BDNF对体外培养的大鼠脊髓前角神经元内突触素Ⅰ与突触囊泡素表达的影响

EFFECTS OF BDNF ON SYNAPSIN I AND SYNAPTOPHYSIN EXPRESSION IN CULTURED RAT SPINAL CORD ANTERIOR HORN NEURONS

探讨BDNF对体外培养的大鼠脊髓前角神经元内突触素I与突触囊泡素(SYN)表达的影响。取孕14d大鼠子宫内胎鼠的脊髓腹侧部分神经元,体外有血清培养。在培养7d后,随机分成对照组、BDNF组和抗BDNF组。BDNF组培养液中加入BDNF(20ng/ml),抗BDNF组培养液中加入BDNF抗体(20μg/ml),对照组加入等量Hanks液。3d后在倒置显微镜下计数三组神经元存活数,并用NF200、MAP2、NSE的免疫组化反应对神经细胞进行鉴定。行突触素I与SYN免疫组化反应,对部分细胞行突触素ImRNA原位杂交反应,运用图像分析系统对突触素I与SYN免疫组织反应阳性产物以及突触素I原位杂交反应阳性产物作光密度分析。结果发现有血清培养时各组脊髓前角神经元的存活数无显著差异(P>0.05);BDNF组突触素I与SYN免疫反应阳性产物的平均光密度值高于其它两组,抗BDNF组最低(P<0.01)。BDNF组突触素ImRNA阳性产物的平均光密度值明显高于其它两组,抗BDNF组突触素ImRNA阳性产物的平均光密度值最低(P<0.01)。本研究结果提示BDNF对有血清培养时脊髓前角神经元的存活没有明显影响,但BDNF可明显上调培养的脊髓前角神经元内突触素I与SYN的表达。

To study the effects of BDNF on synapsin I and synaptophysin expressions in cultured rat spinal cord anterior horn neurons. The neurons from ventral part of spinal cord of embryonic 14 d rats were cultured for 7 d, and then divided randomly into three groups including BDNF group: BDNF (20 ng/ml) was added into culture medium; anti-BDNF group: BDNF antibody (20 μg/ml) was added into culture medium; control group: Hanks solution was added into culture medium. After three days the number of survived neurons in three groups were counted under light microscopy, and some of the cultured neurons were stained with immunohistochemistry by using antibody for NF-200, MAP-2, NSE, synapsin I and synaptophysin. Some neurons were stained with in situ hybridization by using synapsin I oligonucleotide probe. The mean optical densities of synapsin I and synaptophysin immunoreactive products and synapsin I mRNA positive products in neurons were observed. No significant difference in the number of survived neurons between each groups was observed. The mean optical densities of synapsin I-IR and synaptophysin-IR positive products and the mean optical density of synapsin I mRNA positive products in cultured neurons in BDNF group were the highest and anti-BDNF group were the lowest. These results suggests that there is no obvious effect of BDNF on the survival of our cultured spinal cord anterior horn neurons, but BDNF can promote the expression of synapsin I and synaptophysin in cultured spinal cord anterior horn neurons.