探索蛋白酶体抑制剂MG-132对小鼠早期胚胎发育的影响

摘要

目的 探讨泛素-蛋白酶体抑制剂MG-132对小鼠早期胚胎发育细胞周期的影响.方法 通过体外受精的方法,分别收集受精后12h、24 h、36 h、48 h、60 h、72 h、84 h、96 h的胚胎(12 hpi、24 hpi、36 hpi、48 hpi、60 hpi、72 hpi、84 hpi、96 hpi),通过Western Blotting验证在检测12 hpi时总蛋白泛素化水平最高,以此为基准设置不同浓度以及时间梯度,确定MG-132引起2-细胞发育阻滞和延迟的最适浓度和处理时间,最终确定MG-132浓度1μmol/L,在14～18 hpi作用4h可以引起2-细胞发育阻滞和延迟.通过Edu检测、免疫荧光实验,观察分析MG-132是否会引起早期胚胎发育细胞周期改变.结果 用1μmol/L MG-132,在14～18 hpi处理4h可以引起早期胚胎发育细胞周期延迟.结论 MG-132作为泛素-蛋白酶体抑制剂,可以抑制胚胎发育中蛋白质的降解,引起早期胚胎发育细胞周期进程受抑,致使胚胎发育阻滞在G2/M期.%Objective To investigate the effects of ubiquitin proteasome inhibitor MG-132 on early embryonic development.Methods We collected the embryos of 12 hpi,24 hpi,36 hpi,48 hpi,60 hpi,72 hpi,84 hpi and 96 hpi through in vitro fertilization,the result of Western Blotting showed that polyubiquitinated proteins were mainly accumulated in the embryos of 12 hpi.Based on the above,different concentrations and time gradients were set to determine the optimal concentration and processing time of MG-132 induced cell retardation and delayed in two cell development.Utimately,we found that treatment of embroys with 1 μmol/L MG-132 from14 hpi to18 hpi could lead to retardation and delay in two cell development mostly.Edu assay and immunofluorescence assay were used to observe whether MG-132 could influence the cell cycle of embryos.Results Treatment of embryos with 1 μtmol/L MG-132 from 14 hpi to 18 hpi could lead to retardation and delay in two-cell stage development.Conclusion The ubiquitin proteasome inhibitor MG-132 could inhibit the degradation of proteins during embryonic development and it could lead to cell cycle of embryonic development arrested in G2/M phase.