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目的:建立氯化锶(SrCl2)诱导小鼠体细胞核移植胚胎激活的最佳方法。方法:首先构建及鉴定小鼠核移植胚胎,在注核完成后,使用以下实验激活核移植胚胎:实验1,采用5 mmol/L和10 mmol/L SrCl2处理核移植胚胎1～8 h,观察时间对卵裂率和囊胚率的影响;实验2,在4、6 h采用1～20 mmol/L多种浓度SrCl2处理核移植胚胎,观察不同浓度对卵裂率和囊胚率的影响;实验3,研究10 mmol/L SrCl2配合不同培养液激活核移植胚胎的效果;实验4,观察10 mmol/L SrCl2联合蛋白激酶抑制剂6-DMAP、细胞松驰素(CB)对体外培育核移植胚胎的影响。结果:研究发现当浓度一致时(5 mmol/L),6 h组卵裂率(38.9%)与1 h组(6.7%)、2 h组(22.8%)、3 h组(22.8%)、4 h组(25.6%)比较差异均有显著性(P<0.05),但与5 h组(28.9%)、7 h组(34.4%)、8 h组(28.9%)均无显著差异(P>0.05);当时间固定时(6 h),SrCl2浓度为10 mmol/L获得的卵裂率和囊胚率(68.9%和7.2%)较高,较1 mmol/L组(28.3%和0%)、2.5 mmol/L组(35.6%和0%)、5 mmol/L组(37.8%和1.1%)、7.5 mmol/L组(60.6%和2.2%)、15 mmol/L组(51.7%和1.1%)和20 mmol/L组(41.7%和1.1%)均高(P<0.05);不同成分培养液实验显示,含Ca2+和Mg2+KSOM组的胚胎卵裂率较低,仅为27.8%,与不含Ca2+组(69.4%)、不含Ca2+/Mg2+组(66.1%)和添加EDTA组(68.3%)比较差异均显著(P<0.05);虽然SrCl2联合各培养液对核移植胚胎体外发育影响不大,但SrCl2联合CB组囊胚细胞计数(45.40±2.23)较未联合组(30.15±1.12)、联合6-DMAP组(34.95±1.38)、联合6-DMAP+CB组(37.45±1.43)均显著增多(P<0.05)。结论:10 mmol/L SrCl2单独处理6 h或联合CB均能较好地激活小鼠体细胞核移植胚胎。
OBJECTIVE: To establish the best method of SrCl 2-induced embryo transfer in somatic cell nuclear transfer in mice. Methods: Firstly, nuclear transfer embryos were constructed and identified. Nuclear transfer embryos were activated by the following experiments after the completion of the injection. Experiment 1: The nuclear transfer embryos were treated with 5 mmol / L and 10 mmol / L SrCl2 for 1-8 h. Time on the cleavage rate and blastocyst rate; Experiment 2, at 4,6 h using 1 ~ 20 mmol / L SrCl2 treatment of nuclear transfer embryos to observe the different concentrations of cleavage rate and blastocyst rate; In experiment 3, the effects of 10 mmol / L SrCl2 with different culture medium on nuclear transfer embryos were investigated. In experiment 4, the effect of 10 mmol / L SrCl2 combined with protein kinase inhibitor 6-DMAP and cytokinin on the nuclear transfer in vitro Effects of embryos. Results: The cleavage rate (38.9%) in 6 h group was significantly higher than that in 1 h group (6.7%), 2 h group (22.8%), 3 h group (22.8% (P <0.05) in 4 h group (25.6%), but no significant difference compared with 5 h group (28.9%), 7 h group (34.4%) and 8 h group (28.9% > 0.05). When the time was fixed (6 h), the cleavage rate and blastocyst rate (68.9% and 7.2%) were higher than that of 1 mmol / L group (28.3% and 0 (35.6% and 0%), 5 mmol / L group (37.8% and 1.1%), 7.5 mmol / L group (60.6% and 2.2%), 15 mmol / L group (51.7% And 1.1%, respectively) and 20 mmol / L group (41.7% and 1.1%) (P <0.05). The results of different culture media showed that the embryo cleavage rate of Ca2 + and Mg2 + %, Which were significantly different from those in Ca2 + group (69.4%), Ca2 + / Mg2 + group (66.1%) and EDTA group (68.3%) However, the number of blastocyst cells in SrCl2 combined with CB group was significantly lower than that in non-combined group (30.15 ± 1.12), combined with 6-DMAP group (34.95 ± 1.38), combined with 6-DMAP + CB group ± 1.43) were significantly increased (P <0.05). Conclusion: 10 mmol / L SrCl2 alone or combined with CB can activate the somatic cell nuclear transfer embryo.