髓神经细胞凋亡的影响
目的探讨骨髓间充质干细胞移植治疗大鼠脊髓损伤的潜在抗凋亡机制。
方法6周龄雄性SD大鼠8只,用于BMSCs分离培养。成年雄性SD大鼠84只,根据移植术后取材时间点随机分为6组,每组14只:A1.对照组,14天:A2.对照组,28天;B1.模型组,14天;B2.模型组,28天;C1.治疗组,14天;C2.治疗组,28天。模型组与治疗组以改良Allen法复制大鼠下胸段脊髓损伤模型,对照组仅咬除相应节段棘突及椎板,不干预脊髓。造模后7天,于腰椎水平(L4-L5间隙)蛛网膜下腔向对照组及治疗组注射BMSCs,模型组注射同体积的Hank's缓冲液。观察各组动物造模前后及移植前后后肢运动功能,以BBB评分对其进行评估。按组别分别于移植后第14天、第28天处死动物,其中每组8只以损伤节段或相应节段为中心灌注后取脊髓组织,石蜡包埋后行HE染色,原位末端脱氧核苷酸转移酶(TdT)介导的脱氧尿苷三磷酸(d-UTP)缺口末端标记技术(TUNEL)染色,Bcl-2、Bax免疫组织化学染色;5只以损伤节段或相应节段为中心直接取脊髓组织,提取RNA后,行RT-PCR,检测Bcl-2、Bax基因的表达:1只于损伤中心或相应区域直接取1mm×1mm×1mm脊髓组织块,行透射电子显微镜观察。
结果三组动物造模术前BBB评分均为21分(满分)。术后第一天,对照组动物BBB评分恢复至21分。模型组和治疗组动物造模术后第3、7天BBB评分均低于对照组,有极显著性差异(P＜0.01),但两组间比较无显著性差异(P＞0.05)。两组动物自主排尿功能均在造模术后3天内恢复。BMSCs移植后,治疗组动物BBB评分持续增加,但仍低于对照组,有极显著性差异(P＜0.01),模型组动物BBB评分变化不大。从移植术后第7天开始,治疗组动物BBB评分均高于模型组,有极显著性差异(P＜0.01)。脊髓组织HE染色结果表明:移植后14天、28天,对照组切片脊髓结构正常。模型组和治疗组切片均可见脊髓损伤空洞,模型组更为明显。透射电镜观察显示,移植术后14天、28天,对照组凋亡细胞均较少,未见明显坏死细胞。模型组可见大量凋亡细胞及坏死细胞。治疗组凋亡坏死细胞均较模型组少。移植后14天,模型组TUNEL阳性细胞数量多于对照组和治疗组,并有显著性差异(P＜0.01),阳性细胞主要分布于白质中。治疗组亦高于对照组,有显著性差异(P＜0.01)。移植后28天,各组TUNEL阳性细胞数量均减少,模型组仍然高于对照组和治疗组,但无显著性差异。移植后14天,模型组Bax阳性细胞表达多于对照组和治疗组,且有显著性差异(P＜0.01),治疗组Bax阳性细胞表达多于对照组,亦有显著性差异(P＜0.01)。而模型组Bcl-2阳性细胞表达少于对照组和治疗组,但无显著性差异。移植后28天,Bax阳性细胞表达减少,模型组仍然高于对照组和治疗组,但无显著性差异。Bcl-2阳性细胞表达变化不大。RT-PCR结果表明,移植后14天、28天,模型组Bax基因表达均强于对照组和治疗组,而Bcl-2基因表达弱于对照组和治疗组,但其光密度比较均无显著性差异。
结论经蛛网膜下腔注射BMSCs可改善大鼠下胸段脊髓损伤模型的后肢运动功能,同时减少神经细胞的凋亡,降低Bax基因及其蛋白的表达,增加Bcl-2基因及其蛋白的表达。提示BMSCs移植治疗大鼠脊髓损伤具有潜在抗凋亡作用,这一作用很有可能是通过对凋亡调控基因Bax和Bcl-2的影响而实现的。
五、同种异体骨髓间充质干细胞移植对脊髓损伤大鼠应激状态及中枢AMPA受体蛋白表达的影响
目的探讨同种异体骨髓间充质干细胞移植对脊髓损伤大鼠应激状态及海马和杏仁核AMPA受体蛋白表达的影响。
方法成年雄性SD大鼠48只,根据移植术后取材时间点随机分为6组,每组8只:A1.对照组,14天;A2.对照组,28天;B1.模型组,14天;B2.模型组,28天;C1.治疗组,14天;C2.治疗组,28天。模型组与治疗组以改良Allen法复制大鼠下胸段脊髓损伤模型,对照组仅咬除相应节段棘突及椎板,不干预脊髓。造模后7天,于腰椎水平(L4-L5间隙)蛛网膜下腔向对照组及治疗组注射BMSCs,模型组注射同体积的Hank's缓冲液。观察各组动物造模前后及移植前后后肢运动功能,以BBB评分对其进行评估,并记录同时段动物体重。按组别分别于移植后第14天、第28天处死动物,取血,以酶联免疫吸附测定法(ELISA)测定血浆ACTH和血清皮质酮含量;灌注后取脑,冰冻切片后行GluR1、GluR2免疫组织化学染色。
结果三组动物造模术前BBB评分均为21分(满分)。术后第一天,对照组动物BBB评分恢复至21分。模型组和治疗组动物造模术后第3、7天BBB评分均低于对照组,有极显著性差异(P＜0.01),但两组间比较无显著性差异(P＞0.05)。两组动物自主排尿功能均在造模术后3天内恢复。BMSCs移植后,治疗组动物BBB评分持续增加,但仍低于对照组,有极显著性差异(P＜0.01),模型组动物BBB评分变化不大。从移植术后第7天开始,治疗组动物BBB评分均高于模型组,有极显著性差异(P＜0.01)。由于手术的干扰,三组动物在造模手术后体重均下降,术后逐渐恢复,至移植前,对照组体重高于模型组和治疗组,但无显著性差异(P＞0.05)。同样由于手术的干扰,各组动物体重在移植手术后均有明显降低。之后各组动物体重呈增加趋势,对照组体重增加快于同时段模型组和治疗组,从移植术后3天开始,差异有显著性意义(P＜0.05或P＜0.01)。