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本文旨在研究前体脑源性神经营养因子(precursor brain-derived neurotrophic factor,proBDNF)对螺旋神经节神经元(spiral ganglion neurons,SGNs)生长的影响。取新生5天Sprague Dawley(SD)大鼠处死,分离出含有螺旋神经节的蜗轴,将其消化、吹打后用培养液重悬,接种于多聚-D-赖氨酸/层粘连蛋白包被过的8孔腔式载玻片,37°C培养4 h获得贴壁牢固的原代SGNs。将SGNs随机分为对照组、BDNF组(10 ng/mL BDNF处理)、C10组(10 ng/mL proBDNF处理)、C50组(50 ng/mL proBDNF处理)、C100组(100 ng/mL proBDNF处理),无血清培养48 h后行βIII tubulin荧光抗体标记染色,荧光显微镜下观察SGNs的存活数目和神经突再生情况。结果显示,C50组和C100组的SGNs存活数目低于对照组(P<0.001),且C10组、C50组和C100组的SGNs存活数目均低于BDNF组(P<0.001)。根据SGNs的形态,可将其分为无突起和有突起两类。C10组、C50组、C100组的无突起SGNs比例均高于对照组,差异有统计意义(P<0.001)。另外在添加20μmol/L的特异性c-Jun氨基末端激酶(c-Jun Nterminal kinase,JNK)抑制剂SP600125后,C50组的SGNs存活数目提升至(91.5±8.2)/孔,高于没有使用SP600125的C50组(P<0.001)。以上结果说明,proBDNF减少体外培养SGNs的存活数目,并抑制SGNs神经突的出芽生长;使用特异性JNK抑制剂可以部分减轻proBDNF对SGNs存活的抑制作用。
This article aims to investigate the effects of pro brain derived neurotrophic factor (proBDNF) on the growth of spiral ganglion neurons (SGNs). Sprague Dawley (SD) rats were sacrificed on the 5th day of newborn, and the spiral axis of the spiral ganglion was isolated. After digestion, the cells were resuspended in the medium and inoculated into the poly-D-lysine / laminin package Obtained 8-well glass slides were incubated at 37 ° C for 4 h to obtain adherent primary SGNs. The SGNs were randomly divided into control group, BDNF group (10 ng / mL BDNF treatment), C10 group (10 ng / mL proBDNF treatment), C50 group (50 ng / mL proBDNF treatment), C100 group ) Were cultured in serum-free medium for 48 h before being stained with βIII tubulin fluorescent antibody. The number of surviving SGNs and the regeneration of neurites were observed under a fluorescence microscope. The results showed that the number of SGNs in C50 group and C100 group was lower than that in control group (P <0.001), and the number of SGNs in C10 group, C50 group and C100 group was lower than that in BDNF group (P <0.001). According to the morphology of SGNs, it can be divided into two types without protrusions and protrusions. The proportion of non-protuberant SGNs in C10, C50 and C100 groups was higher than that in control group, with statistical significance (P <0.001). In addition, the number of SGNs in C50 group increased to (91.5 ± 8.2) / well after adding 20μmol / L SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, higher than that without SP600125 C50 group (P <0.001). These results indicate that proBDNF can reduce the number of SGNs cultured in vitro and inhibit the growth of SGNs neurite outgrowth. Using specific JNK inhibitors can partially reduce the inhibitory effect of proBDNF on the survival of SGNs.