CAJ | 학술논문

背景:课题前期研究已证实100μg/L 胰岛素样生长因子1能拮抗体外30 mmol/L 高糖毒性引起的凋亡,可改善高糖环境对孕母/胎儿的危害.但这一改善胚胎早期发育的营养环境的发生机制和分子机制却不甚清楚.目的:结合前期工作基础,从表观遗传学角度入手,观察胰岛素样生长因子1拮抗早期高糖环境对小鼠胚胎印迹基因 H19/Igf-2甲基化水平和表达的影响.方法:通过建立孕娠糖尿病小鼠模型,取小鼠2细胞胚胎等分为实验组和对照组;对照组置于含30 mmol/L 葡萄糖的 KSOM 培养基内进行体外高糖逆境培养,实验组额外添加100μg/L 胰岛素样生长因子1处理.体外发育至囊胚期,检测胚胎印迹基因 H19,Igf-2的表达和印迹控制区 DNA 甲基化水平.结果与结论:实时定量 PCR 分析表明,实验组桑椹胚 H19,Igf-2的表达分别是对照组的5.9和5.2倍(P <0.01);实验组囊胚 H19,Igf-2的表达分别是对照组的2.4和1.8倍(P <0.01).BSP 测序法分析 H19,Igf-2印迹控制区的甲基化水平发现,实验组桑椹胚、囊胚的甲基化率分别比对照组下降27.9%和20.9%(P <0.01).结果可见在高糖环境下胰岛素样生长因子1可部分降低 H19/Igf-2印迹控制区 DNA 甲基化水平,使 Igf-2和 H19基因表达增高,能拮抗高糖环境对小鼠早期胚胎发育的损伤.
배경:과제전기연구이증실100μg/L 이도소양생장인자1능길항체외30 mmol/L 고당독성인기적조망,가개선고당배경대잉모/태인적위해.단저일개선배태조기발육적영양배경적발생궤제화분자궤제각불심청초.목적:결합전기공작기출,종표관유전학각도입수,관찰이도소양생장인자1길항조기고당배경대소서배태인적기인 H19/Igf-2갑기화수평화표체적영향.방법:통과건립잉신당뇨병소서모형,취소서2세포배태등분위실험조화대조조;대조조치우함30 mmol/L 포도당적 KSOM 배양기내진행체외고당역경배양,실험조액외첨가100μg/L 이도소양생장인자1처리.체외발육지낭배기,검측배태인적기인 H19,Igf-2적표체화인적공제구 DNA 갑기화수평.결과여결론:실시정량 PCR 분석표명,실험조상심배 H19,Igf-2적표체분별시대조조적5.9화5.2배(P <0.01);실험조낭배 H19,Igf-2적표체분별시대조조적2.4화1.8배(P <0.01).BSP 측서법분석 H19,Igf-2인적공제구적갑기화수평발현,실험조상심배、낭배적갑기화솔분별비대조조하강27.9%화20.9%(P <0.01).결과가견재고당배경하이도소양생장인자1가부분강저 H19/Igf-2인적공제구 DNA 갑기화수평,사 Igf-2화 H19기인표체증고,능길항고당배경대소서조기배태발육적손상.
BACKGROUND: The previous studies have confirmed that 100 μg/L insulin-like growth factor 1 can antagonist apoptosis in vitro induced by 30 mmol/L glucose toxicity, it can relieve harm of high glucose on maternal/fetal. But the mechanisms and molecular mechanisms to improve the nutritional environment of the early embryo development are not clear. OBJECTIVE: To observe the effect of insulin-like growth factor 1 antagonist high glucose on mRNA expression and DNA methylation levels of the development-related imprinted genes H19/Igf-2 in mouse preimplantation embryos in vitro on the basis of preliminary work and starting from epigenetic prospect. METHODS: The diabetic mice models were established. The acquired early period 2-cel embryos were divided into experimental group and control group: the control group was high sugar adversity cultured in KSOM culture medium containing 30 mmol/L glucose, and the experimental group was treated with 100 μg/L insulin-like growth factor 1. Until growth to the blastocyst stage, the mRNA expression level of H19/Igf-2, and the DNA methylation level in imprinting control region of H19/Igf-2 were detected. RESULTS AND CONCLUSION: Real-time PCR analysis showed that the mRNA expression levels of H19 and Igf-2 in morulas were respectively 5.9 and 5.2 times as that of the control group (P < 0.01). Moreover, thus mRNA expression levels of H19 and Igf-2 in blastulas were separately 2.4 and 1.8 folds as that of the control group (P < 0.01). Bisulfite Sequencing PCR analysis demonstrated that the DNA methylation levels of H19 and Igf-2 in mouse morulas and blastulas of the experimental group were respectively declined by 27.9% and 20.9% than that of the control group (P < 0.01). The insulin-like growth factor 1 antagonist high glucose can partial y reduce the DNA methylation level of H19/Igf-2 in imprinting control region in order to increase the mRNA expression levels of H19 and Igf-2, and it can antagonize the damage of high glucose during early mouse embryo development.