从移植术后7天开始,各时段治疗组体重均高于模型组,但无显著性差异(P＞0.05)。移植后14天(即脊髓损伤后21天),模型组血浆ACTH较对照组和治疗组高,皮质酮比正常组要低。移植后28天(即脊髓损伤后35天),模型组ACTH下降,接近正常值,皮质酮持续升高。移植术后14天,GluR1阳性细胞数,模型组和治疗组在CA1区均高于对照组,均有显著性差异(P＜0.05或P＜0.01),在其他区无显著性差异;GluR2阳性细胞数有类似趋势,但均无显著性差异。移植术后28天,GluR1阳性细胞数,模型组在CA1、CA3、DG区均高于对照组,在CA1、CA3区亦高于治疗组,且均有显著性差异(P＜0.05或P＜0.01),模型组和治疗组在DG区均高于各自移植后14天阳性细胞数,均有显著性差异(P＜0.05或P＜0.01);GluR2阳性细胞数,治疗组在BLA区高于对照组,且有显著性差异(P＜0.05),在其他区域各组虽表现出与GluR1表达类似的趋势,但均无显著性差异。
结论BMSCs移植可改善大鼠下胸段脊髓损伤模型的后肢运动功能,同时可缓解脊髓损伤大鼠的慢性应激状态,其可能的机制为通过对AMPA受体蛋白GluR1及GluR2表达的影响而实现。
Part I : Co-culture and multilineage differentiation potentialcharacterization of allogenic rat bone mesenchymal stem cells
Objective To investigate the Co-culture of allogenic SD rat bone mesenchymal stem cells (BMSCs) in vitro, and to perform multilineage differentiation potential characterization.
Methods Rat BMSCs were isolated and cultured by complete marrow direct culture method and density gradient method respectively, and the 3rd passage cells were harvested and co-cultured with same concentration. The passage cells of the co-cultured cells were obtained and Wright-Gemsa staining were performed for morphological observation; and to identify the surface marker of CD29, CD45, CD90 by flow cytometry; moreover, the passage cells were induced to differentiate into osteoblast and adipocyte respectively, then performed alkaline phosphatase, Von Cossa and oil red staining characterization.
Results The primary cells of complete marrow direct culture method are uneven in shape, but with excellent reproductive activity; the cells obtained by density gradient method are fairly uniform in shape, but with lower reproductive activity; after 3 passages, the cells obtained by these two methods both got relative purification. The co-cultured cells were good in reproductive activity and relatively uniform in shape, without cell death because of immunologic rejection. After Wright-Gemsa staining, the cells displayed typical spindle-shaped cells; and the co-cultured cells were positive for CD29 and CD90, but negative for CD45 by flow cytometry. After induction, the co-cultured cells appeared morphological changes of osteoblast and adipocyte, and were confirmed be osteoblast by alkaline phosphatase staining and Von Cossa staining; be adipocyte by oil red staining.
