脊髓源性少突胶质前体细胞在衰老过程中基因组甲基化总体调控模式的变化

Alterations of global DNA methylation profiles of ogliodendrocyte precursor cells derived from spi-nal cord of rats during the aging process

目的观察大鼠脊髓来源的少突胶质前体细胞（OPCs）在衰老过程中基因组DNA甲基化总体调控模式的变化。方法原代培养新生大鼠、4周大鼠和32周大鼠脊髓组织来源的OPCs细胞，并用免疫荧光染色鉴定其表面标志物A285及NG2；采用酶联免疫吸附法检测各组细胞基因组甲基化水平及总体DNA甲基转移酶（DNMTs）活性；采用实时荧光定量聚合酶链反应（RT-qPCR）及Westernblot检测各组细胞DNMTs的3种主要亚型DNMTI、DNMT3a及DNMT3b的mRNA及蛋白表达水平。结果免疫荧光染色结果显示，本研究培养的OPCs纯度达95％以上。新生组、4周组和32组细胞基因组DNA甲基化水平分别为0．87±0．12、0．79±0．14和0．37±0．07，其中32周组与新生组比较差异有统计学意义（P〈0．05）；各组细胞总体DNMTs活性呈下降趋势，差异有统计学意义（P〈0．05）；DNMTs的3种亚型DNMTl、DNMT3a及DNMT3b的变化趋势为：与新生组比较，4周组DNMTl的表达下降43％，DNMT3a和DNMT3b的表达分别增高56％和36％；32周组DNMTl的mRNA表达下降69％，DNMT3b表达增高104％，差异有统计学意义（P〈0．05）。结论大鼠脊髓源性OPCs在衰老过程中，其基因组甲基化水平及总体DNMTs活性呈下降趋势，而在此过程中DNMTl活性的下降发挥了主要作用。

Objective To investigate the change of global DNA methylation profiles of oglioden- drocyte precursor cells （OPCs） derived from spinal cord of rats during the aging process. Methods OPCs were separated and cultured from the spinal cords of neonatal rats, 4-week-old （4w） rats and 32-week-old （32w） rats. The purified OPCs were identified by immunofluorescence staining with NG2 and A2B5. The genome DNA methylation level and the activity of methyhransferaseswas （DNMTs） were measured by using enzyme linked immunosorbent assay （ELISA）. The expression levels of DNMT1, DNMT3a and DNMT3b were detected by using both reverse transcription-polymerase chain reaction （RT-PCR） and Western blot- ting. Results The results of immunofluorescence staining showed that the percentage of OPCs was above 95%. The genome DNA methylation level of neonatal rats, 4w rats and 32w rats was 0. 87 ± 0. 12, 0. 79 ± 0. 14 and 0. 37 ± 0. 07, respectively, with the difference being significant between neonatal rats group and 32w rats group （ P 〈 0.05 ）. The activity of DNMTs in OPCs was decreased gradually during the process of aging （P 〈 0. 05 ）. As compared with neonatal rats group, the expression of DNMT1 was decreased by 43%, and that of DNM33a and DNM33b was increased by 56% and 36% respectively in gw rats group. In 32w rats group, the expression of DNMT1 was decreased by 69%, and that of DNMT3b increased by 104% as compared with neonatal rats group （P 〈 0. 05）. Conclusion The genome DNA methylation level and the activity of DNMTs in OPCs derived from spinal cord of rats are decreased gradually during the process of aging. The activity of DNMT1 played an important role during the aging process.