目的 NT-3-HUMSCs联合基因沉默SOCS3治疗SD大鼠脊髓损伤,以期改善损伤神经运动功能.方法(1)用贴壁法体外培养人脐带间充质细胞(HUMSC),同时进行分离,提纯和鉴定; (2)构建NT-3基因真核表达载体,利用基因转染技术将其转入HUMSC,构建NT-3- HUMSC细胞,体外检测其存活情况及NT-3表达情况; (3)筛选作用于 SOCS3 的特异性靶点,进行序列同源性分析,设立阴性对照,设计并合成SOCS3-siRNA,同时在体外检测功能; (4)建立SD大鼠脊髓损伤模型分为为:假手术组10只; T12全脊髓横断损伤模型 40 只,随机分为 4 组,生理盐水治疗组 10 只; siRNA + NT-3-HUMSCs 治疗组 10 只;NT-3-HUMSCs治疗组10只;SOCS3-siRNA治疗组10只.以上各组造模成功后,分别存活1、2月和3月进行运动功能评价.结果(1) siRNA+NT-3-HUMSCs联合治疗SD大鼠脊髓损伤组BBB评分明显高于NT-3-HUMSCs、siRNA和生理盐水组,差异有统计学意义(P＜0.05); (2) siRNA+NT-3-HUMSCs联合治疗SD大鼠脊髓损伤组爬网格实验表明神经功能恢复明显高于NT-3-HUMSCs、siRNA和生理盐水组,差异有统计学意义(P＜0.05).结论 NT-3-HUMSCs联合基因沉默SOCS3治疗SD大鼠脊髓损伤,可以改善SD大鼠脊髓损伤的运动功能.%Objective To achieve the purpose of promoting movement function of the injury nerve by using the joint therapy of NT- 3- HUMSCs and SOCS3 gene silencing on SD rats'spinal cord injury. Methods (1) We used adherence method in vitro human umbilical cord-derived mesenchymal cells (HUMSC) during separation, purification and identification. (2) Then constructed NT-3 gene eukaryotic expression vector, which was transfected into its HUMSC, and constructed NT-3- HUMSC cell survival in vitro assay conditions and NT-3 expression. (3) We selected specific targets for SOCS3 screening and for sequence homology analysis. A negative control group was established. siRNA was designed and synthesized in vitro detection. (4) SD rats with spinal cord injury model were divided into two categories: (1) sham group with 10 rats; (2) T12 whole spinal cord injury model with 40 rats. The 40 rats were randomly divided into four groups with 10 rats in each group (saline treatment group,siRNA +NT-3-HUMSCs treatment group,NT-3-HUMSCs treatment group and siRNA treated group) . Motor function of the rats were evaluated respectively in 1, 2 and 3 months after the modeling was established successfully.Results(1) siRNA + NT-3-HUMSCs treatment group's BBB scores was significantly higher than NT-3-HUMSCs, SOCS3-siRNA and physiological saline groups ( P＜0.05) . (2) The grid climbing experiments showed that the neural functional recovery performed better in siRNA+the NT- 3- HUMSCs treatment group compared to the NT - 3 - HUMSCs, SOCS3 - siRNA and physiological saline groups (P＜0.05) . Conclusion The NT- 3- HUMSCs joint SOCS3 gene silencing in the treatment of SD rat spinal cord injury can improve the motor function of SD rat spinal cord injury.
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