嗅球成鞘细胞植入大鼠挫伤脊髓内的迁移分布特征

目的 观察嗅球成鞘细胞(olfactory ensheathing cells,OECs)在损伤脊髓内的迁移分布与脊髓功能恢复的关系.方法 用NYU-impactorⅡ装置以10 g·25 mm损伤大鼠T10脊髓,1周后将急性分离、纯化、鉴定的绿色荧光蛋白(GFP)-大鼠OECs植入脊髓损伤部位及其首尾两侧,OECs的移植量为90 000/μl.在移植后13周时间内,镜下定性观察OECs在冰冻切片上的迁移分布特征,然后对其分布面积和长度进行半定量观察.脊髓运动功能用BBB评分法测定.结果 移植后早期OECs主要聚集在移植部位,并逐渐向周围迁移扩散,但主要沿脊髓纵轴向首尾方向扩展,脊髓空腔内也可见移植的OECs.OECs的分布面积和长度分别由术后1周时的1.33 mm2和4.23 mm逐步扩大到13周时的3.30 mm2和7.68 mm.与此同时,大鼠损伤脊髓的运动功能也得到逐步恢复.结论 OECs植入挫伤脊髓后可以迁移游走,且与大鼠脊髓运动功能恢复有一定关系.

Abstract：

Objective To observe the migration and distribution of OECs in injured spinal cord and discuss their relation with the recovery of spinal cord function. Methods The rats were contused by a force of 10 g · 25 mm with NYU-impactor at T10 level. The OECs acutely isolated from green fluorescence protein (GFP) rats were purified, identified and then transplanted into the injured site and the rostral and caudal parts of the spinal cord one week after injury, with total volume of the transplanted OECs for 90 000/μl. Within 13 weeks after transplantation, the migration and distribution of OECs were qualitatively observed on the cryo-sections under fluorescence light microscope. The area and the length of OECs distribution were semi-quantitatively determined. The locomotor function of the spinal cord was appraised by BBB score. Results OECs were located collectively in the transplanted site at early stage after transplantation and then spread gradually mainly along the long axis of the cord. OECs could be found in the cavity of the contused spinal cord. The area and the length of OECs distribution were increased from 1.33 mm2 and 4.23 mm respectively at one week to 3.30 mm2 and 7.68 mm respectively at 13 weeks after transplantation. In the meantime, the locomotor function was gradually improved. Conclusion OECs can migrate within the contused spinal cord, as may contribute to the recovery of locomotor function.     

嗅鞘细胞移植到大鼠损伤脊髓前后的形态学研究

目的:观察体外培养和植入损伤脊髓的自发绿色荧光嗅鞘细胞(Green fluorescent protein-olfactory ensheathing cells,GFP-OECs)的形态结构特征,研究绿色荧光蛋白(Green fluorescent protein,GFP)及脊髓损伤的病理环境对其形态的影响.方法:用酶消化法分离培养来自2.5月龄的绿色荧光蛋白转基因大鼠嗅球最外层的嗅鞘细胞,对培养细胞定期进行光镜、电镜及激光共聚焦显微镜观察并摄片,观察其在体外培养的形态特征.培养第10d,用显微外科技术将嗅鞘细胞移植到脊髓挫伤后的T10节段,分别在移植术后7、15 d取材,观察细胞植入损伤脊髓后光电镜下形态.结果:GFP-OECs在荧光显微镜下呈绿色强荧光,培养第7 d,为双极样(梭形或纺锤形)、三极样和扁圆形3种典型的细胞形态,以双极梭形为主;培养第10d,细胞形态为梭形,植入损伤脊髓后,移植细胞胞体呈长梭形,一端突起细长,与另一嗅鞘细胞接触,呈条索状生长.电镜下嗅鞘细胞核不规则,胞质内有丰富的粗面内质网、游离核糖体及线粒体.结论:GFP不影响嗅鞘细胞(Olfactory ensheathing cells,OECs)形态.在脊髓损伤处,OECs胞体呈长梭形,突起细长,与培养10d移植时比较,形态无显著改变,早期脊髓损伤病理环境不影响GFP-OECs形态.     