Conclusion By use complete marrow direct culture method and density gradient method, after 3～5 passages, can obtain relative purified BMSCs. The co-cultured allogenic BMSCs were good in growth, without immunologic rejection, imply the weak immunogenicity of BMSCs. In vitro, the co-cultured rat BMSCs can be induced osteoblast and adipocyte, indicate the multilineage differentiation potential of BMSCs.
Part II: Neural differentiation potential characterization of allogenicrat bone mesenchymal stem cells
Objective To investigate the Co-culture of allogenic SD rat bone mesenchemal stem cells (BMSCs) in vitro, and to perform neural differentiation potential characterization.
Methods Rat BMSCs were isolated and cultured by complete marrow direct culture method and density gradient method respectively, and the 3rd passage cells were harvested and co-cultured with same concentration. The passage cells of the co-cultured cells were obtained and induced to differentiate into neurocyte, then performed immunocytochemistry of Nestin, NSE, GFAP.
Results After induction, the co-cultured cells appeared morphological changes of neuron and glial cell, and were confirmed by Nestin, NSE, GFAP immunostaining.
Conclusion The co-cultured allogenic BMSCs can be induced into neuron and glial cell in vitro, indicate that in appropriate circumstance, BMSCs can be induced into neurocyte in vivo.
Part III: Migration of allogenic rat bone mesenchymal stem cells toinjured sites through cerebrospinal fluid in rat with spinal cord injury
Objective To investigate the migration of allogenic rat bone mesenchymal stem cells to injured sites through cerebrospinal fluid in rat with spinal cord injury.
Methods Rat BMSCs were isolated and cultured by complete marrow direct culture method and density gradient method respectively, and the 3rd passage cells were harvested and co-cultured with same concentration. The passage cells of the co-cultured cells were labeled by transfection of recombinant adenovirus containing enhanced green fluorescence protein (EGFP) gene (Ad5F35-EGFP). A total of 20 adult male Sprague-Dawley (SD) rats weight 222-289 g were divided into 3 groups: control group, n=4; model group, n=8; treatment group, n=8. The rats in model and treatment group were performed a partial low thoracic spinal cord injury (SCI) by modified Allen's method (weight drop method) at T10 under chloral hydrate anesthesia. Rats in ontrol group received only laminectomy, without spinal cord interference. At day 7 After thoracic SCI, the dura at L4-L5 intervertebral space was exposed with partial removal of the L5 spinous process and L4-L5 ligamentum flavum under chloral hydrate anesthesia; 100μl of Hank's buffered saline solution contained BMSCs (EGFP transfected) or the same amount of Hank's buffered saline solution was injected into the subarachnoid space by single shot with a tip-bent 25 G needle. Then observed the hind limb motor function of all animals, and evaluated by Basso-Beatie-Bresnahan (BBB) scale. Half of the rats in each group had spinal cord tissue harvested at day 7 after cell transplantation. The other were harvested at day 14 after cell transplantation. The spinal cord segments containing the injured sites was removed from the spinal column after perfusion. Spinal cords were postfixed in 4% paraformaldehyde overnight and half-cut longitudinally in the sagittal direction. One half of the spinal cord was transferred to 30% sucrose solution, frozen, and cut in a cryostat at 10μm thickness for the examination of migration of transplanted cells to injured sites under fluorescent microscopy. The other half of the spinal cord was paraffin-embedded and sectioned in the same size as above for hematoxylin and eosin staining for the examination of cell aggregation in injury sites under optical microscope.
Results Before surgery, all animals' BBB scale score was 21 (the maximum value). At day 1 after surgery, the animals of control group reached 21 BBB scale score. Three, seven days after surgery, BBB scale score of the animals in model group and treatment group was lower than control, with significant difference (P<0.01), but without significant difference (P>0.05) between this two groups. All animals' urinary function recovered within 3 days after surgery. 24h after transfection through Ad5F35-EGFP adenovirus, some BMSCs expressed GFP under fluorescent microscope, 48h after transfection, a great many BMSCs expressed GFP. After transplantation of EGFP-BMSCs following a subarachnoid injection, BBB scale score of the animals in treatment group increased progressively. 7 days after grafting procedure, the animals of model group and treatment group reached a mean value of 6.00±1.60 and 8.13±1.96 in the BBB scale respectively, still lower than control group, with significant difference (P<0.01), and treatment group's BBB score higher than those in model group, with significant difference (P<0.05). Under fluorescent microscope, both of control group and treatment group animals' sections expressed GFP 7 and 14 days after grafting procedure, but the GFP expression was negative in model group. HE staining showed that 7 and 14 days after grafting procedure, sections of control group exhibited normal neural morphological change, while huge cell aggregations were observed on the surface of the spinal cord facing the subarachnoid space in treatment group, cell aggregations could not be seen in the model group.
