大鼠衰老过程中脑细胞DNA与c－Ha－ras原癌基因的甲基化

用ＭｓｐⅠ／ＨｐａⅡ酶解电泳法和高效液相色谱（ＨＰＬＣ）两种方法进行比较，研究了不同年龄大鼠的肝、脑细胞基因组ＤＮＡ的甲基化程度。从酶解电泳图谱可观察到，肝、脑细胞基因组ＤＮＡ甲基化在青年鼠和老年鼠之间没有差异。但用具有高分辨率的高效液相色谱测量ＤＮＡ中５－ｍＣ的含量时发现，老年鼠脑细胞ＤＮＡ甲基化程度较大年鼠的下降６２％，而肝细胞ＤＮＡ甲基化程度在老年鼠与青年鼠之间并没有显著差异。这些结果提示：（１）用常规的酶解电泳法所分析的ＤＮＡ甲基化结果并不能反映整个基因组ＤＮＡ甲基化的水平。（２）衰老过程中，不同组织ＤＮＡ甲基化的改变存在差异，引起这种差异的原因可能与组织的增殖和分化程度有关。进一步分析脑细胞原癌基因ｃ－Ｈａ－ｒａｓ的甲基化水平，无论ＭｓｐⅠ酶切图谱，还是ＨｐａⅡ酶切图谱均可观察到分子大小为１９ｋｂ、７．５ｋｂ、１．３ｋｂ、０．９ｋｂ的四条阳性带，说明该基因未发生甲基化，且与年龄无关。

Methylation of Genomic DNA and Proto-oncogene c-Ha-ras in Brain Nuclei of Ageing Rats

The methylation of genomic DNA in liver and brain nuclei of aging rats was analysed By Reverse-phase high-performance chromatography (HPLC) and restriction endonuclease Hpa Ⅱ and Msp Ⅰ. We found that the methylation of genomic DNA in liver and brain nuclei of aging rats was the same as young rats by analysis with methylation-sensitive endonucleases Hpa Ⅱand Msp Ⅰ. However, the difference of genomic DNA methylation of brain nuclei between aging and young rats was shown by Reverse-phase High performance liquid chromatography (HPLC). DNA methylation level of brain nuclei in aging rats was decreased 62% whereas no difference of DNA methylation level was detected in liver-nuclei between aging and young rats. These results suggest: (1)Genomic DNA methylation-sensitive endonuclease analysis does not reflect the overall status of total DNA; (2) Methylation difference in various organs may be related to the proliferation potential and differenciation degree of organ. Southern blot analysis with rat proto-oncogene c-Ha-ras probe showed that both the electrophoretic patterns of rat brain DNA digested with Hpa Ⅱand Msp Ⅰ were similar, containing four bands with the sites of 19kb、 7. 5kb、 1. 3kb and 0. 9kb. It indicates that c-Ha-ras gene was hypomethylated in brain and there was no obvious relationship between c-Ha-ras gene methylation and aging.