NT-3-HUMSCs联合基因沉默SOCS3治疗SD大鼠脊髓损伤后的运动功能分析

An Analysis of Movement Function of SD Rats' Spinal Cord Injury After Joint Therapy Using NT-3-HUMSCs and SOCS3 Gene Silencing

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(<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(<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.