Conclusion EGFP-BMSCs following a subarachnoid injection could migrate from a lumbar site into the injured low thoracic spinal cord through the cerebrospinal fluid, and could improve the hind limb motor function of SCI rats. This would potentially establish a simpler, less invasive procedure for BMSCs transplantation to spinal cord injury by lumbar puncture in clinical application.
Part IV: The influence of allogenic bone mesenchymal stem cellstransplantation to the neurocyte apoptosis of injured rat spinal cord.
Objective To investigate a potential anti-apoptotic mechanism of transplanted BMSCs for rat SCI.
Methods A total of & male SD rats weight 90-102 g were used for BMSCs harvest, and a total of 84 adult male SD rats weight 235-319 g were divided into 6 groups depending on the timing of the lumbar injection after thoracic SCI, n=14 in every group: Group A1, control group, day 14 post; Group A2, control group, day 28 post; Group B1, model group, day 14 post; Group B2, model group, day 28 post; Group C1, treatment group, day 14 post; Group C2, treatment group, day 28 post. The rats in model and treatment group were performed a partial low thoracic spinal cord injury (SCI) by modified Allen's method at T10 under chloral hydrate anesthesia. Rats in ontrol group received only laminectomy, without spinal cord interference. At day 7 After thoracic SCI, the dura at L4-L5 intervertebral space was exposed with partial removal of the L5 spinous process and L4-L5 ligamentum flavum under chloral hydrate anesthesia; 100μl of Hank's buffered saline solution contained BMSCs or the same amount of Hank's buffered saline solution was injected into the subarachnoid space by single shot with a tip-bent 25 G needle. Then observed the hind limb motor function of all animals, and evaluated by BBB scale. Rats were anesthetized at day 14, 28 postoperatively according to the timing point of group. Every 8 rat spinal cord segments containing the injured sites or counterparts in each group were harvested after perfused with normal saline followed 4% paraformaldehyde intracardially. Spinal cords were postfixed in neutral formalin for 24-48h and were paraffin-embedded and sectioned at 4 urn thickness. Hematoxylin and eosin staining were performed for histological observation. Neurocyte apoptosis was examined by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling ( TUNEL) staining, the expression of Bax and Bc1-2 protein were tested with immunohistochemistry. 5 rat spinal cord segments containing the injured sites or counterparts in each group were harvested directly, to investigate the mRNA expression of Bax and Bcl-2 by reverse transcriptase -polymerase chain reaction (RT-PCR). 1 rat spinal cord segments containing the injured sites or counterparts in each group were harvested directly, a 4 1mm×1mm×1mm spinal tissue were cut from the center of injured sites, for ultramicrostructural observation under transmission electron microscope (TEM).
Results Before surgery, all animals' BBB scale score was 21 (the maximum value). At day 1 after surgery, the animals of control group reached 21 BBB scale score. Three, seven days after surgery, BBB scale score of the animals in model group and treatment group was lower than control, with significant difference (P<0.01), but without significant difference (P>0.05) between this two groups. All animals' urinary function recovered within 3 days after surgery. After transplantation of BMSCs, BBB scale score of the animals in treatment group increased progressively, but still lower than control group, with significant difference (P<0.01), and treatment group's BBB score higher than those in model group from 7 day after grafting procedure, with significant difference (P<0.01). HE staining showed that 14 and 28 days after grafting procedure, sections of control group exhibited normal neural morphological change, while cavities of SCI were observed in those of the model and treatment group, the model group was more significant than treatment group. Under TEM, 14 and 28 days after grafting procedure, sections of control group exhibited few apoptotic neurocyte, while in the sections of model and treatment group, a lot of apoptotic and necrotic neurocytes were observed, the model group was more significant than treatment group. The TUNEL staining showed that the TUNEL positive cells in model group were higher than those in control group and treatment group at 14 day after injection, with significant difference (P < 0.01), and positive cells in treatment group were higher than those in control group as well, with significant difference (P<0.01), the TUNEL positive cells mainly distributed in white matter. The positive cells declined at 28 day after injection, model group were still higher than those in the other two groups, but without significant difference. The result of immunostaining against Bax and Bcl-2 demonstrated that model group showed higher Bax positive expression than those in the other two groups, with significant difference (P<0.01) at 14 day after injection, and Bax positive cells in treatment group were higher than those in control group as well, with significant difference (P<0.01), which on the contrary in Bcl-2 positive expression, but without significant difference. The Bax positive cells declined at 28 day after injection, model group were still higher than those in the other two groups, but without significant difference (P>0.05), the change of Bcl-2 positive expression was insignificant. The RT-PCR of Bax and Bcl-2 demonstrated that model group showed higher Bax gene positive expression than those in other two groups, but without significant difference (P>0.05) in relative optical density at 14 and 28 days after injection, which on the contrary in Bcl-2 positive expression, without significant difference (P>0.05) either.
