观察大鼠下丘脑NPY神经元的培养及瘦素对其mRNA表达的影响

Hypothalamus NPY neuron culture and effects of leptin on NPY mRNA in rat

目的观察大鼠下丘脑NPY神经元的培养及瘦素对其mRNA表达的影响。方法用无血清限定性培养基体外培养新生大鼠的下丘脑神经元,观察其生长状况;用NSE、NF、NPY抗血清检测神经元的鉴定和表达,并对三者阳性细胞的胞体和突起做统计学分析。给予Leptin10-10mol/L、10-8mol/L、10-6mol/L3个浓度,观察leptin对NPYm RNA表达的影响。结果培养的NSE和NF阳性细胞生长至第7天时均达高峰;NPY阳性神经元在培养7～10d时,亦处于一稳定时期。NPYmRNA的表达在给予Leptin10-10mol/L时,与对照组相比无显著性差异;10-8mol/L、10-6mol/L浓度时,表达增强,与对照组相比有显著性差异(P<0.05)。结论取材于新生大鼠的下丘脑亦可进行体外培养;培养一周时是研究单因素影响神经元的最佳实验时间;在离体环境下,高浓度的leptin(10-8mol/L、10-6mol/L)能促进NPYmRNA的表达,NPY可能表现为抑制GnRH分泌的作用。

Objective Hypothalamus neurons were obtained from new born rats, cultured in serum-free defined medium, and observed their growth state. Methods Using NSE, NF,NPY antiserum to identify neurons and detect expression of neurons. The cell body and neurits of NSE, NF, NPY positive neuron were analyzed. Neurons were given Leptin 10 -10mol/L? 10 -8 mol/L ? 10 -6 mol/L, and effect of leptin on expression of NPY mRNA was observed. Results The results showed that one week after culture, growth of NSE, NF positive neuron reached to the peak. NPY positive neuron developed to a steady period during culturing of 7～10 days. One week is the best time to study the effect of some factors on hypothalamus neuron. Compared with control group, with Leptin 10 -10mol/L expression of NPY mRNA had no statistical difference, but with 10 -8 mol/L? 10 -6 mol/L expression of NPYmRNA enhanced and had statistical difference. Conclusion The experiment showed that neuron obtained from new born rats can be succeedly cultured. One week later, neuron growed to a steady period and this period is also the best time to study the effect of some factors on hypothalamus neuron. When cultured in vitro, leptin 10 -8 mol/L? 10 -6 mol/L can facilitate expression of NPY mRNA and NPY perhaps suppress secretion of GnRH neuron.