人类胚胎干细胞与小鼠囊胚嵌合的探讨  

Investigation of chimeras of human embryonic stem cells to mouse blastocysts

作　　者：徐小明[1,2] 杜丽丽[1] 林戈[1,2] 段馨[1] 卢光琇[1,2] 

机构地区：[1]中南大学生殖与干细胞工程研究所,湖南长沙410008 [2]中南大学人类干细胞国家工程研究中心,湖南长沙410008

出　　处：《西北农林科技大学学报（自然科学版）》2011年第2期42-46,共5页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家“973”重点基础研究发展计划(2005CB522705);国家自然科学基金项目(30800650);湖南省自然科学基金项目(09JJ4010)

摘　　要：【目的】探讨人类胚胎干细胞（Human embryonic stemcells,hESCs）能否在小鼠胚胎环境中生存并参与其各个组织器官的分化,为hESCs与小鼠囊胚嵌合的可行性研究提供依据。【方法】将154枚受精后3.5 d（3.5days postcoitum,3.5 dpc）ICR品系小鼠囊胚随机分为3组：试验组（注射hESCs）、模拟注射组（注射针仅刺破透明带及滋养层细胞,但不注射细胞和溶液）及对照组（未注射）,于注射后培养24 h统计囊胚完全孵化率。将稳定转染增强绿色荧光蛋白（Enhanced green fluorescent protein,EGFP）的含有9~15个hESCs的小细胞团块,显微注射入3.5 dpcICR品系小鼠囊胚腔内,注射后分别于3,24,48,69,77,94和116 h,在普通荧光显微镜下观察EGFP阳性细胞的定位与动态分布情况,并将嵌合体胚胎移植入假孕母鼠子宫内,分别于移植后第4天和第6天处死孕鼠,普通荧光显微镜下观察EGFP阳性细胞在整个小鼠胚胎中的分布情况。【结果】注射后24 h,试验组和模拟注射组的囊胚完全孵化率分别为73.6%和77.1%,均极显著高于对照组（38.3%）（P〈0.01）,有88.9%（32/36）的胚胎中的hESCs迁移并定位在小鼠内细胞团（ICM）及其临近的滋养层细胞上;体外培养48 h后,EGFP阳性细胞数进一步减少;116 h后,只有1枚胚胎仍残留3~4个EGFP阳性细胞,且散在分布于ICM克隆之外。体内发育试验结果显示,将43枚注射后的嵌合体囊胚移植入6只代孕母鼠子宫内,获得了22枚脱膜,有17枚脱膜中含有胚胎,其中形态正常胎儿14枚,没有胚胎含有EGFP阳性细胞。【结论】hESCs很难与小鼠囊胚正常嵌合。【Objective】 The study was to explore whether hESCs can survive and contribute to different tissues in the mouse embryos,which will provide the basis for the feasibility study of interspecific chimeras using injection of hESCs into mouse blastocysts.【Method】 154 mouse blastocysts of 3.5 days postcoitum（dpc） were randomly divided into 3 groups：experiment group（injection of hESCs）,sham group（needle only pierced the zona pellucida and trophoblast cells,but not injected hESCs and solutions） and control（not injection）.The rate of hatched blastocyst was calculated after embryo cultured 24 h in vitro.3.5 dpc ICR mouse blastocysts were injected with EGFP-hESCs and allowed to culture 3,24,48,69,77,94 and 116 h in vitro,or were transferred to the uterine horns of pseudopregnant recipient mice.By fluorescence microscope observation,the EGFP-hESCs contribution pattern of survival,migration,proliferation,and differentiation was investigated.【Result】 24 h after injection,the blastocyst hatched rate of the experiment group and the sham group were 73.6% and 77.1%,respectively,which was significantly greater than that of the control（38.3%）（P0.01）.At this stage,engrafted cells were localized to the ICM or around ICM of 88.9%（32/36）embryonic chimeras.48 h after culture in vitro,the positive EGFP cells were further decreased.Only 3 to 4 EGFP positive cells scattered out of the ICM in one embryo 116 h after culture in vitro.For chimeric embryos development in vivo,43 hESCs-injected blastocysts were implanted into the uterus of 6 pseudopregnant foster mice and harvested 22 formed deciduaes,of which 17 contained embryos.14 of these embryos were morphous normal and did not contain any EGFP-positive hESCs-137 derivatives.【Conclusion】 It is very difficult for hESCs to chimeras with mouse blastocysts.

关 键 词：人类胚胎干细胞 囊胚 小鼠 嵌合体 

分 类 号：Q813.7[生物学—生物工程]