长期培养人胚神经干细胞对兔脊髓损伤的修复作用

[目的]观察长期培养人胚神经干细胞（hNSCs）的体外生长特性与转染EGFP基因后移植治疗兔脊髓横切损伤模型在体内的生物学活性及对神经结构修复和功能恢复的影响。[方法]体外分离、培养并鉴定hNSCs，用逆转录病毒介导的增强绿色荧光蛋白基因（EGFP）进行转染；制备兔T9全横断脊髓损伤模型；观察hNSCs移植对脊髓损伤后神经结构修复和功能恢复的影响。[结果]从胎龄10～20周的新鲜人胚脑皮层中成功分离出神经干细胞，该细胞具有连续克隆传代能力，诱导分化后表达分化细胞的特异抗原。本实验室已成功连续培养10个月（17代），转染EGFP基因后，仍保持未分化状态，能够自我更新形成新的神经球；移植入兔SCI模型后，hNSCs能在体内存活、迁移、分化并增殖。与对照组相比，hNSCs移植组明显促进了脊髓神经的再生、结构的修复和下肢运动功能的恢复。[结论]hNSCs移植促进了脊髓损伤后神经结构的修复和功能的恢复，是治疗急性脊髓损伤的一种有效方法。     

Bcl-2 and GDNF delivered by HSV-mediated gene transfer after spinal root avulsion provide a synergistic effect

Proximal spinal nerve injury results in the death of motor neurons in ventral horn. We have previously demonstrated this cell death can be prevented by HSV-mediated transfer of the gene coding for the antiapoptotic peptide Bcl-2 7 days prior to injury, but that expression of Bcl-2 does not preserve ChAT expression in the lesioned cells. In the current study, we examined two related issues: whether Bcl-2 delivered by HSV-mediated gene transfer 30 min after injury could similarly protect motor neurons from cell death, and whether the additional HSV-mediated expression of the glial cell derived neurotrophic factor (GDNF) could improve the result. At 30 min after avulsion of the L4, L5, and L6 spinal nerves, replication defective genomic HSV-based vectors coding for Bcl-2, GDNF, a reporter transgene (lacZ), or the Bcl-2 and GDNF vectors together were injected into spinal cord. Transduction of motor neurons with either the Bcl-2-expressing vector or the GDNF-expressing vector resulted in a substantial increase in the number of surviving motor neurons, and coinjection of the two vectors together resulted in cell survival that was similar to the result obtained with either vector alone. Neither the Bcl-2-expressing vector nor the GDNF-expressing vector delivered alone protected choline acetyltransferase (ChAT) expression in lesioned neurons. However, simultaneous injection of the Bcl-2- and the GDNF-expressing vectors together resulted in a substantial increase in the number of ChAT in cells in the lesioned ventral horn. Together, these findings suggest an approach to improving cell survival and regeneration following proximal root injury.     

Cotransplantation of mouse neural stem cells (mNSCs) with adipose tissue-derived mesenchymal stem cells improves mNSC survival in a rat spinal cord injury model

The low survival rate of graft stem cells after transplantation into recipient tissue is a major obstacle for successful stem cell therapy. After transplantation into the site of spinal cord injury, the stem cells face not only hypoxia due to low oxygen conditions, but also a lack of nutrients caused by damaged tissues and poor vascular supply. To improve the survival of therapeutic stem cells after grafting into the injured spinal cord, we examined the effects of cotransplanting mouse neural stem cells (mNSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs) on mNSC viability. The viability of mNSCs in coculture with AT-MSCs was significantly increased compared to mNSCs alone in an in vitro injury model using serum deprivation (SD), hydrogen peroxide (H(2)O(2)), and combined (SD + H(2)O(2)) injury mimicking the ischemic environment of the injured spinal cord. We demonstrated that AT-MSCs inhibited the apoptosis of mNSCs in SD, H(2)O(2), and combined injury models. Consistent with these in vitro results, mNSCs transplanted into rat spinal cords with AT-MSCs showed better survival rates than mNSCs transplanted alone. These findings suggest that cotransplantation of mNSCs with AT-MSCs may be a more effective transplantation protocol to improve the survival of cells transplanted into the injured spinal cord.     