Conclusion Transplantation of BMSCs by a subarachnoid injection could improve the hind limb motor function of low thoracic SCI rats, and could reduce spinal neurocyte apoptosis, decrease the genic and proteinic expression of Bax, increase the genic and proteinic expression of Bcl-2, which implied a potential anti-apoptotic mechanism of transplanted BMSCs for rat SCI, the anti-apoptotic effect maybe achieved by the effect to apoptotic controlling gene Bax and Bcl-2.
Part V: The influence of allogenic bone mesenchymal stem cells transplantation to the stress state and AMFA receptor protein expressionof brain in rat with spinal cord injury.
Objective To investigate the effect of allogenic bone mesenchymal stem cells transplantation to the stress state and AMPA receptors protein expression of hippocampus and basolateral amygdale (BLA) in rat with SCI.
Methods A total of 48 adult male SD rats weight 250-319 g were divided into 6 groups depending on the timing of the lumbar injection after thoracic SCI, n=8 in every group: Group A1, control group, day 14 post; Group A2, control group, day 28 post; Group B1, model group, day 14 post; Group B2, model group, day 28 post; Group C1, treatment group, day 14 post; Group C2, treatment group, day 28 post. The rats in model and treatment group were performed a partial low thoracic SCI by modified Allen's method at T10 under chloral hydrate anesthesia. Rats in ontrol group received only laminectomy, without spinal cord interference. At day 7 After thoracic SCI, the dura at L4-L5 intervertebral space was exposed with partial removal of the L5 spinous process and L4-L5 ligamentum flavum under chloral hydrate anesthesia; 100μl of Hank's buffered saline solution contained BMSCs or the same amount of Hank's buffered saline solution was injected into the subarachnoid space by single shot with a tip-bent 25 G needle. Then observed the hind limb motor function of all animals, and evaluated by BBB scale. Rats were anesthetized at day 14, 28 postoperatively according to the timing point of group, to obtain blood for detection of plasmic ACTH and serumal corticosterone concentration by enzyme linked immunosorbent assay (ELISA), and to harvest brains after perfused with normal saline followed 4% paraformaldehyde intracardially, brains were postfixed in 4% paraformaldehyde overnight and were transferred to 30% sucrose solution, frozen, and cut in a cryostat at 30μm thickness for the immunohistochemistry staining of GluR1 and GluR2.
Results Before surgery, all animals' BBB scale score was 21 (the maximum value). At day 1 after surgery, the animals of control group reached 21 BBB scale score. Three, seven days after surgery, BBB scale score of the animals in model group and treatment group was lower than control, with significant difference (P<0.01), but without significant difference (P>0.05) between this two groups. All animals' urinary function recovered within 3 days after surgery. After transplantation of BMSCs, BBB scale score of the animals in treatment group increased progressively, but still lower than control group, with significant difference (P<0.01), and treatment group's BBB score higher than those in model group from 7 day after grafting procedure, with significant difference (P<0.01). After low thoracic SCI and sham operation, all animals' body weight decreased because of operation interference, but increased gradually from then on. At timing point of day 7 (pre-grafting procedure), animals' body weight in the control group were higher than those in the model and treatment group, but without significant difference (P>0.05). After grafting procedure, all animals' body weight decreased again due to operation interference as before. Then all animals' body weight increased gradually. Animals' body weight in the control group were higher than those in the model and treatment group, and with significant difference (P<0.05或P<0.01) from 3 days after grafting procedure. From 7 days after grafting procedure, animals' body weight in the treatment group were higher than those in the model group, but without significant difference (P>0.05). At the timing point of 14 days after grafting procedure (21 days after SCI), the plasmic ACTH concentrations in the model group were higher than those in the other groups, which were on the contrary in the concentrations of serumal corticosterone. At the timing point of 28 days after grafting procedure (35 days after SCI), the plasmic ACTH concentrations in the model group decreased and near to normal level, but at the same time, the concentrations of serumal corticosterone increased. At the timing point of 14 days after grafting procedure, GluR1 positive cells of the model group increased in CA1, CA3, DG regions of hippocampus and BLA, with significant difference in CA1 (P<0.01); GluR2 positive cells had similar tendency, but without significant difference. At the timing point of 28 days after grafting procedure, GluR1 positive cells of the model group were higher than those of the control group in CA1, CA3, DG regions and of the treatment group in CA1, CA3 regions of hippocampus, with significant difference (P<0.05 or P<0.01, respectively); GluR1 positive cells of the model and treatment group were higher than their counterpart at the timing point of 14 days after grafting procedure, with significant difference (P<0.05 or P<0.01, respectively); GluR2 positive cells of the treatment group were higher than those of the control group in BLA, with significant difference (P<0.05); GluR2 positive cells had similar tendency in the other regions, but without significant difference.
