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目的观察星形胶质细胞(AST)分泌雌激素的规律,研究AST对大脑皮层神经元突触形成的影响及可能的分子机制。方法取新生大鼠大脑皮层进行AST原代及传代培养,分别于传代培养的第0d、7d、14d、21d进行细胞计数,同时用ELISA方法测定AST条件培养液(ACM)中雌二醇(E2)的浓度。以新生大鼠皮层神经元纯培养为模型,实验分成6组:神经元纯培养组;ACM培养组;AST和神经元混合培养组;雌激素培养组;ACM+Tamoxifen(雌激素受体阻断剂)培养组;Tamoxifen培养组。应用免疫荧光技术和突触计数方法观察各组突触形成数量的差别。结果AST数量分别为1×104/ml、1.1×106/ml、1.4×106/ml、1.5×106/m1;ACM中雌二醇浓度分别为(ng L):0、117±22、266±22、252±27。第0d培养液中未检测出雌二醇,随着培养时间的延长雌激素浓度迅速增加,14d左右达高峰,以后逐渐降低,但21d时培养液中雌二醇仍保持较高浓度。各实验组突触荧光颗粒数分别为(个/细胞,培养第9d):14±3;79±5;83±8;80±6;32±3;29±3。显示ACM能增加培养神经元突触形成的数目近6倍,外源性雌激素可基本模拟ACM的效应。Tamoxifen能阻断ACM促突触形成效应的75%左右。结论体外培养新生大鼠大脑皮层AST能合成并分泌雌激素,分泌的雌激素可能参与了胶质细胞调节神经元突触形成的过程,
Objective To observe the regularity of estrogen secretion by astrocytes (AST) and to study the effect of AST on the synapse formation of neurons in cerebral cortex and its possible molecular mechanism. Methods Primary and secondary cultures of AST were subcultured in the cerebral cortex of newborn rats. The cells were counted on the 0d, 7d, 14d and 21d respectively in subculturing culture. The levels of estradiol (E2) in AST conditioned media (ACM) )concentration. Neonatal rat cortical neurons pure culture model was divided into 6 groups: pure neuron culture group; ACM culture group; AST and neuronal mixed culture group; estrogen culture group; ACM + Tamoxifen (estrogen receptor block Agent) culture group; Tamoxifen culture group. Immunofluorescence and synaptic counting methods were used to observe the differences in the number of synapses in each group. Results The numbers of AST were 1 × 104 / ml, 1.1 × 106 / ml, 1.4 × 106 / ml and 1.5 × 106 / ml respectively. The concentrations of estradiol in ACM were (ng L): 0,117 ± 22,266 ± 22,252 ± 27. Estradiol was not detected in the 0d culture medium. With the prolongation of culture time, the estrogen concentration increased rapidly and peaked around 14d, then decreased gradually. However, the estradiol level remained high at 21d. The number of synaptic fluorescent particles in each experimental group was 14 ± 3, 79 ± 5, 83 ± 8, 80 ± 6, 32 ± 3, 29 ± 3, respectively. ACM showed that the number of cultured neurons synapse formation increased nearly 6 times, exogenous estrogen can basically simulate the effect of ACM. Tamoxifen blocked about 75% of ACM-induced synaptic formation. Conclusion AST can synthesize and secrete estrogen in the cerebral cortex of neonatal rats in vitro. The secreted estrogen may be involved in the process of glial cells regulating neuronal synapse formation.