人体细胞绵羊卵细胞克隆胚的构建及类ESc的分离
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摘要
本研究采用组织块培养法建立人胎儿皮肤成纤维细胞系,对培养的F12、F20代细胞进行核型分析,发现85.6%、83.3%的细胞染色体核型保持正常的倍数(2n=46)。
以人胎儿成纤维细胞作为核供体,体外成熟培养的绵羊卵母细胞为核移植受体,构建了人-绵羊异质重构胚。发现H33342荧光辅助去核法与盲吸法对重构胚的融合率及卵裂率无显著差距(P>0.05),但桑椹胚的发育率以盲吸法较高(52.38%对71.85%)。
以带下注射法及胞质内注射法构建重构胚其卵裂率(66.66%、71.42%)及桑椹胚的发育率(52.38%、47.62%),没有明显差别(P>0.05);但与总操作卵比较,胞质内注射效率明显高于带下注射(47.62%、21.78%)。进行带下注射电融合时,发现在电压为1.5 KV/cm时,融合率低于1.6 KV/cm及1.7 KV/cm(41.58%对57.69%、56.52%),但重构配的桑椹胚发育率以1.6KV/cm(40%对52.38%、53.85%)低。
从得到的桑椹胚分离获得了类胚胎干细胞集落,碱性磷酸酶染色,集落呈阳性。
Human Fetal skin fibroblasts were dissociated and cultured by explant tissue culture in vitro; Human fetal fibroblast cell had more normal chromosome content after at least twenty passages(2n=46); Compared with fluorescence method and blindfold method, the blindfold method was more efficient in the cleavage rate (48.83%vs 58.18) and producing reconstituted morula (73.81%vs 87.5% ).
The nuclear of donor was injected into exnuclear occytes by electric fusion method and intracytoplasmic nuclear injection (ICNI), Nuclear transplantation units(NTs) were cultured in vitro . Although there was no difference in the cleavage rate (P>0.05) and the rate of producing reconstituted morula (P>0.05).
Three voltages were entered into computer. It was more efficient in fuse the reconstituted embryos when the voltages were 1.6 KV/cm and 1.7KV/cm (57.69%、56.52%vs 41.58%).But there were not more efficient in producing morulas when the voltage were 1.6KV/cm (40%vs 52.38%、53.85%).
The gather of like Embryonic Stem cell was got and cultured from the morulas. AKP (alkaline phosphatase) dyed the clong, It was positived.
以人胎儿成纤维细胞作为核供体,体外成熟培养的绵羊卵母细胞为核移植受体,构建了人-绵羊异质重构胚。发现H33342荧光辅助去核法与盲吸法对重构胚的融合率及卵裂率无显著差距(P>0.05),但桑椹胚的发育率以盲吸法较高(52.38%对71.85%)。
以带下注射法及胞质内注射法构建重构胚其卵裂率(66.66%、71.42%)及桑椹胚的发育率(52.38%、47.62%),没有明显差别(P>0.05);但与总操作卵比较,胞质内注射效率明显高于带下注射(47.62%、21.78%)。进行带下注射电融合时,发现在电压为1.5 KV/cm时,融合率低于1.6 KV/cm及1.7 KV/cm(41.58%对57.69%、56.52%),但重构配的桑椹胚发育率以1.6KV/cm(40%对52.38%、53.85%)低。
从得到的桑椹胚分离获得了类胚胎干细胞集落,碱性磷酸酶染色,集落呈阳性。
Human Fetal skin fibroblasts were dissociated and cultured by explant tissue culture in vitro; Human fetal fibroblast cell had more normal chromosome content after at least twenty passages(2n=46); Compared with fluorescence method and blindfold method, the blindfold method was more efficient in the cleavage rate (48.83%vs 58.18) and producing reconstituted morula (73.81%vs 87.5% ).
The nuclear of donor was injected into exnuclear occytes by electric fusion method and intracytoplasmic nuclear injection (ICNI), Nuclear transplantation units(NTs) were cultured in vitro . Although there was no difference in the cleavage rate (P>0.05) and the rate of producing reconstituted morula (P>0.05).
Three voltages were entered into computer. It was more efficient in fuse the reconstituted embryos when the voltages were 1.6 KV/cm and 1.7KV/cm (57.69%、56.52%vs 41.58%).But there were not more efficient in producing morulas when the voltage were 1.6KV/cm (40%vs 52.38%、53.85%).
The gather of like Embryonic Stem cell was got and cultured from the morulas. AKP (alkaline phosphatase) dyed the clong, It was positived.
引文
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