proBDNF对培养鼠小脑颗粒细胞增殖和凋亡的影响

proBDNF inhibits cell proliferation and apoptosis in cultured cerebellar granule cells

目的观察脑源性神经营养因子前体(proBDNF)对培养的小鼠小脑颗粒细胞(CGCs)的增殖和凋亡的影响,探讨p75神经营养因子受体(p75NTR)和VPS10P-结构域家族成员sortilin的介导机制。方法采用出生后7 d(P7)的129SV转基因小鼠(p75NTR+/+和p75NTR-/-)进行CGCs的原代培养。分为BSA组(10 ng/mL)、proBDNF干预组(10 ng/mL)和proBDNF(10 ng/mL)+sortilin-Fc(20 ng/mL)干预组。免疫荧光染色法观察p75NTR和sortilin在培养细胞的表达,BrdU标记阳性增殖细胞以及TUNEL法检测凋亡细胞。结果成功培养出高纯度的原代小脑颗粒细胞。免疫荧光显示p75NTR和sortilin共同表达于野生型小鼠培养的小脑神经元胞膜和突起上。给予proBDNF干预后,p75NTR+/+型CGCs的BrdU阳性细胞比例较对照组明显减少(P<0.01),而TUNEL阳性细胞比例较对照组明显增加(P<0.01),且这两种效应都可被sortilin-Fc竞争性抑制并中和;p75NTR-/-型CGCs中各组之间BrdU阳性细胞以及TUNEL阳性细胞比例均无显著性差异(P>0.05)。结论 proBDNF可能通过与p75NTR、sortilin结合形成功能三聚体抑制神经细胞的增殖、促进细胞凋亡,proBDNF具有神经毒性作用。

Objective To determine the effect of brain-derived neurotrophic factor precursor （proBDNF） on the proliferation and apoptosis of cultured eerebellar granule cells （CGCs） and investigate the underlying mechanism of p75 neurotrophin receptor （p75NTR）/VPS10P-domain family member sortilin pathway. Methods CGCs was isolated from 7-day-old transgenic 129SV mice （p75NTR＋/＋ and p75NTR-/-） and then primarily cultured followed by identification. Then the cells were treated with BSA （ 10 ng/mL ） , proBDNF （ 10 ng/mL ） , proBDNF （ 10 ng/mL ） ＋ sortilin-Fc （ 20 ng/mL ） respectively. Immunofluorescent staining was performed to detect the expression of p75NTR and sortilin in the cultured cells. BrdU and TUNEL positive cells were detected to calculate the proportion of proliferation and apoptosis, respectively. Results Primary CGCs were successfully cultured with high purity. Immunofluorescent staining showed a co-expression of p75NTR and sortilin was seen in the cultured p75NTR＋/＋ cerebellar neurons, locating in the cell membrane and neurites, proBDNF treatment resulted in the percentage of BrdU positive cells were significantly decreased （P 〈0.01 ）, but the TUNEL positive cells increased （P 〈0.01 ） in p75NTR＋/＋ cultured CGCs. But these effects were inhibited and neutralized by sortilin-Fc. However, no such change was observed in p75NTR-/- cultured CGCs （P 〉 0.05 ）. Conclusion proBDNF inhibits the proliferation and apoptosis of CGCs probably via forming a triple with p75NTR and sortilin, and exerts a toxic effect on nervous system.