大剂量甲基强的松龙对大鼠急性脊髓损伤后神经细胞Bcl-2表达及细胞凋亡的影响

目的：观察大剂量甲基强的松龙（MP）治疗对大鼠急性脊髓损伤（ASCI）后神经细胞凋亡及凋亡基因Bcl-2的影响。方法：选取48只雌性SD大鼠随机等分为2组,对照组与治疗组,按Nystrom法制备大鼠急性脊髓损伤模型。治疗组伤后30min经腹膜腔注入MP30mg／kg,以后每小时腹膜腔注入MP5．4mg／kg,维持24h;对照组应用生理盐水替代MP,处理方法同治疗组。两组分别于伤后4、8h及1、3、7、14d灌注固定后取材。免疫组织化学检测损伤段脊髓内Bcl-2蛋白表达,TUNEL检测细胞凋亡,染色结果应用图像分析仪进行半定量分析。结果：大鼠ASCI后4h即可见脊髓内TUNEL阳性细胞,8h表达达高峰,此后表达量逐渐下降,14d时仍可见少量阳性细胞。凋亡相关蛋白Bcl-2在伤后4h即可见表达,伤后1d达高峰,伤后14d仍有表达,与对照组相比,治疗组伤后8h、1d和3d时凋亡细胞数减少有统计学意义,伤后8h和1d Bcl-2蛋白表达增高有统计学意义。结论：大剂量甲基强的松龙治疗可抑制大鼠ASCI后神经细胞凋亡,并增加凋亡相关蛋白Bcl-2的表达：     

Repair of acutely injured spinal cord through constructing tissue-engineered neural complex in adult rats

Objective： To construct tissue-engineered neural complex in vitro and study its effect in repairing acutely injured spinal cord in adult rats. Methods： Neural stem cells were harvested from the spinal cord of embryo rats and propagated in vitro. Then the neural stem cells were seeded into polygiycolic acid scaffolds and co-cultured with extract of embryonic spinal cord in vitro. ce histochemistry and scanning electron microscope were used to observe the microstructure of this complex. Animal model of spine semi-transection was made and tissue-engineered neural complex was implanted by surgical intervention. Six weeks after transplantation, functional evaluation and histochemistry were applied to evaluate the functional recovery and anatomic reconstruction. Results： The tissue-engineered neural complex had a distinct structure, which contained neonatal neurons, oligodendrocytes and astrocytes. After tissue-engineered neural complex was implanted into the injured spinal cord, the cell components such as neurons, astrocytes and oligodendrocytes, could survive and keep on developing. The adult rats suffering from spinal cord injury got an obvious neurological recovery in motor skills. Conclusions： The tissue-engineered neural complex appears to have therapeutic effects on the functional recovery and anatomic reconstruction of the adult rats with spinal cord injury.     

