脑皮质星形细胞在1,25-(OH)_2D_3限定培养中表达NGF

Expression of nerve growth factor gene in astrocytes cultured in 1,25-(OH)_2D_3 defined medium

目的 :研究原代中枢神经胶质细胞在 1 ,2 5 -二羟维生素 D3[1 ,2 5 - (OH) 2 D3]化学限定培养中神经生长因子 (NGF)基因转录及表达。方法 :用新生乳鼠脑皮质制备星形胶质细胞 ,采用浓度 0、 1 0 - 7、 1 0 - 8、 1 0 - 9、1 0 - 1 0 、 1 0 - 1 1 及 1 0 - 1 2 mol· L- 1 1 ,2 5 - (OH) 2 D3化学限定无血清培养液培养后 ,RT- PCR法检测 NGF m RNA转录 ,双向夹心 - EL ISA检测 NGF。结果 :1 ,2 5 - (OH ) 2 D3化学限定无血清原代培养的神经胶质细胞在 1 ,2 5 - (OH ) 2 D3作用下可分泌 NGF,实验中 1 ,2 5 - (OH) 2 D3有效浓度范围 1 0 - 1 1 ～ 1 0 - 7mol· L- 1 ;1 ,2 5 - (OH) 2 D3培养 6 h后开始检测到 m RNA的表达 ,1 2 h时表达最高 ,36 h后检测不到 NGF m RNA的表达 ;而 NGF蛋白的 EL ISA反应持续时间大于 4 8h。结论 :星形神经胶质细胞的 NGF基因可被甾体激素 1 ,2 5 - (OH) 2

Objective To investigate the expression of nerve growth factor (NGF) gene in astrocytes cultured in chemical 1,25 (OH) 2D 3 defined medium. Methods The astrocytes prepared from brain of neonatal rats were cultured in a chemical 1,25 (OH) 2D 3 defined serum free medium with various doses of 1,25 (OH) 2D 3 (0,10 -7 ,10 -8 ,10 -9 ,10 -10 ,10 -11 and 10 -12 mol·L -1 ). NGF mRNA transcripts were detected by RNA based polymerase chain reaction method. Secretory NGF protein quantity were measured with a double site ELISA with monoclonal anti NGF antibody. Results NGF synthesis and secretion were induced in astrocytes cultured in chemical 1,25 (OH) 2D 3 defined serum free medium. 1,25 (OH) 2D 3 were effective in the concentration range of 10 -7 to 10 -11 mol·L -1 . The expression of mRNA was detected 6 hours after cultured in 1,25 (OH) 2D 3 medium,the level of expression of mRNA approached to peak value at 12th hour, and NGF mRNA was not found after 36 hours. The reaction period of ELISA of NGF protein was above 48 hours.Conclusion NGF gene in central nervous system (CNS) can be regulated by 1,25-(OH) 2D 3.