电针联合GDNF基因修饰神经干细胞移植对Notch通路影响的实验研究

The Effects on Notch Pathway by Electroacupuncture Combined with GDNF Gene Modified Neural Stem Cell Transplantation

目的通过电针干预GDNF基因修饰的神经干细胞移植治疗大鼠脊髓损伤,探讨其能否对Notch信号通路起负反馈调节作用,对脊髓损伤大鼠起到治疗效果。方法制备GDNF基因修饰神经干细胞,并通过免疫荧光和Q-PCR对转染效果进行鉴定。将40只雄性SD大鼠随机分成A组(正常对照组)、B组(脊髓损伤组+生理盐水)、C组(脊髓损伤+GDNF基因修饰神经干细胞移植组)、D组(脊髓损伤+GDNF基因修饰神经干细胞移植+电针干预组)。各组分别在造模后3天、1周、2周、3周时间点进行取材,通过免疫组化技术、Western Blot技术对各组损伤脊髓局部GFAP、dll 1蛋白含量进行测定;以及通过BBB评分系统分析大鼠造模后各个时间点的运动功能,探讨治疗效果。结果(1)免疫组化检测发现C、D组较B组能明显抑制GFAP蛋白含量的表达,D组抑制更明显。且各时间点、各组之间比较差异明显(P﹤0.05)。(2)WB检测显示与A组比较,其他组脊髓损伤后的dll 1蛋白含量表达在不同时间点均有所增长,在3d和1周时dll 1蛋白含量高表达,1周时达到峰值,之后蛋白含量表达开始降低,在3周时含量表达最低;B组、C组、D组比较:在3d、1周、2周、3周dll1蛋白的表达B组>C组>D组。各组各时间点对比差异显著(P﹤0.05)。(3)BBB评分:B、C、D、三组各节点之间分别进行比较,差异性显著,具有统计学意义(P﹤0.05)。结论成功利用慢病毒载体构建GDNF基因修饰神经干细胞。移植后能够抑制GFAP蛋白及dll 1蛋白在脊髓损伤后的表达,对Notch信号通路起负反馈调节作用,从而使NSCs向目的细胞分化增殖,促进神经再生和功能恢复。并且经电针干预联合治疗后可以对疗效进一步放大。

Objective To investigate whether electroacupuncture can play a negative feedback role in the treatment of spinal cord injury in rats by GDNF gene modified neural stem cell transplantation.Methods Prepared GDNF gene modified neural stem cells,and identify the transfection effect by immunofluorescence and Q-PCR.40 male SD rats were randomly divided into A group(normal control group),B group(spinal cord injury group+saline),C group(spinal cord injury+GDNF gene modified neural stem cell transplantation group)D group(spinal cord injury+GDNF gene modified neural stem cell transplantation+electroacupuncture intervention group).At different time points(3 day,1 week,2 weeks,3 weeks after spinal cord injury modeling successfully).The content of local GFAP,dll 1 protein was determined by immunohistochemical technique and Western Blot technique;And the motor function of rats at all time points was analysed by BBB scoring system to explore the therapeutic effect.Results(1)Immunohistochemical examination showed that C,D group could significantly inhibit the expression of GFAP protein content compared with the B group,and D group was more obvious.The difference between each time point and each group was obvious(P﹤0.05).(2)WB detection showed that compared with A group,after spinal cord injury,the expression of dll 1 protein increased at different time points,dll 1 protein levels were high at 3 d and 1 weeks.After a peak at 1 weeks,protein levels began to decrease,lowest at 3 weeks.Compared betwen B,C,D group:dll1 protein expression in each time,B group>C group>D group.Each group had significant difference in each time points(P﹤0.05).(3)BBB score:B、C、D three groups of nodes were compared,the difference was significant and statistically(P﹤0.05).Conclusion GDNF gene modified neural stem cells were successfully constructed by lentivirus vector.It has a negative feedback regulation on the Notch signal pathway by inhibiting the expression of dll 1 protein and GFAP protein after spinal cord injury.Thus,make NSCs differentiation and proliferation to target cells,promote nerve regeneration and functional recovery.After electroacupuncture intervention combined treatment,it can amplify the curative effect further.