脑源性神经生长因子转基因细胞移植和大剂量甲基强的松龙对脊髓损伤后细胞凋亡的影响

目的：探讨大鼠脊髓损伤后腺病毒介导的脑源性神经生长因子（AxCA-BDNF）体外转基因成肌细胞移植和静脉内注射大剂量甲基强的松龙（MP）对大鼠脊髓损伤后细胞凋亡的影响。方法：120只Wistar大鼠分为：脊髓挫伤组（A组），脊髓挫伤后AxCA-BDNF基因转染的成肌细胞移植组（B组），脊髓挫伤后静脉内注射大剂量MP治疗组（C组），脊髓挫伤后同时应用AxCA-BDNF和MP组（D组）。手术后1、3、7、14、28d用行为学和电生理检查观察大鼠功能恢复情况，并用计算机图像分析技术对脊髓损伤区细胞凋亡（TUNEL法）和Bcl-2蛋白表达（免疫组化法）进行定量分析。结果：四组中均发现凋亡细胞及Bcl-2蛋白阳性表达细胞．图像分析发现四组凋亡细胞核数为A组〉B组〉C组〉D组；Bcl-2免疫反应阳性细胞表达顺序为D组〉C组〉B组〉A组。大鼠后肢功能恢复和电生理检查也有类似的变化趋势。结论：体外转基因成肌细胞移植和大剂量MP都能抑制大鼠脊髓损伤后的细胞凋亡，促进大鼠后肢功能恢复，两者联合应用具有协同作用。     

Secondary degeneration reduced by inosine after spinal cord injury in rats

STUDY DESIGN: Assessment of the potential protective effects of inosine on an animal model of spinal cord injury. OBJECTIVES: Our previous studies have demonstrated that inosine can directly protect neurons in vitro from zinc-induced injury and axotomized retinal ganglion cells of rats in vivo. This investigation was carried out to examine the possible protective effects of inosine on spinal cord secondary degeneration. SETTING: Institute of Neurosciences, The Fourth Military Medical University, Xi'an, China. METHODS: Compressive spinal cord injury (95-g load for 1 min) model was established in rats, and inosine was administrated beginning at different time points (2, 12, or 24 h) after spinal cord injury. RESULTS: Using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) technique and hematoxylin and eosin staining, our study demonstrated that administration of inosine as late as 12 h after injury significantly reduced the total volume of spinal cord degenerative areas and the number of apoptotic cells 3 days following the trauma. CONCLUSION: Inosine can significantly reduce the spread of secondary degeneration and the cell death following spinal cord injury in adult rats. These findings may find a clinical application in the treatment of acute spinal cord injury.     

大鼠脊髓急性损伤后神经细胞凋亡及相关基因表达△

目的研究脊髓急性损伤后神经细胞的凋亡及相关基因的表达.方法大鼠脊髓(T8、T9)经中度压迫损伤后,分别在30min、2h、4h、8h、24h、48h、72h、7d、14d和21d处死取材(各时间组n=4).应用HE染色、免疫组化及凋亡细胞原位末端标记法对脊髓组织进行标记.结果损伤4h后,在损伤段及邻近段可见末端标记阳性神经元,损伤段灰质中阳性细胞数8h达高峰,24h白质中阳性胶质细胞数量达高峰.相邻节段阳性细胞数72h达高峰.损伤后P53及Bax大量表达,而Bcl-2仅少量表达.结论脊髓损伤后神经细胞的凋亡是继发损伤期的重要病理变化.     

大鼠脊髓急性损伤后bax和bcl—2的表达

目的：检测大鼠脊髓损伤后凋亡相关基因的表达,以探讨神经细胞凋亡的分子机制。方法：大鼠脊髓（T8．9）经中度压迫损伤后,分别在30min、2h、4h、8h、24h、48h和72h处死取材（n=6)。主要应用免疫组化及原位杂交技术对脊髓组织进行标记,以检测bcl-2和bax的表达。结果：损伤4h后bax蛋白大量表达,而bcl-2蛋白仅有少量表达,bcl-2 mRNA未见表达。结论：脊髓损伤后凋亡基因bax大量表达,并可能在神经细胞的凋亡过程中起重要作用。     

