尼古丁对培养大鼠基底前脑胆碱能神经元生长存活及TrkA表达的影响

Effects of nicotine on the growth and the TrkA expression of cultured primitive rat basal forebrain cholinergic neurons

目的 探讨不同浓度的尼古丁对原代培养的大鼠基底前脑胆碱能神经元的高亲和力神经生长因子受体(TrkA)表达的影响 ,结合细胞形态学观察和四甲基偶氮唑盐 (MTT)实验了解TrkA表达变化对细胞生长的影响。方法 运用细胞原代培养的方法培养大鼠基底前脑胆碱能神经元并进行鉴定 ,用 0 .0 1,0 .1,1,10和 10 0 μmol/L五个终浓度的尼古丁对原代培养细胞进行干预 ,观察细胞的形态变化 ,MTT实验观察细胞的活力 ,提取细胞总RNA行RT PCR观察神经元TrkAmRNA表达的变化 ,免疫细胞学化观察TrkA表达的变化 ;用 10 μmol/L终浓度的尼古丁以不同的时间 (分别为 8,2 4 ,4 8h)进行干预 ,观察不同时间干预下上述指标的变化。结果 在 0 .0 1～ 10 μmol/L浓度范围内的尼古丁呈剂量依赖性地增加神经元TrkA的表达 ,10 0 μmol/L浓度的尼古丁则引起TrkA表达的下降 ,前浓度范围的尼古丁可以剂量依赖性地增加原代培养的神经元细胞突起数量、平均突起长度和最长突起长度 ,增加MTT法测得的OD值 ,10 0 μmol/L终浓度尼古丁则产生相反的作用。 结论 在一定浓度范围内 ,尼古丁可以增加原代培养的胆碱能神经元TrkA表达 ,对神经元可能具有营养作用。

Objective To investigate the effects of nicotine on the growth and TrkA expression of cultured primitive rat basal forebrain cholinergic neurons. Methods Primitive rat basal forebrain neurons were cultured and evaluated . Morphologic, celluar vigor and TrkA expressing changes were observed by microscop , MTT assay,RT-PCR and immunocytochemistry respectively when the neurons were exposed to nicotine with different ending concentrations(0.01, 0.1, 1, 10, 100 μmol/L) . Results Being exposed to nicotine for 48 h, the neurons showed significant changes in morphology and cellular vigar, and average process quantity of the neurons, average process length and the length of the longest peocess of neurons were increased significantly. The results of MTT assay showed that average OD values were significantly increased. The expression of TrkA in the cultured neurons were increased after the neurons being exposed to nicotine with above four concentrations for 48 h, and the expression were decreased after being exposed to 100 μmol/L nicotine for 48 h. When the neurons were cultured with 10 μmol/L nicotine for different duration (8 h, 24 h, 48 h respectively),the above indexes were increased by time dependency. Conclusion Nicotine may have nutritive effect on rat cholinergic basal forebrain neurons in some extent. It may increase average process quantity of neurons, average process length and the length of the longest peocess, augmenting cellular vigor and increasing expression of TrkA.