Brain-derived neurotrophical factor after olfactory ensheathing cells transplantation in spinal cord injury of rats

<正>Objective To observe the expression of brain - derived neurotrophical factor ( BDNF) in injury spinal cord after transplantation olfactory ensheathing cells ( OECs) , and to investigate the mechanism of OECs repairing spinal cord injury. Methods OECs from GFP transgenic rats were separated and cultured for transplantation. Spinal cord injury rats were separated two groups by random     

Survival and number of olfactory ensheathing cells transplanted in contused spinal cord of rats

Objective： To observe the survival and the number of olfactory ensheathing cells （OECs） transplanted in the contused spinal cord, so as to provide a basis for further studying the biological action of OECs.Methods： The rat spinal cords were contused with NYU-impactor Ⅱ at T10 level by dropping a 10 g rod from a height of 25 mm. At the 1st week after injury, OECs isolated freshly from green fluorecense protein （GFP） of the rats were transplanted into the spinal cord at injured site and other two sites 1 mm apart from the caudal and rostral ends with the OECs number of 30000/μl×3 =90000. The survival and the number of OECs were qualitatively and semi-quantitatively observed under the fluorescense microscope from 1 week to 13 weeks after transplantation. The motor function of the cord was evaluated with BBB score.Results： GFP-OECs could survive at least for 13 weeks within the contused spinal cord. Their arrangement was from tight to loose and their number was decreased from 1 week to 13 weeks after injury. The average number of GFP-OECs was 536 at the 1st week, which was less than 1% of the number as compared with original transplantation. After then, the number of GFP-OECs was continually decreased,but the most obvious decrease was found during 1 week to 2 weeks. The extent of decrease at other time points was relatively mild. In contrast to the cell number, motor function of the cord was gradually recovered after transplantation.Conclusions： The survival and the number of GFPOECs are different between the animals and are affected by the pathological reaction of the host cord. Also it is related to the motor function recovery of the contused cord.     

嗅鞘细胞移植后大鼠损伤脊髓神经生长因子的表达

目的 研究嗅鞘细胞移植对大鼠损伤脊髓内的神经生长因子(NGF)表达的影响,以从NGF角度探讨嗅鞘细胞移植修复大鼠脊髓损伤的机制.方法 48只SD大鼠用NYU -Ⅱ撞击机(10g-25 mm)损伤T10脊髓制作脊髓损伤(SCI)模型,随机分为嗅鞘细胞组、DMEM组各24只;另设立正常对照组6只.将GFP-嗅鞘细胞细胞悬液移植入嗅鞘细胞组大鼠损伤处,DMEM组用单纯的DMEM/F12液代替,正常对照组不做任何处理.移植术后1d、7d、14 d、21 d,用BBB评分法测定脊髓运动功能,RT - PCR方法比较各时间点NGF表达差异.移植术后第21天应用免疫组化比较嗅鞘细胞组、DMEM组、正常对照组大鼠脊髓损伤区NGF的表达差异.结果 移植后1d、7d、14 d、21 d,嗅鞘细胞移植组运动功能评分均高于同期DMEM组.NGF表达量在术后第1天最高,第7天达峰值,之后缓慢降低.移植后第21天,嗅鞘细胞移植组脊髓NGF表达量高于同期DMEM组和正常对照组.结论 嗅鞘细胞移植可上调损伤脊髓NGF的表达,从而促进了损伤脊髓的修复.     

嗅鞘细胞在神经系统损伤修复中的作用

嗅鞘细胞是一种特殊的神经胶质细胞,也是目前唯一应用到临床治疗脊髓损伤的细胞。在神经系统损伤修复中,嗅鞘细胞具有改善损伤微环境、促进轴突再生、穿越胶质瘢痕、亲和神经元和导向神经再生方面的作用,为神经系统损伤修复及病变治疗提供了广阔的临床应用前景。     