钙蛋白酶抑制剂对缺血再灌注损伤脊髓的保护作用

目的 观察大鼠脊髓缺血再灌注损伤后应用钙蛋白酶特异性抑制剂E-64-D,对脊髓神经细胞组织学改变和凋亡的影响及对大鼠后肢运动功能的保护作用.方法 选用纯种雄性成年SD大鼠106只,夹闭右肾动脉分支下腹主动脉30 min,再灌注即刻静脉应用钙蛋白酶特异性抑制剂E-64-D,观察再灌注后3、24、72 h和7 d脊髓损伤节段神经细胞的凋亡及再灌注后24、72h组织病理学改变;对再灌注后72 h的大鼠后肢功能进行评分.结果 脊髓缺血再灌注24 h开始出现神经细胞凋亡现象,脊髓组织出现病理学改变,神经元死亡,胶质细胞增生.应用E-64-D后,凋亡现象和细胞坏死得到抑制,差异有统计学意义(P<0.01).再灌注后72 h后肢功能也得到一定程度的保护.结论 脊髓再灌注损伤后静脉应用E-64-D治疗,可以明显抑制脊髓神经细胞的凋亡,有利于神经元的存活,损伤后3 d大鼠后肢运动功能得到一定程度的改善.     

成年脊髓损伤大鼠脊髓神经干细胞的体外培养及分化研究

目的 探讨大鼠脊髓损伤后脊髓神经干细胞的分离培养方法及分化情况.方法 采用Allen法制作大鼠脊髓损伤模型,利用无血清培养和单细胞克隆技术在成年脊髓损伤7 d大鼠脊髓中分离具有单细胞克隆能力的神经干细胞,并进行培养鉴定.结果 从成年脊髓损伤7 d大鼠脊髓中成功分离出神经干细胞,该细胞具有连续克隆能力,可传代培养,表达神经巢蛋白抗原.分化后的细胞表达神经元细胞、星形胶质细胞和少突胶质细胞的特异性抗原.结论 致伤7 d的成年大鼠脊髓组织体外町培养出神经十细胞,并分化为神经无细胞、星形胶质细胞和少突胶质细胞,有可能参与脊髓损伤的修复过程.     

脊髓急性损伤后神经细胞凋亡的时相和空间分布特点

目的 研究脊髓急性损伤后神经细胞的凋亡及其时相和空间特点。方法 大鼠脊髓（T8,9）经中度压迫损伤后,分别在30min、2h、4h、8h、24h、48h、72h、7d、14d、和21d处死取材（n=4)。应用HE、Nissl染色及凋亡细胞原位末端标记法对脊髓组织进行标记。结果 损伤4h后,在损伤段及邻近段可见末端标记阳性神经细胞,损伤段灰质中阳性细胞数8h达高峰,24h白质中阳性胶质细胞数量达高峰。相邻节段阳性细胞数量在72h达高峰。阳性细胞以白质中胶质细胞为主,主要分布于相邻节段。结论 脊髓损伤后神经细胞凋亡是继发损伤期的重要病理变化,并有其时相和空间分布特点。     

Neurogenesin-1基因促进脊髓损伤后功能修复的实验研究

目的 探讨Neurogenesin-1(Ng1)基因对脊髓损伤后功能恢复的影响.方法 将36只大鼠随机分为实验组和对照组,每组18只.采用改良Allen法制备大鼠胸10脊髓损伤模型后,通过Alzet微泵分别向实验组和对照组持续转染入Ng1重组质粒和空白质粒.术后各时间点BBB评分系统监测大鼠运动功能恢复情况,并于术后第2周和第4周时分别取材,应用神经细胞特异性免疫荧光双标染色和组织学观察Ng1基因对内源性神经干细胞分化的影响以及脊髓组织病理变化情况.结果 自损伤后1周起实验组大鼠的BBB评分明显高于对照组.组织学观察实验组脊髓形态恢复较好逐渐趋于正常.实验组脊髓组织中新分化的神经元细胞数较对照组明显增多,同时新分化的星型胶质细胞数显著减少.结论 Ng1基因能够诱导脊髓损伤后内源性神经干细胞分化为神经元,促进脊髓运动功能的修复.     

