嗅鞘细胞移植对脊髓全横断损伤大鼠大脑运动皮质胰岛素样生长因子1和睫状神经营养因子表达的影响

背景：多项研究已证实嗅鞘细胞能促进脊髓损伤大鼠神经功能的恢复，但其分子机制还不清楚。 目的：观察嗅鞘细胞移植对脊髓全横断大鼠大脑运动皮质神经营养因子胰岛素样生长因子1和睫状神经营养因子mRNA表达的影响。 设计、时间及地点：随机对照动物实验，于2007-07/2008-12在昆明医学院神经科学研究所完成。 材料：清洁级健康成年雌性SD大鼠88只，随机分成3组：假手术组8只、模型组40只、细胞移植组40只。另取新生一两天的GFP转基因小鼠5只用于分离培养嗅鞘细胞。 方法：模型组、细胞移植组大鼠建立T9脊髓全横断损伤模型，假手术组仅行T8椎板切除。造模后，细胞移植组吸取嗅鞘细胞悬液15 μL(约3×105个细胞)滴加到约2 mm3的明胶海绵上，将含有嗅鞘细胞的明胶海绵植入大鼠脊髓两横断面之间的间隙处。分别于移植后3，7，14，21，28 d取大脑运动皮质行RT-PCR检测。 主要观察指标：采用免疫细胞化学法对培养的嗅鞘细胞进行鉴定，RT-PCR法检测大脑运动皮质胰岛素样生长因子1和睫状神经营养因子mRNA的表达变化。 结果：培养的嗅鞘细胞p75-NGFR阳性率>90%，主要为双极细胞，突起较长。与假手术组比较，模型组术后3 d睫状神经营养因子表达明显升高，7，14，21 d降至无明显差异，至28 d明显降低(P < 0.05)；术后各时间模型组胰岛素样生长因子1的表达无明显差异(P > 0.05)。与模型组比较，移植后14 d细胞移植组胰岛素样生长因子1表达明显增多(P < 0.05)；移植后3 d睫状神经营养因子的表达明显减少(P < 0.05)，但移植后7，14，21 d表达逐渐回升，至28 d明显高于模型组水平(P < 0.05)。 结论：嗅鞘细胞移植能够促进脊髓损伤修复，其机制可能与促进大脑运动皮质胰岛素样生长因子1 mRNA的表达上调，并在初期抑制睫状神经营养因子mRNA的表达有关。

Effect of olfactory ensheathing cells transplantation on the expressions of insulin-like growth factor 1 and ciliary neurotrophic factor in the motor cortex of rats subjected to cord transection

BACKGROUND: Many studies demonstrated that olfactory ensheathing cells (OECs) transplantation has promotion effect on the recovery from spinal cord injury. However, the underlying molecular mechanism is poorly understood. OBJECTIVE: To observe the effect of OECs transplantation on expressions of ciliary neurotrophic factor (CNTF) and insulin-like growth factor 1 (IGF-1) mRNA in the motor cortex of rats subjected to spinal cord transection. DESIGN, TIME AND SETTING: The randomized control animal experiment was performed at Institute of Neuroscience, Kunming Medical College from July 2007 to December 2008. MATERIALS: Totally 88 adult, female, Sprague Dawley rats were randomly divided into sham operation (n=8), model (n=40) and OECs transplantation (n=40) groups. Additional 5 GFP transgenic mice with 1-2 days old were used to prepare OECs. METHODS: Model of spinal cord transection was established by cut transversely rats T9 segment in the model and OECs transplantation groups. Rats in the sham operation group only subjected to laminectomy at T8. After the spinal cord was exposed at the lesion site, a small piece of 2 mm3 gel foam soaked with 3×105 OECs were implanted into the gap to fill the lesion site of the T9 level in rats. RT-PCR was performed at the days 3, 7, 14, 21 and 28 after operation. MAIN OUTCOME MEASURES: Inverted microscope was used to detect the morphology and growth patterns of cultured OECs. RT-PCR was employed to detect gene expression of IGF-1 and CNTF. RESULTS: The purity of OECs was estimated more than 90% by p75 immunostaining, which were bipolar cell with long cell process. Compared to the sham operation group, the gene expression of CNTF in the motor cortex of model group increased significantly at 3 days, decreased at days 7, 14 and 21, especially obviously decreased at 28 days after operation (P < 0.05). However, there was no statistical difference in the expression of IGF-1 (P > 0.05). The expression of IGF-1 in motor cortex of OECs transplantation group was dramatically increased than that of the model group (P < 0.05). While CNTF decreased at day 3 after transplantation, which increased at days 7, 14 and 21, ultimately, greater than the model group at day 28 after operation (P < 0.05). CONCLUSION: The changes of CNTF and IGF-1 mRNA in the motor cortex may contribute to the functional neurological improvement of the rats subjected to spinal cord transection following OECs transplantation.