猪骨髓间充质干细胞的分离培养及核移植后重构胚胎发育能力的研究  

CULTURE OF PORCINE MESENCHYMAL STEM CELLS IN VITRO AND DEVELOPMENTAL CAPACITY OF RECONSTRUCTED EMBRYOS BY NUCLEAR TRANSFER

作　　者：王振坤[1] 田江天[1] 李秋明[2] 武美玲[1] 孔庆然[1] 郑重[1] 于学武[3] 雷蕾[2] 刘忠华[1] 

机构地区：[1]东北农业大学生命科学学院,哈尔滨150030 [2]哈尔滨医科大学基础医学院组织学与胚胎学教研室,哈尔滨150086 [3]东北农业大学动物医学学院,哈尔滨150030

出　　处：《分子细胞生物学报》2008年第4期275-282,共8页Journal of Molecular Cell Biology

基　　金：国家自然科学基金资助项目(30671025)

摘　　要：不同类型的细胞核移植效率不同,原因之一可能是不同类型细胞核移植后进行重编程的潜力不同。本实验对猪骨髓间充质干细胞(porcine bone marrow mesenchymal stem cells,pMSCs)体外分离培养的方法进行了优化,对猪骨髓间充质干细胞的增殖及生长特性进行了观察分析,并以其作为供体细胞进行核移植,对此类型细胞进行重编程的潜力进行了评估。结果表明用密度梯度离心法分离猪骨髓间充质干细胞优于全骨髓贴壁法;猪骨髓间充质干细胞数目在培养第6天达到峰值,传代培养10h时,贴壁率达到78.50%;传代培养后第4天分裂指数最高,为24.00‰;以猪骨髓间充质干细胞(pMSCs)和猪胎儿成纤维细胞(PF)分别作为供核细胞构建核移植胚胎,其体外囊胚发育率分别为14.63%与15.07%(P>0.05),孤雌对照组囊胚发育率为30.91%(P<0.05);而三组囊胚细胞数分别为30.67±17.7、24.1±6.5和25.8±11.4(P>0.05)。实验表明,体外培养的猪骨髓间充质干细胞生长增殖旺盛,生物学性状稳定,并适合作为核移植供体细胞。The developmental potential of reconstructed embryos varied according to the source of donor cells, it was thought that the donor cells capabilities to be reprogrammed were different. We established the method of culturing porcine bone marrow mesenchymal stem cells （pMSCs）, identified and observed the growth characteristics of pMSCs, and determined pMSCs reprogramming potential as donor cells for nuclear transfer （SCNT） . We found that the method of gradient centrifugation to isolate pMSCs from porcine bone marrow was better than the method of anchoring culture; the number of pMSCs achieved peak at day 6, the adhesive rate of cultured cells was 78.50% at 10h and the division index of cultured cells was 24.00‰ at day 4. The developmental competence were compared among three kinds of embryos, reconstructed embryos with PF and pMSCs, Parthenogenetic. The blastocysts rate and total cell number of blastocysts were 15.07%, 14.63% vs 30.91% and 24.1±6.5, 30.67±17.7 vs 25.8±11.4. These results indicated that pMSCs could be high proliferation and stable growth characters in vitro, and were suitable donor cells type for nuclear transfer.

关 键 词：猪骨髓间充质干细胞 生长特性 核移植 

分 类 号：Q813[生物学—生物工程]