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目的探讨香烟烟雾暴露对小鼠卵母细胞染色质构型、全基因组DNA甲基化及相关基因表达的影响。方法将清洁级健康ICR雌鼠分成3组(对照组和香烟烟雾低剂量组、高剂量组),分别置于点燃0、1、2支香烟的染毒柜(容积18 L)中,每天2次,每次1 h,共4周。利用Hoechst 33342对细胞核染色,观察卵母细胞质量及染色质构型;间接免疫荧光法分析卵母细胞全基因组DNA甲基化模式,实时荧光定量PCR测定卵母细胞DNA甲基转移酶(DNA methyltransferases,DNMT)1、DNMT3a及DNMT3b基因的mRNA表达水平。结果随着烟雾浓度的升高,去透明带卵母细胞直径显著减小(P<0.05),核仁未包围(non surrounded nucleolus,NSN)染色质构型比例明显升高(P<0.05)。高剂量组卵母细胞DNA甲基化水平显著下降(P<0.05)。随烟雾浓度升高DNMT1表达量降低,DNMT3b在高剂量组中表达水平显著降低(P<0.05)。结论香烟烟雾暴露可能通过改变卵母细胞染色质构型,降低DNA甲基转移酶的表达,使DNA甲基化水平下降,从而降低卵母细胞质量。
Objective To investigate the effects of cigarette smoke exposure on chromatin structure, whole genome DNA methylation and related gene expression in mouse oocytes. Methods Clean healthy ICR female rats were divided into 3 groups (control group and low dose cigarette smoke group, high dose group), were placed in ignited 0, 1, 2 cigarettes exposure cabinet (volume 18 L), 2 Times, 1 h each time, a total of 4 weeks. The nuclei were stained with Hoechst 33342 to observe the oocyte quality and chromatin configuration. The methylation patterns of oocyte genome DNA were analyzed by indirect immunofluorescence method. The oocyte DNA methyltransferase (DNA methyltransferase) , DNMT) 1, DNMT3a and DNMT3b mRNA expression levels. Results With the increase of smoke concentration, the diameter of oocytes in de-zona pellucida decreased significantly (P <0.05) and the proportion of chromatin in non-surrounded nucleolus (NSN) increased significantly (P <0.05). High-dose oocyte DNA methylation levels were significantly decreased (P <0.05). The expression of DNMT1 decreased with the increase of smoke concentration, while the expression of DNMT3b decreased significantly in high dose group (P <0.05). Conclusion Cigarette smoke exposure may reduce oocyte quality by changing the chromatin configuration of oocytes, decreasing the expression of DNA methyltransferase, decreasing the DNA methylation level.