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

中文关键词:  触液神经元  神经干细胞  Notch1信号通路  增殖  大鼠

英文关键词:Cerebrospinal fluid-contacting neurons  Neural stem cells  Notch1 signal pathway  Proliferation  Rat

中文摘要:

  【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, CSF-cNs）。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（P72h=0.03，P96h=0.02，P120h=0.01）；Ki67蛋白A.U.值较对照组显著性升高（P＜0.01）。DAPT组细胞Notch1、NICD、Hes1蛋白表达量较对照组均显著性减低（P＜0.01）；72h、96h、120h的OD值与对照组比较均显著性降低（P<0.01）；Ki67蛋白A.U.值较对照组显著性降低（P<0.01）。DMSO组各项指标与对照组比较均无统计学差异（P>0.05）。结论：CSF-cNs在体外具有神经干细胞潜能；激活Notch1信号通路可增强CSF-cNs的增殖能力，抑制Notch1信号通路可降低CSF-cNs的增殖能力。

英文摘要:

  【Abstract】 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 0h, 24h, 48h, 72h, 96h, and 120h. 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 + 50umol/L DAPT(0.05% DMSO configuration); Jagged-1 group: serum-free nerve culture medium + 5umol/L Jagged-1. The OD value of CSF-cNs was detected by CCK-8 at 0h, 24h, 48h, 72h, 96h, and 120h. After 5 days of culture, the expression of PKD2L1, Notch1, NICD, Hes1, and βactin 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 0h, 24h, 48h, 72h, and 96h(P<0.05), but there was no significant difference between 96h and 120h(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 different groups(P=0.27). In Jagged-1 group, the expression of Notch1, NICD and Hes1 proteins was higher than control group(P<0.01). At 72h, 96h and 120h, the OD value was significantly higher than control group(P72h=0.03, P96h=0.02, P120h=0.01). The A.U. value of Ki67 protein was significantly higher than control group(P<0.01). In DAPT group, the expression of Notch1, NICD and Hes1 proteins was lower than control group(P<0.01). At 72h, 96h and 120h, the OD value was significantly lower than control group(P<0.01). The A.U. value of Ki67 protein was significantly lower than control group(P<0.01). There was no statistical difference in all indexes between the DMSO group and the control group(P>0.05). Conclusions: Activating Notch1 signal pathway can enhance the proliferation ability of CSF-cNs, while silencing Notch1 signal pathway can reduce the proliferation ability of CSF-cNs.