Notch1信号通路对小鼠触液神经元体外增殖能力的调节作用  

Notch1 signal pathway promotes the proliferation of cerebrospinal fluid-contacting neurons in vitro

作　　者：何宇祺 林宗龙 王硕 陈立 王洪超 施晓会 豆晓伟 李青 HE Yuqi;LIN Zonglong;WANG Shuo

机构地区：[1]贵州医科大学临床医学院,贵阳市550025 [2]湖北省恩施土家族苗族自治州中心医院骨科,恩施市445000 [3]山东省日照市莒县人民医院骨科,276500 [4]四川省达州市中心医院骨科,635001 [5]贵州医科大学附属医院临床研究中心,贵阳市550004 [6]贵州医科大学附属医院创伤骨科,贵阳市550004

出　　处：《中国脊柱脊髓杂志》2021年第3期262-270,共9页Chinese Journal of Spine and Spinal Cord

基　　金：国家自然科学基金(81960234);贵州省自然科学基金[QKH-J(2020)1Y323]。

摘　　要：目的:探讨Notch1信号通路在调节小鼠触液神经元增殖中的作用。方法:(1)取出生24h内C57BL/6小鼠延髓组织,消化后提取细胞,经流式细胞分选得到触液神经元(cerebrospinal fluid-contacting neurons, CSFcNs)。将CSF-cNs悬浮培养并传代。(2)将CSF-cNs传至第2代,培养0h、24h、48h、72h、96h、120h时应用CCK-8法检测CSF-cNs光密度(optical density,OD)值,培养5d时应用免疫荧光检测CSF-cNs特异性标志物多囊肾病2型1通道蛋白(polycystic kidney disease 2 like 1,PKD2L1)与神经干细胞标志物Nestin、Sox2及增殖标志物Ki67共表达情况。(3)将第3代CSF-cNs分为4组:对照组、Jagged-1组、二甲基亚砜(dimethyl sulfoxide,DMSO)组、γ-分泌酶抑制剂(3,5-二氟苯乙酰基)-L-丙氨酰基-L-2-苯基甘氨酸叔丁酯[(3,5-difluorophenylacetyl)-L-alanyl-L-2-phenylglycine tert-butyl ester,DAPT]组。对照组采用无血清神经培养液培养;Jagged-1组采用无血清神经培养液+5μmol/L Jagged-1培养;DMSO组采用无血清神经培养液+0.05%DMSO培养;DAPT组采用无血清神经培养液+50μmol/L DAPT培养。培养0h、24h、48h、72h、96h、120h时应用CCK-8法检测各组CSF-cNs的OD值;培养5d时应用Western Blot检测各组CSF-cNs PKD2L1、Notch1、Notch受体胞内段(NICD)、Hes1、β-actin蛋白表达情况,应用免疫荧光法检测各组CSF-cNs增殖标志物Ki67蛋白荧光强度(arbitrary unit,A.U.)值。结果:(1)提取的细胞经流式细胞分选后得到的CSF-cNs纯度为(94.5±2.03)%,存活率为(93.64±2.35)%。CSF-cNs可连续传代并形成神经球。(2)第2代CSF-cNs培养0h、24h、48h、72h及96h时的OD值具有统计学差异(P<0.05),96h与120h的OD值无统计学差异(P=0.44)。CSF-cNs中PKD2L1可与Nestin、Sox2或Ki67共表达。(3)第3代CSF-CNS分组处理后各组细胞PKD2L1蛋白表达量无统计学差异(P=0.27)。Jagged-1组细胞Notch1、NICD、Hes1蛋白表达量均较对照组升高(P<0.01);72h、96h、120h的OD值较对照组均显著性升高(P_(72h)=0.03,P_(96h)=0.Objectives:To explore whether Notch1 signaling pathways regulate the proliferation of cerebrospinal fluid-contacting neurons(CSF-cNs).Methods:(1)The peripheral nerve tissue of the upper central canal of the cervical spinal cord of C57BL/6 mice was extracted within 24 hours after birth,and the CSF-cNs were sorted and purified by fluorescence-activated cell sorter(FACS).CSF-cNs were cultured in suspension and subcultured.(2)The CSF-cNs was passaged to the second generation,and the CSF-cNs’optical density(OD)value was detected by CCK-8 at 0 h,24 h,48 h,72 h,96 h,and 120 h.After 5 days of culture,the co-expression of CSF-cNs specific marker polycystic kidney disease-2-like-1(PKD2L1)and neural stem cell markers(Nestin and Sox2),and proliferation marker(Ki67)was detected by immunofluorescence.(3)The third generation CSF-cNs suspended in vitro were divided into 4 groups.Control group:serum-free nerve culture medium;Dimethyl sulfoxide(DMSO)group:serum-free nerve culture medium+0.05%DMSO;(3,5-difluorophenylacetyl)-L-alanyl-L-2-phenylglycine tert-butyl ester(DAPT)group:serum-free nerve culture medium+50 umol/L DAPT(0.05%DMSO configuration);Jagged-1 group:serum-free nerve culture medium+5 umol/L Jagged-1.The OD value of CSF-cNs was detected by CCK-8 at 0 h,24 h,48 h,72 h,96 h,and 120 h.After 5 days of culture,the expression of PKD2L1,Notch1,NICD,Hes1,andβactin proteins was detected by Western Blot.The fluorescence intensity arbitrary unit(A.U.)value of Ki67 protein,a marker of cell proliferation,was detected by immunofluorescence.Results:(1)After FACS,the purity of CSF-CNS was(94.5±2.03)%,and the survival rate was(93.64±2.35)%.CSF-cNs can be continuously passaged down and form the neurospheres.(2)The OD value of the second-generation CSF-cNs was statistically different at 0 h,24 h,48 h,72 h,and 96 h(P<0.05),but there was no significant difference between 96 h and 120 h(P=0.44).PKD2L1 can be co-expressed with Nestin,Sox2 or Ki67 in CSF-cNs.(3)There was no statistical difference in the expression of PKD2L1 protein among

关 键 词：触液神经元 神经干细胞 Notch1信号通路 增殖 大鼠 

分 类 号：Q507[生物学—生物化学] Q813