Conclusions Transplantation of BMSCs could improve the hind limb motor function of low thoracic SCI rats, and could relieve the stress state of SCI rats, which implied a potential anti-chronic stress mechanism of transplanted BMSCs for rat SCI, the anti-chronic stress effect maybe achieved by influence to the expression of AMPA receptor protein of GluR1 and GluR2.
引文
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4 Wang PP, Wang JH, Yan ZP, et al. Expression of hepatocyte-like phenotypes in bone marrow stromal cells after HGF induction. Biochem Biophys Res Commun,2004,320(3):712-716.
5 Luk JM, Wang PP, Lee CK, et al. Hepatic potential of bone marrow stromal cells:development of in vitro co-culture and intra-portal transplantation models. J Immunol Methods, 2005,305(1):39-47.
6 Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999,284(5411):143-147.
7 Hassan HT, El-Sheemy M. Adult bone-marrow stem cells and their potential in medicine. J R Soc Med, 2004,97(10):465-471.
8 Rickard DJ, Sullivan TA, Shenker BJ, et al. Induction of rapid osteoblast differentiation in rat bone marrow stromal cell cultures by dexamethasone and BMP-2.DevBiol, 1994,161(1):218-228.
9 Ferrari G, Cusella-De Angelis G, Coletta M, et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science, 1998,279(5356): 1528-1530.
10 Woodbury D, Schwarz EJ, Prockop DJ, et al. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res, 2000,61(4):364-370.
11 Sanchez-Ramos J, Song S, Cardozo-Pelaez F, et al. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol, 2000,164(2):247-256.
12 Deng W, Obrocka M, Fischer I, et al. In vitro differentiation of human marrow stromal cells into early progenitors of neural cells by conditions that increase intracellular cyclic AMP. Biochem Biophys Res Commun, 2001,282(1): 148-152.
13 Pereira RF, Halford KW, O'Hara MD, et al. Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice.Proc Natl Acad Sci U S A, 1995,92(11):4857-4861.
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15 Cianferotti L, Demay MB. VDR-mediated inhibition of DKK1 and SFRP2 suppresses adipogenic differentiation of murine bone marrow stromal cells. J Cell Biochem,2007,101(1):80-88.
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14 Suva D, Garavaglia G, Menetrey J, et al. Non-hematopoietic human bone marrow contains long-lasting, pluripotential mesenchymal stem cells. J Cell Physiol,2004,198(1):110-118.
15 Reyes M, Dudek A, Jahagirdar B, et al. Origin of endothelial progenitors in human postnatal bone marrow. J Clin Invest, 2002,109(3):337-346.
16 Kim BJ, Seo JH, Bubien JK, et al. Differentiation of adult bone marrow stem cells into neuroprogenitor cells in vitro. Neuroreport, 2002,13(9): 1185-1188.
17 Tohill M, Mantovani C, Wiberg M, et al. Rat bone marrow mesenchymal stem cells express glial markers and stimulate nerve regeneration. Neurosci Lett,2004,362(3):200-203.
18 Alexanian AR. Neural stem cells induce bone-marrow-derived mesenchymal stem cells to generate neural stem-like cells via juxtacrine and paracrine interactions. Exp Cell Res, 2005,310(2):383-391.
19 Arnhold S, Klein H, Klinz FJ, et al. Human bone marrow stroma cells display certain neural characteristics and integrate in the subventricular compartment after injection into the liquor system. Eur J Cell Biol, 2006,85(6):551-565.
20 Hofstetter CP, Schwarz EJ, Hess D, et al. Marrow stromal cells form guiding strands in the injured spinal cord and promote recovery. Proc Natl Acad Sci U S A,2002,99(4):2199-2204.
21 Satake K, Lou J, Lenke LG. Migration of mesenchymal stem cells through cerebrospinal fluid into injured spinal cord tissue. Spine, 2004,29(18):1971-1979.
22 Zurita M, Vaquero J. Bone marrow stromal cells can achieve cure of chronic paraplegic rats: Functional and morphological outcome one year after transplantation.Neurosci Lett, 2006,402(1-2):51-56.