Neurogenesin-1基因促进脊髓损伤后功能修复的实验研究

目的 探讨Neurogenesin-1(Ng1)基因对脊髓损伤后功能恢复的影响.方法 将36只大鼠随机分为实验组和对照组,每组18只.采用改良Allen法制备大鼠胸10脊髓损伤模型后,通过Alzet微泵分别向实验组和对照组持续转染入Ng1重组质粒和空白质粒.术后各时间点BBB评分系统监测大鼠运动功能恢复情况,并于术后第2周和第4周时分别取材,应用神经细胞特异性免疫荧光双标染色和组织学观察Ng1基因对内源性神经干细胞分化的影响以及脊髓组织病理变化情况.结果 自损伤后1周起实验组大鼠的BBB评分明显高于对照组.组织学观察实验组脊髓形态恢复较好逐渐趋于正常.实验组脊髓组织中新分化的神经元细胞数较对照组明显增多,同时新分化的星型胶质细胞数显著减少.结论 Ng1基因能够诱导脊髓损伤后内源性神经干细胞分化为神经元,促进脊髓运动功能的修复.     

Neurogenesin-1基因促进脊髓损伤后功能修复的实验研究

目的 探讨Neurogenesin-1(Ng1)基因对脊髓损伤后功能恢复的影响.方法 将36只大鼠随机分为实验组和对照组,每组18只.采用改良Allen法制备大鼠胸10脊髓损伤模型后,通过Alzet微泵分别向实验组和对照组持续转染入Ng1重组质粒和空白质粒.术后各时间点BBB评分系统监测大鼠运动功能恢复情况,并于术后第2周和第4周时分别取材,应用神经细胞特异性免疫荧光双标染色和组织学观察Ng1基因对内源性神经干细胞分化的影响以及脊髓组织病理变化情况.结果 自损伤后1周起实验组大鼠的BBB评分明显高于对照组.组织学观察实验组脊髓形态恢复较好逐渐趋于正常.实验组脊髓组织中新分化的神经元细胞数较对照组明显增多,同时新分化的星型胶质细胞数显著减少.结论 Ng1基因能够诱导脊髓损伤后内源性神经干细胞分化为神经元,促进脊髓运动功能的修复.     

Detection of gene expression pattern in the earyly stage after spinal cord injury by gene chip

Objective:To study the changes of the gene expression pattern of spinal cord tissues in the early stage after injury by DNA microarray (gene chip).Methods:The contusion model of rat spinal cord was established according to Allen‘s falling strike method and the gene expression patterns of normal and injured spinal cord tissues were studied by gene chip.Results:The expression of 45 genes was significantly changed in the early stage after spinal cord injury, in which 22 genes up-regulated and 23 genes down-regulated.Conclusions:The expression of some genes changes significantly in the early stage after spinal cord injury, which indicates the complexity of secondary spinal cord injury.     

Detection of gene expression pattern in the early stage after spinal cord injury by gene chip

Ｓｐｉｎａｌｃｏｒｄｉｎｊｕｒｙ (ＳＣＩ )ｉｓａｍｕｌｔｉｆａｃｔｏｒｐａｔｈｏｌｏｇｉｃａｌ ｐｒｏｃｅｓｓ ,ｗｈｉｃｈｄｅｔｅｒｍｉｎｅｓｔｈｅｃｏｍｐｌｅｘｉｔｙｉｎｉｔｓｅｖｏｌｕｔｉｏｎａｎｄｔｈｅｄｉｆｆｉｃｕｌｔｙｉｎｔｈｅｒａｐｙ ,ｓｏｔｈｉｓｉｓｗｈｙｗｅｃａｎｎｏｔｅｘｐｅｃｔｓａｔｉｓｆａｃｔｏｒｙｒｅｓｕｌｔｉｆｏｎｌｙａｆｅｗｆａｃｔｏｒｓａｒｅｃｏｎｓｉｄｅｒｅｄ .Ｇｅｎｅｃｈｉｐ(ＤＮＡｍｉｃｒｏａｒｒａｙ)ｉｓａｎｅｗｌｙｄｅｖｅｌｏｐｅｄｔｅｃｈｎｉｑｕｅｃｈａｒ…     

