DNA甲基转移酶1的表达对小鼠精原干细胞分化的影响  

Impact of the expression of DNA methyltransferase 1 on the differentiation of spermatogonial stem cells in mice

作　　者：李娜[1] 苗聪秀[1] 毕慧玲[1] 苗卉[1] 李丹[1] 李敏[1] LI Na;MIAO Cong-xiu;BI Hui-ling;MIAO Hui;LI Dan;LI Min(Department of Reproductive Genetics/Changzhi Medical College Research Institute of Reproduction and Genetics,Heping Hospital Affiliated to Changzhi Medical College,Changzhi,Shanxi 046000,China)

机构地区：[1]长治医学院附属和平医院生殖遗传科/山西省卫健委生殖工程委级重点实验室/长治医学院生殖遗传研究所,山西长治046000

出　　处：《中华男科学杂志》2022年第5期395-401,共7页National Journal of Andrology

基　　金：国家自然科学基金(31971073);山西省自然科学基金(201901D111325);山西省卫健委科研项目(2017162)。

摘　　要：目的:探讨DNA甲基转移酶1(DNMT1)的表达对小鼠精原干细胞(SSC)分化的影响。方法:采用两步酶消化法从1周龄BALB/c雄性小鼠睾丸组织中分离获取SSC,将DNMT1-siRNA、siRNA阴性对照(NC-siRNA)转染入分离纯化后第三代SSC,分别命名为DNMT1-Si组、DNMT1-NC组,另取未转染细胞命名为对照组;采用实时荧光定量PCR(RT-qPCR)法和Western印迹检测转染24 h后各组DNMT1 mRNA和蛋白表达水平,并检测转染后细胞基因组甲基化水平。干细胞生长因子诱导SSC向精母细胞方向分化,采用RT-qPCR法和Western印迹检测诱导分化48 h生殖细胞增殖相关蛋白(Nanos2)、早幼粒细胞白血病锌指蛋白(PLZF)、维甲酸刺激蛋白(Stra8)mRNA和蛋白表达水平。结果:转染24 h后,DNMT1-Si组DNMT1 mRNA(0.13±0.03)和蛋白相对表达量(0.44±0.08)、DNA甲基化水平[(14.16±2.62)%]均低于对照组[0.94±0.08、1.21±0.11、(27.15±3.54)%]和DNMT1-NC组[0.93±0.09、1.19±0.10、(26.85±3.55)%,P<0.05],对照组与DNMT1-NC组比较,差异无统计学意义(P>0.05)。分化48 h后,与对照组和DNMT1-NC组比较,DNMT1-Si组Nanos2、PLZF mRNA和蛋白相对表达量降低,Stra8 mRNA和蛋白相对表达量升高(P<0.05);对照组与DNMT1-NC组Nanos2、PLZF、Stra8 mRNA和蛋白相对表达量比较,差异均无统计学意义(P>0.05)。结论:敲低DNMT1可促进小鼠SSC向精母细胞方向分化,其机制可能与降低基因组甲基化水平,抑制Nanos2、PLZF表达,促进Stra8表达有关。Objective:To investigate the influence of the expression of DNA methyltransferase 1(DNMT1)on the differentiation of spermatogonial stem cells(SSC)in mice.Methods:SSCs were isolated from the testis tissue of 1-week-old BALB/c male mice by two-step enzyme digestion.DNMT1-siRNA and negative control siRNA(NC-siRNA)were transfected into the third-generation SSCs after isolation and purification,and the untransfected cells were used as the control.At 24 hours after transfection,the mRNA and protein expressions of DNMT1 were detected by real-time quantitative PCR(RT-qPCR)and Western blot,respectively,and the methylation level of DNMT1 was determined.The SSCs were induced to differentiate into spermatocytes using the stem cell growth factor,and the expressions of the germ cell proliferation-related protein(Nanos2),promyelocytic leukemia zinc finger protein(PLZF)and retinoic acid-stimulated protein 8(Stra8)were measured by RT-qPCR and Western blot after 48 hours of differentiation.Results:At 24 hours after transfection,the relative mRNA and protein expressions of DNMT1 and the DNA methylation level were significantly decreased in the DNMT1-siRNA group compared with those in the control and DNMT1-NC groups(P<0.05),but showed no statistically significant difference between the latter two(P>0.05).The relative mRNA and protein expressions of Nanos2 and PLZF were also decreased while those of Stra8 increased in the DNMT1-siRNA group in comparison with those in the control and DNMT1-NC groups after 48 hours of differentiation(P<0.05),but none exhibited any statistically significant difference between the control and DNMT1-NC groups(P>0.05).Conclusion:Knockdown of DNMT1 promotes the differentiation of SSCs into spermatocytes in mice,which may be related to the reduction of the genome methylation level,inhibition of the expressions of Nanos2 and PLZF,and promotion of the expression of Stra8.

关 键 词：DNA甲基转移酶1 精原干细胞 分化 小鼠 

分 类 号：R321.1[医药卫生—人体解剖和组织胚胎学]