猪单倍体孤雌胚胎和体外受精胚胎干细胞培养的研究
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摘要
从基因组大小、代谢生理和个体大小来看,猪是最接近人类的哺乳动物。以猪为研究对象,进行胚胎干细胞培养的研究,可以为人类疾病研究和治疗提供借鉴。单倍体孤雌胚胎干细胞或类胚胎干细胞系的成功建立,可实现雌性基因组的复制(即卵子克隆),从而为治疗人类由配子发生障碍导致的不孕不育疾病提供有效途径,同时也可能成为研究基因组印迹以及提高克隆动物生产效率的主要工具。体外受精(IVF)胚胎干细胞或类干细胞系的建立,不但可以用于研究人类器官病变或损伤的细胞移植治疗和药物的细胞代谢水平筛选,还可以为基因敲除介导的动物转基因育种提供靶细胞。
本研究主要探讨不同的基础培养液和小分子化合物、LIF及bFGF因子的添加以及不同种类的饲养层细胞对猪孤雌单倍体胚胎和体外受精胚胎类胚胎干细胞建系的影响。本研究共分四部分：一、比较不同的卵母细胞激活方法,确定能获得高比率单倍体胚胎的孤雌激活方法；二、在实验一的基础上,用能获得高比率单倍体的激活方法生产孤雌胚胎,进行猪单倍体孤雌胚胎类胚胎干细胞的培养；三、通过常规体外受精方法生产猪IVF胚胎,进行猪IVF胚胎类胚胎干细胞的培养；四、比较孤雌胚胎和IVF胚胎的发育率和质量,分析导致两种胚胎具有不同贴壁和生长能力的可能原因。结果如下：
一、采用不同的物理或化学方法激活猪卵母细胞。试验一分别采用电激活、电激活结合细胞松弛素B(CB)、不同浓度乙醇以及不同作用时间对成熟卵母细胞进行激活处理,试验二检测电激活结合MG-132或Thimerosal和DTT对成熟卵母细胞的激活效果。试验一结果显示电激活结合CB组(27.34%)处理后的囊胚率显著高于电激活处理组(16.92%)(P<0.05),且二者均显著高于所有的乙醇组(P<0.05),乙醇处理组中以9%乙醇作用11 mmin效果最佳(10.20%)。试验二结果显示电激活结合MG-132组的囊胚发育率(24.69%)与电激活结合CB组(27.50%)没有显著差异,二者显著高于电激活结合Thimerosal和DTT组(14.10%)及电激活处理组(17.5%)(P<0.05)。对所得囊胚进行染色体倍性分析后得出,电激活处理后的囊胚单倍体率(41.7%)与9%乙醇处理组(40%)和电激活结合Thimerosal和DTT处理组(35.3%)无显著差异,但却显著高于电激活结合CB(O)及电激活结合MG-132(6.7%)处理组(P<0.05)。综合囊胚发育率和囊胚单倍体比率,电激活、9%乙醇、电激活结合Thimerosal、DTT和电激活结合MG-132处理组获得单倍体囊胚的总效率分别为7.3%(17.5%×41.7%)、4.1%(10.2%×40%)、5.0%(14.1%×35.3%)和1.7%(24.69%×6.7%)。电脉冲处理可能是比较理想的获得猪单倍体孤雌胚胎的激活方法。
二、将发育到囊胚的猪孤雌胚胎去透明带后,分别接入不同的培养液和不同的饲养层细胞中。将胚胎接入培养液I(DMEM培养液：DMEM+ bFGF+LIF+nucleoside mix+FBS)中,以MEF作为饲养层细胞,胚胎贴壁率为0；将胚胎接入培养液Ⅱ(BRL细胞条件化的TCM-199),在没有饲养层细胞的情况下,胚胎贴壁率达到21.7%,但贴壁后的细胞有明显的分化现象且未形成克隆。在培养液中添加LIF因子,胚胎贴壁率降至9.3%,没有形成克隆。加上MEF饲养层后,胚胎无一贴壁；将胚胎接入培养液Ⅲ(小分子培养液：DMEM/ F12+Neurobasal medium+N2B27+PD0325901+CHIR99021)中,分别以MEF和L-cell为饲养层,胚胎均未贴壁,以MEF和L-cell等比混合作为饲养层,获得23.1%的贴壁率,贴壁后的细胞虽无分化却未形成克隆；胚胎接入培养液Ⅳ(DMEM/F12培养液：DMEM/F12+KSR+bFGF)中,以MEF作为饲养层,贴壁率为O；胚胎接入培养液V(DMEM/HAM' S-F10培养液：DMEM +HAM' S-F 10+NUTRIENT MIX+LIF+bFGF+KSR+FBS)中,以MEF作为饲养层,贴壁率为10%,但贴壁后的细胞也未形成克隆。
三、采用4种不同的培养液,即上述培养液Ⅰ.Ⅱ.Ⅲ.Ⅳ,以MEF、L-cell或STO细胞为饲养层,培养猪IVF囊胚。将胚胎接入培养液Ⅰ中,以MEF为饲养层,胚胎贴壁率为0；胚胎接入BRL条件化的培养液,在无饲养层细胞的情况下,其贴壁率达到37.5%,但贴壁后的细胞有明显的分化现象且未形成克隆。胚胎接入培养液Ⅲ中,以MEF和L-cell等比混合作为饲养层,获得27.8%的贴壁率,贴壁后的细胞未形成克隆；将胚胎接入培养液Ⅳ中,以MEF为饲养层,贴壁率为22%,贴壁后的胚胎能形成克隆的比率为63.6%,且有3个胚胎贴壁后已传至第三代。以STO为饲养层,其贴壁率为25%,贴壁后的胚胎形成克隆率为75%,且有一个胚胎贴壁后传至第一代。将于MEF及STO上贴壁生长的细胞用碱性磷酸酶鉴定均为阳性,将MEF上贴壁生长的细胞用Oct-4、SSEA-1免疫荧光染色,结果显示为阳性,证实贴壁后生长的细胞为猪类胚胎干细胞。
四、比较了孤雌胚胎及IVF胚胎的发育率、囊胚质量和免疫荧光染色效果。结果显示二者的发育率没有显著差异,分别为28.7%和25.9%,囊胚总细胞数上也无显著差异,分别为51和49。两种胚胎的Oct-4免疫荧光染色结果均显示阳性。由此可见,猪孤雌胚胎发育到囊胚阶段时的卵裂球全能性和胚胎质量不是导致其不能获得类胚胎干细胞的主要原因。
Pigs are more similar to humans regarding their genomic, metabolic physiology and body size. The investigation on porcine embryonic stem cell could be helpful for therapy and research in human disease. Establishment of porcine haploid parthenogenetic embryonic stem cell could make the clone of oocyte come true, then make an approach in therapy for human sterility and yeld disease resulted from gamets ungenesis. It also could become a tool for research on genomic imprinting and improvement of animals cloning efficiency. Establishment of embryonic stem cell from porcine IVF embryos could be used not only for investigation on cells transfer to recipients with organ pathological changes and damnification but also for medicine screening. It also could offer target cells for producing gene knockout animals.
