脑源性神经营养因子前体对小鼠海马神经元存活和突起生长的抑制作用

Inhibitory effects of brain-derived neurotrophic factor precursor on viability and neurite growth of murine hippocampal neurons

目的探讨脑源性神经营养因子前体(proBDNF)对小鼠海马神经元存活和突起生长的影响,以及p75神经营养素受体(p75NTR)在其中的介导作用。方法取p75NTR转基因18日龄胎鼠(野生型p75NTR+/+,基因敲除型p75NTR–/–)海马组织,进行新生海马神经元原代培养。p75NTR+/+基因型神经元设对照组、proBDNF(30ng/ml)组和proBDNF(30ng/ml)+p75/Fc(30μg/ml)组,p75NTR–/–基因型神经元设对照组和proBDNF(30ng/ml)组。采用MTT法检测各组处理24h后新生海马神经元的存活情况,免疫荧光染色观察各组处理72h后的突起长度。结果经MAP2和DAPI双重荧光染色鉴定,成功培养出高纯度新生海马神经元。MTT检测结果显示,在p75NTR+/+型海马神经元中,proBDNF组570nm处吸光度值(A570)明显小于对照组(P<0.05),加入p75NTR受体竞争性抑制剂p75/Fc后与对照组相比差异无统计学意义(P>0.05);在p75NTR–/–型海马神经元中,proBDNF组A570值与对照组差异无统计学意义(P>0.05)。免疫荧光染色显示,p75NTR+/+神经元proBDNF组神经突起长度明显小于对照组(P<0.05),proBDNF(30ng/ml)+p75/Fc(30μg/ml)组与对照组比较差异无统计学意义(P>0.05);p75NTR–/–神经元proBDNF组神经突起长度与对照组比较差异无统计学意义(P>0.05)。结论 proBDNF通过p75NTR受体途径抑制神经元的存活和突起生长,具有毒性作用。

Objective To explore the mediation effect of p75 neurotrophin receptor （p75NTR） in the effect of brain- derived neurotrophic factor precursor （proBDNF） on viability and neurite growth of murine hippocampal neurons. Methods Hippocampal neurons were obtained from p75NTR^＋/^＋ and p75NTR/ 18-day mice and primarily cultured. For p75NTR^＋/^＋ neurons, three experimental groups were set, i.e. control, proBDNF （30ng/ml）, and proBDNF （30ng/ml）＋p75/Fc （30μg/ml） groups. For p75NTR-/- neurons, two experimental groups were set, i.e. control and proBDNF （30ng/ml） groups. MTT assays were performed after 24h to examine the viability of neonatal primary neurons. Immunofluorescent staining was conducted after 72h to investigate the neurite length. Results With MAP2 and DAPI double fluorescent staining it was identified that the neonatal hippocampal neurons were successfully cultured in vitro with high purity. For viability assay of p75NTR^＋/^＋ neurons, it was found that the absorbance value at 570nm （A_570） in proBDNF group was significantly lower than that in control group （P〈0.05）, but no difference was observed when co-cultured with p75/Fc. For viability assay of p75NTR-/- neurons, no difference was observed between proBDNF group and control group （P〉0.05）. With neurite growth assay ofp7SNTR^＋/^＋ neurons, it was found that the neurite length in proBDNF group was significantly shorter than that in control group （P〈0.05）, but no difference was observed when co-cultured with p75/Fc （P〉0.05）. With neurite growth assay of p75NTR-/- neurons, no difference in neurite length was observed between proBDNF group and control group. Conclusion proBDNF may inhibit the neuronal viability and neurite growth via p75NTR.