23 Akiyama Y, Radtke C, Kocsis JD. Remyelination of the rat spinal cord by transplantation of identified bone marrow stromal cells. J Neurosci,2002,22(15):6623-6630.
24 Deng YB, Yuan QT, Liu XG, et al. Functional recovery after rhesus monkey spinal cord injury by transplantation of bone marrow mesenchymal-stem cell-derived neurons. Chin Med J (Engl), 2005,118(18):1533-1541.
25 Koda M, Okada S, Nakayama T, et al. Hematopoietic stem cell and marrow stromal cell for spinal cord injury in mice. Neuroreport, 2005,16( 16): 1763-1767.
26 Kamada T, Koda M, Dezawa M, et al. Transplantation of bone marrow stromal cell-derived Schwann cells promotes axonal regeneration and functional recovery after complete transection of adult rat spinal cord. J Neuropathol Exp Neurol,2005,64(1):37-45.
27 Vaquero J, Zurita M, Oya S, et al. Cell therapy using bone marrow stromal cells in chronic paraplegic rats: systemic or local administration?. Neurosci Lett,2006,398(1-2):129-134.
28 Rosenfeld JV, Gillett GR. Ethics, stem cells and spinal cord repair. Med J Aust,2004,180(12):637-639.
29 Moviglia G, Fernandez Vina R, Brizuela J, et al. Combined protocol of cell therapy for chronic spinal cord injury. Report on the electrical and functional recovery of two patients. Cytotherapy, 2006,8(3):202-209.
2 Carlo-Stella C, Gianni MA. Biology and clinical applications of marrow mesenchymal stem cells. Pathol Biol (Paris), 2005,53(3):162-164.
3 Hassan HT, El-Sheemy M. Adult bone-marrow stem cells and their potential in medicine. J R Soc Med, 2004,97(10):465-471.
4 Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999,284(5411):143-147.
5 Rickard DJ, Sullivan TA, Shenker BJ, et al. Induction of rapid osteoblast differentiation in rat bone marrow stromal cell cultures by dexamethasone and BMP-2.Dev Biol, 1994,161(1):218-228.
6 Pereira RF, Halford KW, O'Hara MD, et al. Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice.Proc Natl Acad Sci U S A, 1995,92(11):4857-4861.
7 Lee RH, Kim B, Choi I, et al. Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell Physiol Biochem, 2004,14(4-6):311-324.
8 Park J, Gelse K, Frank S, et al. Transgene-activated mesenchymal cells for articular cartilage repair: a comparison of primary bone marrow-, perichondrium/periosteum- and fat-derived cells. J Gene Med, 2006,8(1):112-125.
9 Wakitani S, Saito T, Caplan Al. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve,1995,18(12):1417-1426.
10 Ferrari G, Cusella-De Angelis G, Coletta M, et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science, 1998,279(5356): 1528-1530.
11 Cianferotti L, Demay MB. VDR-mediated inhibition of DKK1 and SFRP2 suppresses adipogenic differentiation of murine bone marrow stromal cells. J Cell Biochem,2007,101(1):80-88.
12 Annabi B, Naud E, Lee YT, et al. Vascular progenitors derived from murine bone marrow stromal cells are regulated by fibroblast growth factor and are avidly recruited by vascularizing tumors. J Cell Biochem, 2004,91(6): 1146-1158.
13 Li H, Yu B, Zhang Y, et al. Jaggedl protein enhances the differentiation of mesenchymal stem cells into cardiomyocytes. Biochem Biophys Res Commun,2006,341(2):320-325.
14 Schwartz RE, Reyes M, Koodie L, et al. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J Clin Invest,2002,109(10):1291-1302.
15 Wang PP, Wang JH, Yan ZP, et al. Expression of hepatocyte-like phenotypes in bone marrow stromal cells after HGF induction. Biochem Biophys Res Commun,2004,320(3):712-716.
16 Luk JM, Wang PP, Lee CK, et al. Hepatic potential of bone marrow stromal cells:development of in vitro co-culture and intra-portal transplantation models. J Immunol Methods, 2005,305(1):39-47.
17 Kopen GC, Prockop DJ, Phinney DG. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci U S A, 1999,96(19):10711-10716.
18 Woodbury D, Schwarz EJ, Prockop DJ, et al. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res, 2000,61(4):364-370.
19 Sanchez-Ramos J, Song S, Cardozo-Pelaez F, et al. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol, 2000,164(2):247-256.
20 Deng W, Obrocka M, Fischer I, et al. In vitro differentiation of human marrow stromal cells into early progenitors of neural cells by conditions that increase intracellular cyclic AMP. Biochem Biophys Res Commun, 2001,282(1):148-152.
