【摘要】 背景:现有研究表明嗅鞘细胞移植可促进脊髓半横断损伤大鼠后肢运动功能的恢复,但多数实验是以动物行为学评分作为观察指标。目的:实验拟进一步观察嗅鞘细胞移植后6周脊髓半横断损伤大鼠后肢小腿三头肌组织形态学的变化。设计、时间及地点:细胞观察,于2006-10/2007-03在四川大学华西基础医学与法医学院神经生物学开放实验室完成。材料:清洁级2.5月龄雌性SD大鼠40只,由自四川大学华西医学中心提供。细胞培养过程中使用的阿糖胞苷为ROCHE产品。方法:5只大鼠麻醉后取出脑双侧嗅球,采用差速贴壁与阿糖胞苷抑制相结合的方法分离培养纯化嗅鞘细胞,第14天分别收集培养上清液和密度为1×1010L-1细胞悬液备用。剩余35只大鼠设立两组:正常组5只,不做任何处理;模型组30只,于T11~12节段建立脊髓半横断损伤模型后,又分为5个亚组:8只移植纯化的嗅鞘细胞悬液,6只移植纯化的嗅鞘细胞培养上清液,6只移植未纯化的嗅鞘细胞悬液(主要是嗅神经成纤维细胞),5只移植未纯化的嗅鞘细胞培养上清液,5只移植DMEM/F12培养液。移植量12μL/只,在脊髓半横断损伤上、下各1mm处多点注射移植4μL,同时在半横断处填充的明胶海绵内注入8μL。主要观察指标:移植后6周取左侧后肢小腿三头肌,苏木精-伊红染色观察肌细胞形态变化,图像分析软件测量肌细胞横截面积和直径。结果:①正常组肌细胞近似圆形或多边形,多核,核分布于外周。各移植组肌细胞均变小,核深染,其中移植DMEM/F12培养液组变化最为明显。②各移植组肌细胞横截面积及直径较正常组均明显减少(P<0.05),其中移植纯化的嗅鞘细胞组减少程度最轻(P<0.05),移植DMEM/F12培养液组减少程度最严重(P<0.05),移植未纯化的嗅鞘细胞、纯化/未纯化的嗅鞘细胞培养上清液3组间无明显差异(P>0.05)。结论:移植纯化的嗅鞘细胞悬液脊髓半横断损伤大鼠后肢小腿三头肌萎缩的程度最轻,肌细胞组织学变化最小。更多还原

【Abstract】 BACKGROUND: Olfactory ensheathing cell (OEC) transplantation can repair motor function in hind limbs of rats with hemisection of spinal cord injury (hSCI). Animal ethology was considered to be observational indexes in most trials. OBJECTIVE: To observe the histomorphological changes in hind limb triceps surae in rats with hSCI 6 weeks following OEC transplantation. DESIGN, TIME AND SETTING: The cell experiment was performed at the Opening Laboratory of Neurobiology, West China School of Preclinical and Forensic Medicine, Sichuan University from October 2006 to March 2007. MATERIALS: Forty 2.5-month-old female SD rats of clean grade were provided by West China Medical Center of Sichuan University. Arabinosylcytosine was purchased from ROCHE. METHODS: Bilateral bulbus olfactorius was collected from the brain of 5 rats after anaesthesia. Purified OECs were isolated and cultured by differential adherence and Arabinosylcytosine inhibition. At day 14, the transplanted cells were collected at a density of 1×1010 L-1. Remaining 35 rats were divided into 2 groups. Five rats in the normal group were intact. Thirty rats in the model group were used to establish hSCI at T11-12 and assigned into 5 subgroups. Eight rats were transplanted with purified OEC suspension. Six rats were transplanted with purified OEC culture supernatant. Six rats were transplanted with unpurified OEC suspension (mainly olfactory nerve fibroblasts). Five rats were transplanted with unpurified OEC culture supernatant. Five rats were transplanted with DMEM/F12 medium. 12 μL were transplanted into each rat. 4 μL was injected into many regions 1 mm upper and lower hSCI. 8 μL was injected into gelatin sponge at semisection. MAIN OUTCOME MEASURES: Six weeks after transplantation, the left triceps surae were dissected. Morological changes in myocytes were observed by hematoxylin-eosin staining. The cross section area and diameter of muscle fibers were measured with image analysis system. RESULTS: Myocytes were round and polygonal, with many nuclei around periphery. Myocytes became smaller, with dark stained nuclei in the transplanted groups, especially in the group transplanted with DMEM/F12 medium. The cross section area and diameter of skeletal muscle fibers in left triceps surae were lesser than normal group (P < 0.05). The decreasing extent was the least in the group transplanted with OECs (P < 0.05), whereas the greatest in the group transplanted with DMEM/F12 (P < 0.05). No significant difference was found in groups transplanted with unpurified OECs, purified/unpurified OEC culture supernatant (P > 0.05). CONCLUSION: The atrophy of triceps surae is relieved and histological changes in myocytes are small by transplantation of OECs in rats with hSCI.更多还原