In this study, the effects of different basic medium, addition of small moleculars, LIF and bFGF and different feeder cells on the establishment of porcine haploid parthenogenetic embryonic stem cells and IVF embryonic stem cells were investigated. This study consisted of four parts.1. We compared different methods for activating porcine oocyte, in order to obtain high efficient method to produce porcine haploid parthenogenetic embyos.2. The method which could result in high percentage haploid parthenogenetic embyos was used to produce parthenogenetic embyos, and these embryos were used for culture of haploid parthenogenetic embryonic stem cell.3. The IVF embryos were produced with conventional method and used for culture of porcine embryonic stem cell.4. The development rate and quality of parthenogenetic and IVF blastocysts were compared, in order to elucidate the potential reasons resulted in different competences of these two kinds of embryos to attach the dish and outgrowth cell clone. The results were as follows:
1. The different physical and chemical methods were applied to activate MII porcine oocytes. In experiment 1, electrical activation, a combination of electrical activation and cytochalasin B (CB), and ethanol activation at different concentrations and durations were used to activate porcine oocytes. In experiment 2, electrical activation followed by MG-132 or Thimerosal/DTT treatments were investigated for their effects on porcine oocytes activation. The results of experiment 1 indicated that the development rates of blastocysts in CB group (27.34%) were significantly higher than that of electrical activation alone (16.92%) (P<0.05), and both of them were significantly higher than that of all ethanol groups (P<0.05). In all ethanol groups, 9% ethanol treatment for 11min got the highest blastocyst rate (10.20%). The results of experiment 2 showed that the rate of blastocysts formation in MG-132 group (24.69%) was not significantly different with that of CB group (27.50%), but they were significantly higher than that of Thimerosal/DTT (14.10%) and electrical activation (17.5%) groups (P< 0.05). The ploidy analysis of blastocysts derived from different groups indicated that the percentages of haploid blastocysts in electrical activation,9% ethanol and Thimerosal/DTT group were 41.7%,40% and 35.3% respectively, they were significantly higher than that of CB (0) and MG-132 (6.7%) groups (P<0.05).Considering both blastocysts formation rate (out of oocytes) and haploid rate (out of total blastocysts), the final haploid blastocyst percentages (out of oocytes) in electrical activation,9% ethanol, Thimerosal/DTT and MG-132 groups were 7.3%(17.5%*41.7%)、4.1%(10.2%*40%)、5.0%(14.1%*35.3%) and 1.7% (24.69%*6.7%), respectively. The electrical activation might be the best activation way to obtain haploid embryos.
