山羊体细胞核移植的研究
摘要
本实验研究了绒山羊体细胞核移植中供体成纤维细胞的的原代和传代培养、受体卵母细胞的体外成熟以及重构胚的激活和培养,并将重构胚移植给代孕受体。实验采用组织块培养法,在37℃、5%CO2、饱和湿度条件下,DMEM/F12+10%FBS培养液适宜于绒山羊皮肤成纤维细胞的体外生长,通过组织块培养法可以有效地建立绒山羊成纤维细胞系;传代培养时,酶消化法可以分离纯化绒山羊皮肤成纤维细胞;通过生长曲线测定和染色体计数可确定分离培养的绒山羊皮肤成纤维细胞基本符合体外培养细胞生长特性(2n=60);在山羊卵母细胞的采集方法上比较了切剖法与抽吸法对卵母细胞成熟的影响,结果发现,切剖法所获得的卵母细胞的成熟率比抽吸法的高(P<0.05);研究了在卵母细胞体外成熟液中,添加不同血清对卵母细胞体外成熟的影响。结果表明,自然发情的山羊血清对卵母细胞的成熟效果最好(P<0.05),PG诱导发情的山羊血清对卵母细胞的成熟效果也要比FBS好(P<0.05);在对山羊卵母细胞孤雌激活的研究上,采用了电激活和化学激活两种方法,结果显示,这两种方法都可以激活山羊卵母细胞,其中离子霉素联合6-DMAP激活山羊卵母细胞的卵裂率、囊胚率为78.1%和20.4%,显著高于7%乙醇联合6-DMAP的卵裂率28.7%、囊胚率3.2%和电激活的卵裂率55.3%、囊胚率9.3%;山羊孤雌胚在6-DMAP中作用2～4 h在囊胚率上没有差异(P>0.05);采用山羊卵丘细胞单层共培养孤雌胚,共培养囊胚率17.5%,非共培养囊胚率15.4%,对胚胎发育没有显著差异(P>0.05);融合电压从2400～3200V/cm、脉冲时间30μs、脉冲次数2时,都可以使重构胚融合,并且都能发育到卵裂,但是只有2800V/cm电压融合时,能发育到囊胚,囊胚率为13.2%;将所得的重构胚140枚移植到11只受体羊中,没有羊妊娠。
The study investigated somatic cell nuclear transfer in cashmere goat, including donor cell culture, recipient oocyte in vitro maturation, reconstructed oocyte electrofusion and activation, embryo in vitro culture and embryo transfer. The condition of 37℃、100% humidity and 5% CO2, DMEM/F12 supplemented with 10% FBS was used to support the goat skin fibroblasts growth in vitro. The method of explanted tissue culture can effectively establish the goat fibroblast cell line. The goat skin fibroblasts could be separated and purified by enzyme digestion. Growth curve analysis and chromosome counting can determine that the the isolated and cultured goat skin fibroblasts is similar with the growth characteristics of cultured cells in vitro (2n=60). The experiment also studied the collection methods of oocytes, the different serum. The result showed that there was significant difference between surface cutting and aspiration (P<0.05), EGS (natural estrus), EGS (PG-inducing estrus) and FBS (P<0.05). The conclusion was that surface cutting was more helpful for goat oocytes maturation; EGS (natural estrus) was significantly useful for oocytes maturation, homologous serum was better than heterologous serum for oocytes maturation. Matured goat oocytes were efficiently activated by Ionomycin together with 6-DMAP and 20.4% of blastocyst were achieved. It was higher than the rate of blastocyst achieved by 7% ethanol (3.2%) and electroactivation (9.3%). The goat parthenogenesis embryos were cultured 2～4h in 6-DMAP,we found that the blastocyst rate had no significant difference(P>0.05). When the reconstructed embryos were co-cultured with goat cumulus cell monolayer, the blastocyst development was 17.5%, but the blastocyst development was 15.4% without co-cultured, there is no significant difference. The fusion voltage 2400～3200V/cm, 30μs pulse duration, 2 pulse was suitable for the fusion of reconstructed embryos. A total of 140 reconstructed embryos were transferred into 11 recipients, no goat gestation.
The study investigated somatic cell nuclear transfer in cashmere goat, including donor cell culture, recipient oocyte in vitro maturation, reconstructed oocyte electrofusion and activation, embryo in vitro culture and embryo transfer. The condition of 37℃、100% humidity and 5% CO2, DMEM/F12 supplemented with 10% FBS was used to support the goat skin fibroblasts growth in vitro. The method of explanted tissue culture can effectively establish the goat fibroblast cell line. The goat skin fibroblasts could be separated and purified by enzyme digestion. Growth curve analysis and chromosome counting can determine that the the isolated and cultured goat skin fibroblasts is similar with the growth characteristics of cultured cells in vitro (2n=60). The experiment also studied the collection methods of oocytes, the different serum. The result showed that there was significant difference between surface cutting and aspiration (P<0.05), EGS (natural estrus), EGS (PG-inducing estrus) and FBS (P<0.05). The conclusion was that surface cutting was more helpful for goat oocytes maturation; EGS (natural estrus) was significantly useful for oocytes maturation, homologous serum was better than heterologous serum for oocytes maturation. Matured goat oocytes were efficiently activated by Ionomycin together with 6-DMAP and 20.4% of blastocyst were achieved. It was higher than the rate of blastocyst achieved by 7% ethanol (3.2%) and electroactivation (9.3%). The goat parthenogenesis embryos were cultured 2～4h in 6-DMAP,we found that the blastocyst rate had no significant difference(P>0.05). When the reconstructed embryos were co-cultured with goat cumulus cell monolayer, the blastocyst development was 17.5%, but the blastocyst development was 15.4% without co-cultured, there is no significant difference. The fusion voltage 2400～3200V/cm, 30μs pulse duration, 2 pulse was suitable for the fusion of reconstructed embryos. A total of 140 reconstructed embryos were transferred into 11 recipients, no goat gestation.
引文
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