Lombardi Valter R M, Etcheverría Ignacio, Fernández-Novoa Lucía, Cacabelos Ramón
Ebiotec Biotechnology Division, La Coruña, Spain.
Neurotox Res. 2003;5(3):201-12. doi: 10.1007/BF03033140.
The cell culture approach to the study of the nervous system attempts to reduce cellular complexity to various extents and to characterize the influences of extrinsic molecules on the cell population under study. To date, the main source of culture model systems to explore CNS function and dysfunction is fetal brain material from experimental animals, typically rodents. We have developed primary microglial cell cultures and focused on the concentration-dependent effects of different amino acids and growth promoting additives on microglial morphology and function. We used Basal Medium Eagle (BME) with 1g/L of glucose instead of Dulbecco's modified Eagle medium (DMEM) as serum-free condition, since BME does not contain L-Glycine (Gly) and L-Serine (Ser), and investigated the effects of these two amino acids on microglial morphology and functions by adding various concentrations of the amino acids to BME and different concentrations of ascorbic acid (10-75 micro g/ ml), hydrocortisone (1-7.5 nM) and DL-alpha-tocopherol (0.01-0.5 micro g/ml) as growth promoters. Under Gly/Ser-free, serum-free condition, and growth promoters-free conditions, the majority of rat microglial cells displayed round morphology, whereas in the presence of 5 micro M Gly and 25 micro M Ser, which correspond to the concentrations of Gly and Ser in the cerebrospinal fluid, they extended multiple branched processes and formed clusters of rough endoplasmic reticulum. Ascorbic acid (25 micro g/ml), 2.5 nM hydrocortisone and 0.05 micro g/ml of DL-alpha-tocopherol elicited the highest level of microglial activation as measured by an increased expression of MHC class-I and MHC class-II antigens. Neuron culture experiments using the conditioned medium obtained from the different microglial culture conditions indicate neurotoxic and neurotrophic effects depending on the concentrations of amino acids as well as on the concentration of the growth promoters. These findings suggest that resting ramified microglial cells with neurotrophic activity can be induced with the combination of BME medium and small amounts of extracellular matrix growth promoters.
用于研究神经系统的细胞培养方法试图在不同程度上降低细胞复杂性，并表征外在分子对所研究细胞群体的影响。迄今为止，探索中枢神经系统功能和功能障碍的培养模型系统的主要来源是实验动物（通常是啮齿动物）的胎脑材料。我们已经开发了原代小胶质细胞培养物，并专注于不同氨基酸和生长促进添加剂对小胶质细胞形态和功能的浓度依赖性影响。我们使用含1g/L葡萄糖的伊格尔基础培养基（BME）而非杜尔贝科改良伊格尔培养基（DMEM）作为无血清条件，因为BME不含L-甘氨酸（Gly）和L-丝氨酸（Ser），并通过向BME中添加不同浓度的这些氨基酸以及不同浓度的抗坏血酸（10 - 75μg/ml）、氢化可的松（1 - 7.5nM）和DL-α-生育酚（0.01 - 0.5μg/ml）作为生长促进剂，研究了这两种氨基酸对小胶质细胞形态和功能的影响。在无Gly/Ser、无血清以及无生长促进剂的条件下，大多数大鼠小胶质细胞呈现圆形形态，而在存在5μM Gly和25μM Ser（这与脑脊液中Gly和Ser的浓度相对应）时，它们伸出多个分支突起并形成粗面内质网簇。抗坏血酸（25μg/ml）、2.5nM氢化可的松和0.05μg/ml的DL-α-生育酚通过MHC I类和MHC II类抗原表达增加，引发了最高水平的小胶质细胞活化。使用从不同小胶质细胞培养条件获得的条件培养基进行的神经元培养实验表明，根据氨基酸浓度以及生长促进剂浓度的不同，存在神经毒性和神经营养作用。这些发现表明，具有神经营养活性的静息分支小胶质细胞可以通过BME培养基和少量细胞外基质生长促进剂的组合来诱导。
