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目的:探讨正常人的前列腺移行带(PZ)和外周带(TZ)基质成纤维细胞中MEST(mesoderm specific transcript)基因的甲基化水平变化和表达差异,及DNA甲基化抑制剂(5-Aza-CdR)对其表达水平的影响。方法:用硫化测序PCR(bisulfite sequencing PCR,BSP)和实时荧光定量PCR方法分别检测5-Aza-CdR处理前、后在前列腺PZ和TZ原代成纤维细胞中MEST基因的甲基化水平和相应的mRNA表达。结果:MEST基因在PZ成纤维细胞中为低甲基化高表达,在TZ成纤维细胞中为高甲基化低表达。加入5-Aza-CdR后,MEST在PZ和TZ成纤维细胞中都为去甲基化,而表达水平上升,但是TZ成纤维细胞表达水平的改变比PZ成纤维细胞大。结论:MEST基因的DNA甲基化差异可能是前列腺外周带和移行带成纤维细胞生物学行为差异的分子基础。
OBJECTIVE: To investigate the changes and differences of methylation levels of mesoderm-specific transcript (MEST) gene in prostate stromal zone (PZ) and peripheral stroma (TZ) stromal fibroblasts in normal subjects and to explore the role of DNA methylation inhibitor 5- Aza-CdR) on its expression level. Methods: Methylation level of MEST gene in prostate PZ and TZ primary fibroblasts before and after 5-Aza-CdR treatment was detected by bisulfite sequencing PCR (BSP) and real-time fluorescence quantitative PCR MRNA expression. Results: The MEST gene was hypomethylated in PZ fibroblasts and hypermethylated in TZ fibroblasts. After adding 5-Aza-CdR, MEST was demethylated in both PZ and TZ fibroblasts and the expression level was increased, but the change of TZ fibroblast expression level was larger than that of PZ fibroblasts. Conclusion: The difference of DNA methylation of MEST gene may be the molecular basis of the biological behavior difference between peripheral and transitional zone fibroblasts.