21 Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science,1997,276(5309):71-74.
22 Sammons J, Ahmed N, El-Sheemy M, et al. The role of BMP-6, IL-6, and BMP-4 in mesenchymal stem cell-dependent bone development: effects on osteoblastic differentiation induced by parathyroid hormone and vitamin D(3). Stem Cells Dev,2004,13(3):273-280.
23 Sammons J, Ahmed N, El-Sheemy M, et al. The Role of BMP-6, IL-6, and BMP-4 in Mesenchymal Stem Cell-Dependent Bone Development: Effects on Osteoblastic Differentiation Induced by Parathyroid Hormone and Vitamin D(3). Stem Cells Dev,2004,13(3):273-280.
24 Caplan Al, Bruder SP. Mesenchymal stem cells: building blocks for molecular medicine in the 21st century. Trends Mol Med, 2001,7(6):259-264.
25 Erices A, Conget P, Rojas C, et al. Gp130 activation by soluble interleukin-6 receptor/interleukin-6 enhances osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells. Exp Cell Res, 2002,280(1):24-32.
26 Bonnet D. Biology of human bone marrow stem cells. Clin Exp Med,2003,3(3): 140-149.
27 Suva D, Garavaglia G, Menetrey J, et al. Non-hematopoietic human bone marrow contains long-lasting, pluripotential mesenchymal stem cells. J Cell Physiol,2004,198(1):110-118.
1 Dumont RJ, Okonkwo DO, Verma S, et al. Acute spinal cord injury, part I:pathophysiologic mechanisms. Clin Neuropharmacol, 2001,24(5):254-264.
2 Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science, 1992,255(5052): 1707-1710.
3 Eriksson PS, Perfilieva E, Bjork-Eriksson T, et al. Neurogenesis in the adult human hippocampus. Nat Med, 1998,4( 11): 1313-1317.
4 Wang PP, Wang JH, Yan ZP, et al. Expression of hepatocyte-like phenotypes in bone marrow stromal cells after HGF induction. Biochem Biophys Res Commun,2004,320(3):712-716.
5 Luk JM, Wang PP, Lee CK, et al. Hepatic potential of bone marrow stromal cells:development of in vitro co-culture and intra-portal transplantation models. J Immunol Methods, 2005,305(1):39-47.
6 Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999,284(5411):143-147.
7 Hassan HT, El-Sheemy M. Adult bone-marrow stem cells and their potential in medicine. J R Soc Med, 2004,97(10):465-471.
8 Rickard DJ, Sullivan TA, Shenker BJ, et al. Induction of rapid osteoblast differentiation in rat bone marrow stromal cell cultures by dexamethasone and BMP-2.DevBiol, 1994,161(1):218-228.
9 Ferrari G, Cusella-De Angelis G, Coletta M, et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science, 1998,279(5356): 1528-1530.
10 Woodbury D, Schwarz EJ, Prockop DJ, et al. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res, 2000,61(4):364-370.
11 Sanchez-Ramos J, Song S, Cardozo-Pelaez F, et al. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol, 2000,164(2):247-256.
12 Deng W, Obrocka M, Fischer I, et al. In vitro differentiation of human marrow stromal cells into early progenitors of neural cells by conditions that increase intracellular cyclic AMP. Biochem Biophys Res Commun, 2001,282(1): 148-152.
13 Pereira RF, Halford KW, O'Hara MD, et al. Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice.Proc Natl Acad Sci U S A, 1995,92(11):4857-4861.
14 Li H, Yu B, Zhang Y, et al. Jagged1 protein enhances the differentiation of mesenchymal stem cells into cardiomyocytes. Biochem Biophys Res Commun,2006,341(2):320-325.
15 Cianferotti L, Demay MB. VDR-mediated inhibition of DKK1 and SFRP2 suppresses adipogenic differentiation of murine bone marrow stromal cells. J Cell Biochem,2007,101(1):80-88.
16 Park J, Gelse K, Frank S, et al. Transgene-activated mesenchymal cells for articular cartilage repair: a comparison of primary bone marrow-, perichondrium/periosteum-and fat-derived cells. J Gene Med, 2006,8(1): 112-125.
17 Lee RH, Kim B, Choi I, et al. Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell Physiol Biochem, 2004,14(4-6):311-324.
18 Annabi B, Naud E, Lee YT, et al. Vascular progenitors derived from murine bone marrow stromal cells are regulated by fibroblast growth factor and are avidly recruited by vascularizing tumors. J Cell Biochem, 2004,91 (6):1146-1158.
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