摘要
本研究利用TSA(Trichostatin A)、ROS(Roscovitine)、血清饥饿和接触抑制方法处理牛卵丘/颗粒细胞,检测处理前后细胞乙酰化水平、周期分布及其对重组胚发育能力的影响,为提高核移植效率提供理论基础。分别采用上述方法处理牛卵丘/颗粒细胞,首先对处理的细胞形态及活率进行观察,并利用流式分选技术检测处理细胞的周期分布,再通过间接免疫荧光法检测经上述不同处理前后细胞乙酰化水平变化,最后以上述处理细胞作为核供体构建重组胚,比较重组胚发育水平的不同。试验结果显示,经上述处理的卵丘/颗粒细胞形态良好,活率均保持在94%以上;TSA处理卵丘/颗粒细胞后,其乙酰化水平较对照组和其他处理组明显增加(P<0.05),经ROS处理的卵丘/颗粒细胞乙酰化表达水平同样高于对照组(P<0.05),但仍低于TSA处理组(P<0.05),但经血清饥饿处理的卵丘/颗粒细胞乙酰化水平较对照组明显降低(P<0.05);利用TSA处理卵丘/颗粒细胞24 h后,细胞被明显抑制在G0/G1期,且较其他处理组的抑制现象更加明显(P<0.05);并且,经TSA处理后的卵丘/颗粒细胞充当供体细胞,其卵裂率和囊胚率较对照组明显增加(P<0.01,(85.2±3.4)%vs(68.6±6.7)%;(30.2±5.7)%vs(10.4±8.3)%)。经TSA处理的牛卵丘/颗粒细胞,与其他处理组相比,乙酰化水平较高,细胞形态良好,细胞周期被明显抑制在G0/G1期,且获得了较高的卵裂率和囊胚率,作为供体细胞的处理方式较为适合。
In this study, the cumulus cells were dealed with Trichostatin A（TSA）, Roscovitine （ROS）, serum starvation and contact inhibition, we detect the change of level of acetylation, the cell cycle, and the development of cloning embryos after the treatments, which provided academic foundation for raising efficiency of cloning. Using the above methods, the cell morphology change and the motility rate were observed, then the levels of acetylation and cell cycle were detected through the indirect immunofluorescence and the flow cytometry respectively, and the motility rate were more than 94% all. TSA obviously enhanced the level of acetylation （P〈0.05） ; ROS also made a high level of acetylation, but lower than the TSA treatment （P〈0.05）, the serum starvation treatment was lower than the control. The cumulus cells after treatments had good morphology, TSA made the cell cycle stop at the G0/G1 and the highest cleavage and blastula rate （P〈0.01, （85.2±3.4）% vs （68.6±6.7）%;（30.2±5.7）% vs （10.4±8.3）%）. These results indicated that the cumulus cells treated with TSA not only had a high level of acetylation and better morphology change compared with the other groups, but also had the highest percent of blastula, so it was more suitable method to deal with the donor cells.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2009年第5期645-651,共7页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
“863”高新技术发展计划(2006AA10Z198)
国家自然科技平台建设项目(2005DKA21101)
关键词
牛
卵丘/颗粒细胞
乙酰化
克隆胚
bovine
cumulus cells
acetylation
cloning embryos