嗅鞘细胞移植对大鼠损伤脊髓组织中BDNF表达的影响及意义

目的 观察嗅鞘细胞(OEC)移植治疗大鼠脊髓损伤的作用以及脊髓损伤组织中脑源性神经营养因子(BDNF)表达的变化,从神经营养因子角度探讨OEC移植修复脊髓损伤的机制.方法 分离和培养绿色荧光蛋白转基因大鼠的OEC,备移植用.取SD大鼠制备脊髓损伤模型,用随机数字表法将建模成功后的SD大鼠分为2组.实验组:建模成功后立即进行OEC移植,移植部位为脊髓损伤处及其首尾正中血管的左右两侧;对照组:用DMEM/F12培养液替代OEC悬液,操作同实验组.移植后对各组大鼠的运动功能进行评分,每周1次.采用实时逆转录聚合酶链反应检测BDNF mRNA的表达,采用免疫组织化学法检测BDNF蛋白的表达,取正常SD大鼠BDNF mRNA和蛋白的检测水平作为正常对照.结果 移植后两组大鼠的运动功能均逐渐改善,移植后21 d时,实验组大鼠运动功能评分明显高于对照组(P＜0.05).移植后OEC存活良好,实验组损伤的脊髓组织周围分布有大量的呈绿色荧光的OEC.移植后21 d时,实验组BDNF mRNA和蛋白的表达水平显著高于对照组和正常对照组(P<0.05),而对照组也显著高于正常对照组(P<0.05).结论 OEC移植可明显修复大鼠的脊髓损伤,其机制可能与OEC通过增强损伤脊髓组织中BDNF mRNA和蛋白的表达,改善局部微环境有关.

Abstract：

Objective To observe the expression of brain-derived neurotrophical factor (BDNF) in injury spinal cord after transplantation olfactory ensheathing cells (OECs), and to investigate the mechanism of OECs repairing spinal cord injury.Methods OECs from GFP transgenic rats were separated and cultured for transplantation. Spinal cord injury rats were separated two groups by random digits table. In experimental group, OECs suspension were transplanted into injured spinal cord following spinal cord injury. In control group, DMEM was transplanted into the injured spinal cord after spinal cord injury. Motor function was evaluated per week after transplantation. The expression levels of BDNF mRNA and protein were detected by using RT-PCR and immunohistochemistry respectively, and compared with those from normal SD rats.Results Motor function of two groups was improved gradually after transplantation. The motor function scores in experimental group was obviously higher than in control group at 21st day after transplantation (P<0.05). A lot of survival GFP OECs distributed around impaired myeloid tissue. At 21st day after transplantation, BDNF mRNA and protein expression in experimental group were strongest (P<0.05), and stronger in control group than in normal group (P<0.05).Conclusion The transplantation of OECs can repair the injured spinal cord by increasing the expression of BDNF mRNA and protein to improve local microenvironment.     

嗅鞘细胞移植到损伤脊髓存活时间的研究

目的观察植入损伤脊髓的自发绿色荧光的嗅鞘细胞（green fluorescent protein-olfactory ensheathing cells,GFP-OECs）,研究移植后嗅鞘细胞的存活时间。方法用酶消化法分离培养来自2.5月龄的绿色荧光蛋白转基因大鼠嗅球最外层的嗅鞘细胞,培养第10天,用显微外科技术将嗅鞘细胞移植到脊髓挫伤后的T10节段,对照组移植术后未作特殊处理,实验组移植术后腹腔注射环孢菌素A,两组分别在移植术后1、2、4、8周取材,观察细胞植入损伤脊髓后存活情况。结果对照组在移植术后2周内可见存活的嗅鞘细胞;实验组在移植术后4周仍可见存活的嗅鞘细胞,8周未见存活的嗅鞘细胞。结论嗅鞘细胞移植后存活时间小于8周。     

年轻成年GFP大鼠嗅鞘细胞的分离培养与形态学特征

目的 建立年轻成年GFP大鼠嗅鞘细胞(OECs)的分离培养方法 ,为OECs移植治疗脊髓损伤奠定基础. 方法 解剖显微镜下、无菌环境取2.5月龄的GFP大鼠嗅球最外层,经酶消化法分散后将细胞接种于含20%胎牛血清的DMEM/F12培养基中.定期进行光电镜观察并摄片.在培养第10天进行S-100和NGFRp75的免疫细胞化学染色鉴定,并计算OECs的纯度. 结果 培养的GFP-OECs在荧光显微镜下呈绿色强荧光,形态以双极、多突起形为主,呈S-100、NGFR p75阳性,纯度在95%以上.电镜下形态与光镜下一致,但细胞结构更为精细. 结论 本文所用方法 简便易行,可分离出高纯度的GFP-OECs,其形态学特征与生物学活性无变异.源自GFP转基因大鼠的OECs可以作为一种有效的工具细胞,广泛应用于OEC在脊髓损伤修复中的研究.