人参皂苷Rg1对大鼠坐骨神经冷冻保存后SCs活性及异体移植后神经再生的影响  

Effect of Ginsenoside Rg_(1) on Biological Activity of Cryopreserved Schwann Cells and Nerve Regeneration After Allograft in Rat Sciatic Nerve

作　　者：刘云霄 黄英如[1,2] 石一峰 张松 杨康 冼华 LIU Yunxiao;HUANG Yingru;SHI Yifeng;ZHANG Song;YANG Kang;XIAN Hua(College of Traditional Chinese Medicine(TCM),Chongqing Medical University,Chongqing 400016,China;Chongqing Key Laboratory of TCM for Prevention and Cure of Metabolic Diseases,Chongqing 400016,China)

机构地区：[1]重庆医科大学中医药学院,重庆400016 [2]中医药防治代谢性疾病重庆市重点实验室,重庆400016

出　　处：《中国实验方剂学杂志》2022年第13期52-61,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(81373668,81674002)。

摘　　要：目的:观察人参皂苷Rg(1 G-Rg1)对冷冻保存大鼠坐骨神经施万细胞(SCs)生物学活性影响,探讨G-Rg1减轻冷冻保存大鼠坐骨神经SCs损伤的可行性。方法:取SD大鼠双侧坐骨神经,随机分为7组:新鲜组、空白组、G-Rg1组(1×10^(-7)、1×10^(-6)、1×10^(-5)、1×10^(-4)、1×10^(-3)mol·L^(-1)G-Rg1),除新鲜组神经外,将其余各组神经依次置于含0、1×10^(-7)、1×10^(-6)、1×10^(-5)、1×10^(-4)、1×10^(-3)mol·L^(-1)G-Rg1的冷冻保存液中液氮保存4周。原位末端标记法(TUNEL)/S100检测各组神经SCs凋亡,蛋白免疫印迹法(Western blot)检测胱天蛋白酶(Caspase)-9、Caspase-3和主要组织相容性复合体(MHC)-Ⅰ、MHC-Ⅱ表达。将新鲜组和液氮保存4周的6组神经,置于37℃、5%CO_(2)培养箱中培养7 d,Western blot检测胶质细胞源性神经营养因子(GDNF)、神经生长因子(NGF)表达。用液氮保存4周的空白组和1×10^(-6)、1×10^(-5)、1×10^(-4)mol·L^(-1)G-Rg1组坐骨神经,同种异体移植修复Wistar大鼠坐骨神经10 mm缺损(空白移植组,1×10^(-6)、1×10^(-5)、1×10^(-4)mol·L^(-1)G-Rg1移植组),同时设新鲜坐骨神经同种异体移植、同系移植对照组。移植术后16周,电生理检测肌肉复合动作电位(CMAP)和神经传导速度(NCV),神经丝(NF)-200免疫荧光单染、透射电镜和甲苯胺蓝染色分析再生神经组织学。结果:与新鲜组比较,空白组和G-Rg1各浓度组Caspase-9、Caspase-3表达、SCs凋亡明显升高(P<0.05,P<0.01),GDNF、NGF表达及MHC-Ⅰ、MHC-Ⅱ表达显著降低(P<0.01)。与空白组比较,1×10^(-7)、1×10^(-6)、1×10^(-5)、1×10^(-4)mol·L^(-1)G-Rg1组Caspase-9、Caspase-3表达显著降低(P<0.01);1×10^(-7)、1×10^(-6)、1×10^(-5)、1×10^(-4)mol·L^(-1)G-Rg1组SCs凋亡降低(P<0.05,P<0.01),1×10^(-3)mol·L^(-1)组升高(P<0.05);1×10^(-7)、1×10^(-6)、1×10^(-5)、1×10^(-4)mol·L^(-1)G-Rg1组GDNF、NGF表达升高(P<0.05,P<0.01),1×10^(-3)mol·L^(-1)组降低(P<0.01);G-Rg1各浓度组MHC-Ⅰ、MObjective:To observe the effect of ginsenoside Rg1(G-Rg1)on the biological activity of cryopreserved Schwann cells(SCs)of the rat sciatic nerve and explore the feasibility of G-Rg1 in reducing the cryopreservation-induced injury in SCs.Method:Bilateral sciatic nerves of SD rats were randomly divided into a fresh group,a blank group,and five G-Rg1 groups of different doses(1×10^(-7),1×10^(-6),1×10^(-5),1×10^(-4),and 1×10-3 mol·L^(-1)).The nerves in the blank group and the G-Rg1 groups were preserved in liquid nitrogen solutions containing 0,1×10^(-7),1×10^(-6),1×10^(-5),1×10^(-4),and 1×10^(-3)mol·L^(-1)G-Rg1 for four weeks.The apoptosis of SCs was detected by TdT-mediated dUTP-biotin nick end labeling(TUNEL)/S100 immunofluorescence staining.The expression of cysteinyl aspartate-specific protease(Caspase)-9,Caspase-3,major histocompatibility complex(MHC)-Ⅰ,and MHC-Ⅱwas detected by Western blot.Subsequently,all nerves were cultured in the incubator at 37℃with 5%CO_(2) for 7 days.The expression of glial cell line-derived neurotrophic factor(GDNF)and nerve growth factor(NGF)was detected by Western blot.In addition,the above cryopreserved nerves in the blank group and the 1×10^(-6),1×10^(-5),and 1×10^(-4)mol·L^(-1)G-Rg1 groups were transplanted to the Wistar rats by allografting(blank transplantation group and the 1×10^(-6),1×10^(-5),and 1×10^(-4)mol·L^(-1)G-Rg1 transplantation groups),and fresh sciatic nerve allograft and isograft control group were set up.Sixteen weeks after transplantation,compound muscle action potential(CMAP)and nerve conduction velocity(NCV)were measured by electrophysiology.Nerve filament(NF)200 immunofluorescence staining,transmission electron microscopy,and toluidine blue staining were used to analyze the histology of the regenerated nerves.Result:Compared with the fresh group,the blank group and the G-Rg1 groups showed increased expression of Caspase-9,Caspase-3,and the apoptosis of SCs(P<0.05,P<0.01)and decreased expression of GDNF,NGF,MHC-Ⅰ,and MHC-Ⅱ(P<0.01).Co

关 键 词：人参皂苷Rg_(1) 周围神经冷冻保存 冷冻保存损伤 异体神经移植 神经再生 

分 类 号：R2-0[医药卫生—中医学] R33R289