2. The parthenogenetic embryos were removed of zona pellucida and cultured in different media with different feeder cells. The attachment rates of embryos cultured in mediumⅠ(DMEM+bFGF+LIF+nucleoside mix+FBS) with MEF as feeder cells were 0. The attachment rate of embryos cultured in mediumⅡ(bafflo rat liver cell conditioned TCM-199) without feeder cells was 21.7%, but the attached cells differentiated apparently and they did not form clone. Adding LIF in this medium reduced the attachment rate to 9.3%, and no clone was formed. Embryos did not attach when cultured in this medium with MEF as feeder cells. When the embryos were cultured in mediumⅢ(DMEM/F12+Neurobasal medium+N2B27+ PD0325901+CHIR99021) with MEF or L-cell as feeder cells, the attachment rates were 0. When the mixation of 1/2MEF+1/2L-cell was used as feeder cells, attachment rate was 23.1%. The attached cells did not form clone, though they did not differentiate. The attachment rate of embryos in mediumⅣ(DMEM/F12 +KSR+bFGF) with MEF as feeder cells was 0. The attachment rate of embryos in mediumⅤ(DMEM+HAM'S-F10+NUTRIENT MIX+LIF+bFGF+KSR+FBS) with MEF as feeder cells was 10%, but the attached cells did not form clone.
3. Four kinds of different media (Ⅰ,Ⅱ,ⅢandⅣmentioned above) were used to culture IVF embryos. The attachment rate of IVF embryos cultured in mediumⅠwith MEF as feeder cells were 0. The attachment rate of IVF embryos cultured in BRL conditioned medium without feeder cells was 37.5%, but the attached cells differentiated apparently and they did not form clone. The attachment rate of embryos cultured in mediumⅢwith 1/2MEF+1/2L-cell as feeder cells was 27.8%, the attached cells did not form clone. The IVF embryos were cultured in mediumⅣwith MEF or STO as feeder cells. The attachment rate of embryos cultured were 22% and 25% respectively. The rate of embryos formed primary clone was 63.6% and 75.0% respectively. Three attached embryos in MEF group passaged for 3 times. One attached embryo in STO group passaged for 1 time. The cells attached on both MEF and STO were detected positive by AKP staining. The attached cells on MEF were detected positive by Oct-4 and SSEA-1 staining.The results shown that the attached cells were porcine embryonic stem cell-like cells.
4. The parthenogenetic and IVF embryos were compared for their development rates, quality and immuno fluorescence staining of blastocyst. The blastocyst formation percentages were not significantly different between parthenogenetic and IVF embryos, they were 28.7% and 25.9% respectively. The cell number of blastocysts did not show significant difference either, they were 51 and 49.Both parthenogenetic and IVF embryos shown positive Oct-4 staining.The results shown that neither poor quality nor the lack of pluripotence could result in the poor attachment and outgrowth competence of parthenogenetic embryos.
