Hobi R, Studer M, Ruch F, Kuenzle C C
Brain Res. 1984 Jul 9;305(2):209-19. doi: 10.1016/0006-8993(84)90427-x.
Previous work from our laboratories has indicated that the DNA content of rat cerebral cortex neurons increases postnatally to a level of slightly above 3c, where 2c denotes the diploid DNA complement. We have re-evaluated this concept by using various cytophotometric assays and a novel high performance liquid chromatography (HPLC) technique. The latter consists of digesting the DNA in isolated neuronal nuclei by a mixture of DNA-degrading enzymes followed by analysis of the resulting deoxynucleosides by HPLC. We find that the various methods fall into two groups. The first gives evidence of a postnatal DNA (or histone) increase, while the second does not. The first group (DNA increase) comprises cytofluorometry for DNA following Schiff-type staining with fluorochromes 2,5-bis-(4-aminophenyl)-1,3,4-oxadiazole (BAO) and pararosaniline, ultraviolet absorption scanning for DNA and cytofluorometry for histones following staining with sulfaflavine at pH 8. The second group (no DNA increase) consists of cytofluorometry for DNA following staining with the DNA-complexing agents mithramycin, chromomycin A3, 4',6-diamidino-2-phenylindole (DAPI) and bisbenzimide (Hoechst 33258), as well as the newly developed HPLC technique. Since the HPLC technique measures DNA by a direct chemical approach without interference from other nuclear constituents or from higher order packaging in the chromatin, and detects at least 94-95% of the total DNA contained in neuronal nuclei independent of the developmental stage, we infer that the HPLC technique and, by consequence, the cytochemical assays of the second group reflect true DNA values. Therefore, we propose that cerebral cortex neurons retain a diploid DNA level throughout development.
我们实验室之前的研究表明，大鼠大脑皮层神经元的DNA含量在出生后增加至略高于3c的水平，其中2c表示二倍体DNA含量。我们通过使用各种细胞光度测定法和一种新型高效液相色谱（HPLC）技术重新评估了这一概念。后者包括用DNA降解酶混合物消化分离的神经元核中的DNA，然后通过HPLC分析产生的脱氧核苷。我们发现各种方法分为两组。第一组提供了出生后DNA（或组蛋白）增加的证据，而第二组则没有。第一组（DNA增加）包括用荧光染料2,5-双-（4-氨基苯基）-1,3,4-恶二唑（BAO）和副蔷薇苯胺进行席夫型染色后对DNA进行细胞荧光测定、对DNA进行紫外吸收扫描以及在pH 8下用磺胺黄酮染色后对组蛋白进行细胞荧光测定。第二组（无DNA增加）包括用DNA络合剂光神霉素、色霉素A3、4',6-二脒基-2-苯基吲哚（DAPI）和双苯甲酰亚胺（Hoechst 33258）染色后对DNA进行细胞荧光测定，以及新开发的HPLC技术。由于HPLC技术通过直接化学方法测量DNA，不受其他核成分或染色质中高级包装的干扰，并且无论发育阶段如何都能检测到神经元核中至少94-95%的总DNA，我们推断HPLC技术以及第二组的细胞化学测定反映了真实的DNA值。因此，我们提出大脑皮层神经元在整个发育过程中保持二倍体DNA水平。