促红细胞生成素在大鼠脊髓缺血再灌注损伤中的表达及其意义

目的:观察促红细胞生成素(EPO)在大鼠脊髓缺血再灌注损伤(SCII)中的表达和重组人促红细胞生成素(rhuEPO)预处理对再灌注损伤脊髓神经细胞的作用。方法:将W ister大鼠分为正常组、假手术组、rhuEPO处理组和生理盐水对照组;rhuEPO处理组和生理盐水对照组于术前3h腹腔注射rhuEPO和生理盐水,制备大鼠脊髓缺血再灌注损伤模型。以免疫组化和W estern blot法检测脊髓组织中EPO的表达变化;以原位末端脱氧核糖核苷酸转移酶介导dUTP标记法(TUNEL法)检测细胞的凋亡情况。结果:EPO在无损伤脊髓中即有少量的表达,SCII后8h表达显著上调,于12、24h(12h与24h组比较差异无显著性意义,P>0.05)达高峰,伤后3d表达逐渐下调,5d仍保持较高水平。rhuEPO处理组SCII后8h、12h及24h时神经细胞凋亡水平明显低于生理盐水对照组,差异有显著性意义(P<0.01)。结论:在脊髓缺血再灌注损伤中EPO呈现时序性表达变化,可能是机体内源性神经保护的机制之一;EPO预处理能明显抑制SCII后神经细胞的凋亡。     

神经干细胞静脉移植治疗脊髓损伤的实验研究

[目的]观察神经干细胞静脉移植对损伤大鼠脊髓功能的治疗作用。[方法]取孕14—16dSD胎鼠的脑室下区组织，体外培养后鉴定细胞。制作脊髓全切模型，伤后1周将Brdu标记好的神经干细胞通过尾静脉注射移植到大鼠体内，移植后及8周行皮层体感诱发电位（CSEP）检测和BBB功能评分，并留损伤脊髓处作病理切片及免疫组化染色。[结果]（1）移植后8周BBB评分损伤组、移植组都有所恢复，但都未达到正常水平，移植组恢复较好；（2）模型制作后，CSEP波均消失，细胞移植后8周移植组的波形有不同程度的恢复，但潜伏期延长；（3）移植组大鼠脊髓损伤处存在大量Brdu染色阳性细胞，表明移植的细胞在体内可到达损伤脊髓处并能存活；脊髓损伤部位NF-200及GFAP染色阳性的细胞表明移植的细胞可以分化为具有神经元和胶质细胞特性的细胞。[结论]静脉移植的神经干细胞能到达损伤区代替受损的神经元及神经胶质细胞，使损伤的脊髓功能得到一定程度的恢复。     

神经干细胞对脊髓前角运动神经元保护作用的实验研究

目的观察臂丛根性撕脱伤后神经干细胞脊髓内移植对前角运动神经元的保护作用。方法取孕龄15～18d胎鼠脑组织，分离获得神经干细胞，在体外培养、扩增，并用5溴-2脱氧尿苷(BrdU)标记。取Wistar大鼠72只，随机分成实验组与对照组。先将C5～T1神经根撕脱，实验组把体外培养的神经干细胞移植于C5～T1脊髓节段前角附近，而对照组则用缓冲液替代神经干细胞。术后1、2、4、6、8、12周取脊髓标本进行组织学与免疫组化染色观察。结果臂丛根性撕脱伤后脊髓前角运动神经元数目明显减少，到术后12周时，对照组运动神经元减少达80．3％，实验组达63．7％。并且，各时间点实验组运动神经元的存活率均高于对照组。实验组脊髓前角内可见散在但仍保持未分化特征的神经干细胞。结论神经干细胞在植入臂丛根性撕脱伤的脊髓后能存活，并能明显减少前角运动神经元的继发性死亡。