Sajdel-Sulkowska E M, Majocha R E, Salim M, Zain S B, Marotta C A
Neuroscience Program, Harvard Medical School, Boston, MA 02115.
J Neurosci Methods. 1988 Mar;23(2):173-9. doi: 10.1016/0165-0270(88)90189-6.
Although the precise role of astrocytes in the pathogenesis of Alzheimer's disease (AD) is currently undefined, studies carried out at the molecular level may lead to new insights into the functioning of this class of brain cells in dementia. In order to facilitate such investigations, methods are described that establish that structurally and functionally intact messenger RNA (mRNA) for an astrocytic marker, glial fibrillary acidic protein (GFAP), is present in the postmortem Alzheimer's disease brain after long postmortem intervals. Rapid preparative procedures were used to obtain poly(A)+ RNA from postmortem control and AD cortices. In vitro protein synthesis was carried out in a reticulocyte system. Relative to controls, AD mRNA synthesized a two-fold higher level of a 50,000 mol.wt. protein that was immunologically identified as GFAP. High levels of GFAP synthesis by purified mRNA from AD cortices was independent of age at death and postmortem interval up to 24 h. Northern blot hybridization using a cloned human GFAP riboprobe was used to evaluate postmortem GFAP mRNA stability. No appreciable degradation products of GFAP mRNA were observed on Northern blots for at least 10 h postmortem in poly(A)+ RNA extracted from the AD brain. The described methodology demonstrates that the postmortem AD brain is an excellent source of functionally and structurally intact astrocyte-specific mRNA.
虽然星形胶质细胞在阿尔茨海默病(AD)发病机制中的精确作用目前尚不清楚,但在分子水平上开展的研究可能会为这类脑细胞在痴呆症中的功能带来新的见解。为了便于此类研究,本文描述了一些方法,这些方法证实了在较长的死后间隔时间后,阿尔茨海默病患者的尸检大脑中存在用于星形胶质细胞标志物——胶质纤维酸性蛋白(GFAP)的结构和功能完整的信使核糖核酸(mRNA)。采用快速制备程序从尸检对照和AD皮质中获取多聚腺苷酸加尾(poly(A)+)RNA。在网织红细胞系统中进行体外蛋白质合成。相对于对照,AD的mRNA合成了免疫鉴定为GFAP的50,000道尔顿分子量的蛋白质,其水平高出两倍。来自AD皮质的纯化mRNA高水平合成GFAP与死亡年龄和长达24小时的死后间隔时间无关。使用克隆的人GFAP核糖探针进行Northern印迹杂交,以评估死后GFAP mRNA的稳定性。从AD大脑中提取的poly(A)+ RNA在死后至少10小时的Northern印迹上未观察到GFAP mRNA有明显的降解产物。所描述的方法表明,AD尸检大脑是功能和结构完整的星形胶质细胞特异性mRNA的极佳来源。