Engele J, Schubert D, Bohn M C
Department of Neurobiology and Anatomy, University of Rochester Medical Center, NY 14642.
J Neurosci Res. 1991 Oct;30(2):359-71. doi: 10.1002/jnr.490300212.
Neuronal differentiation is influenced by extracellular factors; however, only a few such factors have been identified for central neurons. To address this issue, we have screened media conditioned (CM) by several glial cell lines for neurotrophic effects on dopaminergic neurons in dissociated cell cultures of the E14.5 rat mesencephalon grown in serum-free conditions. To establish culture conditions under which dopaminergic cell survival depends on the exogenous support from neurotrophic factors, cell suspensions were seeded at varying densities and the number of tyrosine hydroxylase-immunoreactive (TH-IR) neurons was determined. This number was maximal at plating densities greater than 175,000 cells/cm2 and was 10-fold lower at the plating density of 80,000 cells/cm2. Cell density had only a minimal effect on [3H]dopamine uptake per TH-IR neuron. Treatment of cultures plated at 80,000 cells/cm2 with CM derived from the glial cell line, B49, the neural retina glial cell line, R33, and the Schwannoma cell line JS1, increased the number of surviving TH-IR neurons 160-330%. These effects were dose dependent and heat sensitive. All CM stimulated neurite elongation of TH-IR neurons, while only the B49-CM increased [3H]dopamine uptake. The neurotrophic effects of these media were not confined to dopaminergic neurons but increased overall neuronal density in culture by 50-100%. Moreover, all three CM were mitogenic for mesencephalic glia as demonstrated by glial fibrillary acidic protein (GFAP)-immunocytochemistry in combination with [3H]thymidine-autoradiography. By contrast, medium conditioned by the pheochromocytoma cell line, PC12, did not increase the number of astrocytes or promote the survival of dopaminergic neurons. Inhibition of glial proliferation reduced the neurotrophic effects of the B49-, R33-, and JS1-CM by 40-80%. These observations suggest that the glial cell lines B49, R33, and JS1 secrete factors that promote the survival of dopaminergic neurons and induce proliferation of glial precursors. The partial decrease of the survival-promoting effects of these CM on dopaminergic neurons in glial-free mesencephalic cultures further suggests that the observed neurotrophic effects result from the combined action of cell line-derived substances directly on neurons and indirectly via effects on mesencephalic astrocytes or astrocyte precursors.
神经元分化受细胞外因子影响；然而，对于中枢神经元而言，仅鉴定出少数此类因子。为解决这一问题，我们筛选了几种神经胶质细胞系的条件培养基（CM），以研究其对在无血清条件下培养的E14.5大鼠中脑解离细胞培养物中多巴胺能神经元的神经营养作用。为建立多巴胺能细胞存活依赖于神经营养因子外源支持的培养条件，将细胞悬液以不同密度接种，并测定酪氨酸羟化酶免疫反应性（TH-IR）神经元的数量。该数量在接种密度大于175,000个细胞/cm²时最大，在接种密度为80,000个细胞/cm²时低10倍。细胞密度对每个TH-IR神经元的[³H]多巴胺摄取仅有最小影响。用源自神经胶质细胞系B49、神经视网膜神经胶质细胞系R33和施万细胞瘤细胞系JS1的CM处理接种密度为80,000个细胞/cm²的培养物，可使存活的TH-IR神经元数量增加160 - 330%。这些作用呈剂量依赖性且对热敏感。所有CM均刺激TH-IR神经元的神经突伸长，而只有B49-CM增加了[³H]多巴胺摄取。这些培养基的神经营养作用不仅限于多巴胺能神经元，还使培养物中的总体神经元密度增加了50 - 100%。此外，通过胶质纤维酸性蛋白（GFAP）免疫细胞化学结合[³H]胸腺嘧啶核苷放射自显影证明，所有三种CM对中脑神经胶质细胞均有促有丝分裂作用。相比之下，嗜铬细胞瘤细胞系PC12的条件培养基并未增加星形胶质细胞数量或促进多巴胺能神经元的存活。抑制神经胶质细胞增殖可使B49-、R33-和JS1-CM的神经营养作用降低40 - 80%。这些观察结果表明，神经胶质细胞系B49、R33和JS1分泌促进多巴胺能神经元存活并诱导神经胶质前体细胞增殖的因子。这些CM对无神经胶质的中脑培养物中多巴胺能神经元的存活促进作用部分降低，进一步表明观察到的神经营养作用是细胞系衍生物质直接作用于神经元以及通过对中脑星形胶质细胞或星形胶质前体细胞的作用间接产生的联合作用的结果。
