文献类型：期刊文献

中文题名：电针联合HIF-1α、GDNF基因修饰NSCs移植对大鼠脊髓损伤治疗作用

英文题名：Effect of Electroacupuncture Combined with HIF-1αand GDNF Gene Modified NSCs Transplantation on Spinal Cord Injury in Rats

作者：徐斌[1];李江[2];谭龙旺[2]

第一作者：徐斌

机构：[1]甘肃中医药大学,甘肃兰州730000;[2]陕西中医药大学附属医院,陕西咸阳712000

第一机构：甘肃中医药大学

年份：2022

卷号：24

期号：7

起止页码：91

中文期刊名：辽宁中医药大学学报

外文期刊名：Journal of Liaoning University of Traditional Chinese Medicine

收录：CSTPCD

基金：国家自然科学基金(81774349)。

语种：中文

中文关键词：电针治疗;脊髓损伤;DLL1;胶质纤维酸性蛋白;神经干细胞移植;低氧诱导因子-1α;胶质源性神经生长因子

外文关键词：electroacupuncture therapy;spinal cord injury;DLL1;GFAP;neural stem cell;HIF-1α;GDNF

摘要：目的研究电针联合低氧诱导因子-1α(HIF-1α)、胶质源性神经生长因子(GDNF)基因修饰的神经干细胞(NSCs)移植对急性脊髓损伤(SCI)大鼠模型的治疗作用及与Notch信号通路的相关性。方法提取神经干细胞,传代培养,并运用免疫荧光染色法鉴定;HIF-1α、GDNF基因修饰NSCs,并运用qRT-PCR法鉴定。SD大鼠(SPF级)48只,随机分为4组:A组(对照组)、B组(模型组)、C组(B组+HIF-1α、GDNF基因修饰NSCs移植)、D组(C组+电针治疗),每组12只大鼠。A组:正常饲养,不做任何处理;B组、C组、D组:采用改良Allen's法制作T11脊髓损伤模型,造模成功后,B组损伤部位注射生理盐水;C组、D组损伤部位进行HIF-1α、GDNF基因修饰的NSCs移植,且D组取双侧心、肺俞穴予电针治疗,1次/d,直至取材为止。于造模前及治疗的每个时间点采用背髓损伤的行为学评分(BBB评分)对大鼠进行行为学评分,实验结束后取脊髓损伤段运用HE染色法观察各组脊髓病理改变;免疫组化法检测胶质纤维酸性蛋白(GFAP)的表达;Western Blot法检测DLL1蛋白表达。结果巢蛋白(Nestin)免疫荧光染色结果示,所提取的细胞符合神经干细胞特征;运用qRT-PCR鉴定结果示,GDNF、HIF-1α转染后mRNA表达明显,表明NSCs双基因修饰成功。与A组比较,B组BBB评分明显降低,HE染色示,大量神经细胞坏死、变性,可见轴突肿胀和空泡出现,大量炎症细胞浸润,GFAP、DLL1蛋白表达明显增加(P<0.05,P<0.01);随时间推移,与B组比较,C组、D组BBB评分增加,HE染色示,神经细胞萎缩凋亡减少,神经纤维开始恢复,GFAP、DLL1蛋白表达均降低(P<0.05,P<0.01),D组在第3周时效果最显著(P<0.01)。结论电针督脉联合GDNF、HIF-1α基因修饰NSCs移植疗法通过调控Notch信号通路改善微环境,阻止瘢痕形成,防止神经细胞凋亡、萎缩,促进轴突生长及神经通路的再通,提高神经干细胞存活率,为其分化修复神经细胞创造条件,从而对SCI大鼠起治疗作用。
Objective To investigate the therapeutic effect of electroacupuncture combined with HIF-1αand GDNF gene modified NSCs transplantation on acute spinal cord injury(SCI)rat model and its correlation with Notch signaling pathway.Methods Neural stem cells were extracted,subcultured and identified by immunofluorescence staining.NSCs were modified with HIF-1αand GDNF genes and identified by qRT-PCR.Forty-eight SD rats(SPF grade)were randomly divided into 4 groups:group A(control group),group B(model group),group C(group B+HIF-1α,GDNF gene modified NSCs transplantation),and group D(group C+electroacupuncture treatment),with 12 rats in each group.Group A:normal feeding without any treatment;Group B,C,and D:T11 spinal cord injury model was established by modified Allen's method.NSCs modified by HIF-1αand GDNF gene were transplanted into the injured sites in group C and D,and electroacupuncture was performed at bilateral heart and lung acupoints once a day in group D until the samples were collected.BBB score was used to score the behavior of rats before modeling and at each time point of treatment,and HE staining was used to observe the pathological changes of spinal cord in each group.The expression of GFAP was detected by immunohistochemistry.DLL1 protein expression was detected by Western Blot.Results The results of Nestin immunofluorescence staining showed that the extracted cells were consistent with the characteristics of neural stem cells.The results of qRT-PCR showed that the mRNA expression of GDNF and HIF-1αwas obvious after transfection,indicating that NSCs double gene modification was successful.Compared with group A,BBB score of group B was significantly lower,HE staining showed A large number of nerve cell necrosis and degeneration,visible axon swelling and vacuole,a large number of inflammatory cell infiltration,GFAP,DLL1 protein expression was significantly increased(P<0.05,P<0.01).Compared with group B,BBB score of group C and D increased with time,HE staining showed that nerve cell atrophy and apoptosis decreased,nerve fibers began to recover,GFAP and DLL1 expression decreased(P<0.05,P<0.01),and group D was the most significant on 3 week(P<0.01).Conclusion Acusector dumai joint GDNF,HIF-1αgene-modified NSCs transplantation therapy through regulating the Notch signal pathway to improve the micro environment,to prevent scarring,prevent nerve cells apoptosis,contraction,promote axon growth and recanalization of neural pathways,to improve the survival rate stem cells,repair nerve cells to create conditions for its differentiation,treatment of SCI in rats.