Vick R S, Chen S J, DeVries G H
Department of Biochemistry, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond 23298-0614.
J Neurosci Res. 1990 Apr;25(4):524-34. doi: 10.1002/jnr.490250409.
Investigation into CNS demyelinating diseases, which usually occur in adults, can be facilitated by the use of a good in vitro model. We have established a methodology whereby oligodendrocytes from adult rat CNS can be cultured in vitro, and we have characterized these cultures morphologically and immunologically. Approximately 1 g of spinal cord and brainstem per adult rat was removed and dissociated mechanically and enzymatically. After filtration of the white matter homogenate, myelin was removed by 0.9 M sucrose density centrifugation. The cells were further purified by centrifugation through a 0.3%/4% discontinuous gradient of bovine serum albumin (BSA). The pellet was resuspended and placed in an untreated 6-well culture dish overnight to allow the astrocytes to attach. The non-adherent cells were replated on poly-l-lysine-treated coverslips. Approximately 8.25 x 10(5) cells were recovered per animal. The adult oligodendrocytes initially appeared as rounded cell bodies, but after 2-5 days in vitro (DIV), the oligodendrocytes extended 6-10 thick processes. A membrane sheath between these processes was immunostainable with either anti-galactocerebroside (GC), anti-O4, anti-myelin basic protein (MBP), or anti-2'3' cyclic nucleotide 3' phosphohydrolase (CNPase) and was also evident in scanning EM. Older cultures (up to 60 DIV) maintained whorls of myelin and transmission EM revealed a major dense line distance of approximately 103 A with up to 11 concentric layers of membrane. Immunologically, the adult oligodendrocytes are GC+, O4+, MBP+, CNPase+, and GFAP-. The method described will allow adult rat oligodendrocytes to be isolated and maintained in culture; these cultures retain the characteristics of differentiated adult oligodendrocytes.
对通常发生在成年人中的中枢神经系统脱髓鞘疾病的研究，可以借助良好的体外模型来推动。我们已经建立了一种方法，通过该方法可以在体外培养成年大鼠中枢神经系统的少突胶质细胞，并且我们已经从形态学和免疫学方面对这些培养物进行了表征。每只成年大鼠大约取1克脊髓和脑干，进行机械解离和酶解。对白质匀浆进行过滤后，通过0.9M蔗糖密度离心去除髓鞘。细胞通过在0.3%/4%不连续牛血清白蛋白（BSA）梯度中离心进一步纯化。将沉淀重悬并置于未处理的6孔培养皿中过夜，以使星形胶质细胞附着。将未贴壁的细胞重新接种到经聚-L-赖氨酸处理的盖玻片上。每只动物大约回收8.25×10⁵个细胞。成年少突胶质细胞最初表现为圆形细胞体，但在体外培养2至5天（DIV）后，少突胶质细胞伸出6至10条粗大的突起。这些突起之间的膜鞘可用抗半乳糖脑苷脂（GC）、抗O4、抗髓鞘碱性蛋白（MBP）或抗2'3'环核苷酸3'磷酸二酯酶（CNPase）进行免疫染色，并且在扫描电子显微镜下也很明显。较老的培养物（长达60 DIV）维持着髓鞘的螺旋结构，透射电子显微镜显示主要致密线间距约为103埃，有多达11层同心膜层。在免疫学上，成年少突胶质细胞为GC⁺、O4⁺、MBP⁺、CNPase⁺且GFAP⁻。所描述的方法将允许分离并在培养中维持成年大鼠少突胶质细胞；这些培养物保留了分化的成年少突胶质细胞的特征。
