Rho相关蛋白激酶抑制剂Y-27632对猪诱导多能干细胞冻存和传代效率的影响  

Effected of Rho-associated Protein Kinase Inhibitor Y-27632 on Improving the Cryopreserved Survival and Passage of Porcine(Sus scrofa) Induced Pluripotent Stem Cells

作　　者：成德[1] 高星[1] 李珍珍[1] 高祎[1] 刘亚军[1] 鲍建昌[1] 王华岩[1] 

机构地区：[1]西北农林科技大学动物医学院,陕西省干细胞工程技术研究中心,杨凌712100

出　　处：《农业生物技术学报》2012年第7期726-734,共9页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究发展规划(973)项目(No.2009CB941002;No.2011CBA01002)

摘　　要：猪作为重要的疾病模型动物,目前猪诱导多能干细胞(iPS 细胞)已有建系,但是细胞的冻存和传代的效率较低,影响后续的实验研究。Rho 相关蛋白激酶(ROCK)抑制剂 Y-27632 可提高人胚胎干细胞的解冻存活率,促进其克隆形成。本实验以猪(Sus scrofa)iPS 细胞为研究对象,通过在猪 iPS 细胞冻存和解冻过程中添加Y-27632,发现其可以减少猪 iPS 细胞在冻存和解冻复苏过程中的凋亡。在细胞的传代过程中,使用 Y-27632 可以促进猪 iPS 细胞的贴壁和克隆形成。虽然高浓度 Y-27632 会对猪 iPS 克隆的形态产生一定的影响,但并未影响细胞的碱性磷酸酶(AP)活性和多能性基因 Oct4、Sox2 和 Nanog 的表达水平。最后将转座子报告质粒 PB[Act-RFP]DS 导入猪 iPS 细胞中,经流式筛选后得到的带有红色荧光的细胞,并将其注射于猪孤雌胚胎中进行发育能力检测,发现 Y-27632 的处理能减少细胞在流式筛选和胚胎注射中的凋亡,促进其在胚胎内的嵌合发育。研究结果说明,ROCK 抑制剂 Y-27632 可以提高猪 iPS 细胞冻存和传代的效率,促进其在胚胎中的嵌合。该研究有助于猪 iPS 细胞及其他多能干细胞的保存,传代和筛选等相关研究。Pig is used as an important model animal, and induced pluripotent stem cells （iPS cells） have been established in pig now. However, the efficiency of cryopreserved survival and passage is very low. Rho-associated protein kinase （ROCK） inhibitor Y-27632 enhanced the survival of cryopreserved of human embryonic stem （ES） cells, and improved the ES colony formation. In this study, we used Y-27632 for porcine （Sus scrofa） induced pluripotent stem cells. 5 and 10 tzmol/L Y-27632 was used for cryopreservation and passage of porcine iPS cells, and it suggested that Y-27632 could greatly suppress cryopreserved-induced apoptosis and increase thaw-survival rates of porcine iPS cells. Meanwhile, it was able to helo the adhesion of porcine iPS cell in passage when 5 and 10 μmol/L Y-27632 was mixed into culture medium, and it also contributed to porcine iPS colonies formation in 24 h. Furthermore, 10 μmol/L Y-27632 would change the morphology of porcine iPS cells, and made the colonies become flat and loose, but it did not affect alkaline phosphatase （AP） activity and the expression level ofpluripotenct genes, octamer-binding transcription factor-4 （Oct4）, SRY-related high-mobility-group （HMG）-box protein-2 （Sox2） and homeobox transcription factor （Nanog） in porcine iPS cells treated with Y-27632. Besides, PB [Act-RFP] DS, the transposon reporter construct, was introduced into porcine iPS cells through electric transfected, and RFP positive cells were sorted by flow-cytometry. After treated with 10 μmol/L Y-27632 in culture medium, these sorted cells were easier to grow. After these sorting porcine iPS cells injected into porcine early embryos through micromanipulation, the cells treated with Y-27632 were easier to integrate into porcine parthenogenetic embryos than that without treated cells. Above all Y-27632 were able to improve the cryopreserved survival and passage of porcine iPS cells, and suppress the apoptosis induced by single cell dissociated and fluorescence-activated cell sor

关 键 词：Y-27632 猪诱导多能干细胞(iPS细胞) Rho相关蛋白激酶(ROCK) 冻存 凋亡 

分 类 号：S635[农业科学—蔬菜学]