用快速自动比色显微分析法探讨巨噬细胞条件性培养基对小脑皮质神经元生存的影响

USING A RAPID AUTOMATED COLORIME-TRIC MICROASSAY TO STUDY THE EFFECT OF MACROPHAGE CONDI-TIONED MEDIUM ON THE SUR-VIVAL OF CEREBELLAR CORTICAL NEURON

用快速自动比色显微分析法检测巨噬细胞条件性培养基(MΦCM)对体外培养的生后7天SD大鼠小脑皮质神经元的作用。实验结果表明,MΦCM有支持神经元生存及增强其活性的作用。此作用以细胞密度1×10~5/孔,MΦCM浓度10μl/孔为显著(P<0.05)。不同分子量的MΦCM对神经元的作用亦有不同,>10kdaltons(kDa)组分比<10KDa的作用强。

Macrophage culture was prepared and its conditioned medium (MφCM) was collected to measure its neurotrophic activities by a rapid automated colorimetric microassay. Cerebel-lar cortical neuron was dissociated from 7-day-old Sprague-Dawley rats and seeded in poly-L-ornithine coated 96-well culture plates at a density of1×104, 2×104, 4×104, 6×104, 8×104, and 1X105 cells per well. The, culture medium consists of MEM plus 6% fetal calf serum. Various concentrations and molecular weight fractions of M<j>CM were added to the medium in experimental groups. The period of culture lasted 24, 48 or. 72 hours. A yellow tetrazolium derivative, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 diph-enyl tetrazolium bromide] was added to each culture at the last 4 h of culture. MTT was taken up selectively by active neuron and converted to a blue formazan product. The amount of blue color development could be rapidly quantified using an automatic microplate reader. The resulting optical density was directly proportional to the number of active neurons. Our experimental results show that the number of active neurons at 43 and 72 h of culture markedly decrease as compared with that at 24 h of culture, whereas M^CM plays a role in supporting the neuronal survival' and enhancing the neuronal activity, especially at a density of 1×108 cells and a concentration of 10 Ul MφCM per well (P< 0.05). The effect of various molecular weight fractions of MφCM on neuronal growth and activity was different in which MφCM fraction of>10kDa was significantly better than that of<10kDa.