本研究主要探讨不同的基础培养液和小分子化合物、LIF及bFGF因子的添加以及不同种类的饲养层细胞对猪孤雌单倍体胚胎和体外受精胚胎类胚胎干细胞建系的影响。本研究共分四部分：一、比较不同的卵母细胞激活方法,确定能获得高比率单倍体胚胎的孤雌激活方法；二、在实验一的基础上,用能获得高比率单倍体的激活方法生产孤雌胚胎,进行猪单倍体孤雌胚胎类胚胎干细胞的培养；三、通过常规体外受精方法生产猪IVF胚胎,进行猪IVF胚胎类胚胎干细胞的培养；四、比较孤雌胚胎和IVF胚胎的发育率和质量,分析导致两种胚胎具有不同贴壁和生长能力的可能原因。结果如下：
一、采用不同的物理或化学方法激活猪卵母细胞。试验一分别采用电激活、电激活结合细胞松弛素B(CB)、不同浓度乙醇以及不同作用时间对成熟卵母细胞进行激活处理,试验二检测电激活结合MG-132或Thimerosal和DTT对成熟卵母细胞的激活效果。试验一结果显示电激活结合CB组(27.34%)处理后的囊胚率显著高于电激活处理组(16.92%)(P<0.05),且二者均显著高于所有的乙醇组(P<0.05),乙醇处理组中以9%乙醇作用11 mmin效果最佳(10.20%)。试验二结果显示电激活结合MG-132组的囊胚发育率(24.69%)与电激活结合CB组(27.50%)没有显著差异,二者显著高于电激活结合Thimerosal和DTT组(14.10%)及电激活处理组(17.5%)(P<0.05)。对所得囊胚进行染色体倍性分析后得出,电激活处理后的囊胚单倍体率(41.7%)与9%乙醇处理组(40%)和电激活结合Thimerosal和DTT处理组(35.3%)无显著差异,但却显著高于电激活结合CB(O)及电激活结合MG-132(6.7%)处理组(P<0.05)。综合囊胚发育率和囊胚单倍体比率,电激活、9%乙醇、电激活结合Thimerosal、DTT和电激活结合MG-132处理组获得单倍体囊胚的总效率分别为7.3%(17.5%×41.7%)、4.1%(10.2%×40%)、5.0%(14.1%×35.3%)和1.7%(24.69%×6.7%)。电脉冲处理可能是比较理想的获得猪单倍体孤雌胚胎的激活方法。
二、将发育到囊胚的猪孤雌胚胎去透明带后,分别接入不同的培养液和不同的饲养层细胞中。将胚胎接入培养液I(DMEM培养液：DMEM+ bFGF+LIF+nucleoside mix+FBS)中,以MEF作为饲养层细胞,胚胎贴壁率为0；将胚胎接入培养液Ⅱ(BRL细胞条件化的TCM-199),在没有饲养层细胞的情况下,胚胎贴壁率达到21.7%,但贴壁后的细胞有明显的分化现象且未形成克隆。在培养液中添加LIF因子,胚胎贴壁率降至9.3%,没有形成克隆。加上MEF饲养层后,胚胎无一贴壁；将胚胎接入培养液Ⅲ(小分子培养液：DMEM/ F12+Neurobasal medium+N2B27+PD0325901+CHIR99021)中,分别以MEF和L-cell为饲养层,胚胎均未贴壁,以MEF和L-cell等比混合作为饲养层,获得23.1%的贴壁率,贴壁后的细胞虽无分化却未形成克隆；胚胎接入培养液Ⅳ(DMEM/F12培养液：DMEM/F12+KSR+bFGF)中,以MEF作为饲养层,贴壁率为O；胚胎接入培养液V(DMEM/HAM' S-F10培养液：DMEM +HAM' S-F 10+NUTRIENT MIX+LIF+bFGF+KSR+FBS)中,以MEF作为饲养层,贴壁率为10%,但贴壁后的细胞也未形成克隆。
三、采用4种不同的培养液,即上述培养液Ⅰ.Ⅱ.Ⅲ.Ⅳ,以MEF、L-cell或STO细胞为饲养层,培养猪IVF囊胚。将胚胎接入培养液Ⅰ中,以MEF为饲养层,胚胎贴壁率为0；胚胎接入BRL条件化的培养液,在无饲养层细胞的情况下,其贴壁率达到37.5%,但贴壁后的细胞有明显的分化现象且未形成克隆。胚胎接入培养液Ⅲ中,以MEF和L-cell等比混合作为饲养层,获得27.8%的贴壁率,贴壁后的细胞未形成克隆；将胚胎接入培养液Ⅳ中,以MEF为饲养层,贴壁率为22%,贴壁后的胚胎能形成克隆的比率为63.6%,且有3个胚胎贴壁后已传至第三代。以STO为饲养层,其贴壁率为25%,贴壁后的胚胎形成克隆率为75%,且有一个胚胎贴壁后传至第一代。将于MEF及STO上贴壁生长的细胞用碱性磷酸酶鉴定均为阳性,将MEF上贴壁生长的细胞用Oct-4、SSEA-1免疫荧光染色,结果显示为阳性,证实贴壁后生长的细胞为猪类胚胎干细胞。
四、比较了孤雌胚胎及IVF胚胎的发育率、囊胚质量和免疫荧光染色效果。结果显示二者的发育率没有显著差异,分别为28.7%和25.9%,囊胚总细胞数上也无显著差异,分别为51和49。两种胚胎的Oct-4免疫荧光染色结果均显示阳性。由此可见,猪孤雌胚胎发育到囊胚阶段时的卵裂球全能性和胚胎质量不是导致其不能获得类胚胎干细胞的主要原因。
Pigs are more similar to humans regarding their genomic, metabolic physiology and body size. The investigation on porcine embryonic stem cell could be helpful for therapy and research in human disease. Establishment of porcine haploid parthenogenetic embryonic stem cell could make the clone of oocyte come true, then make an approach in therapy for human sterility and yeld disease resulted from gamets ungenesis. It also could become a tool for research on genomic imprinting and improvement of animals cloning efficiency. Establishment of embryonic stem cell from porcine IVF embryos could be used not only for investigation on cells transfer to recipients with organ pathological changes and damnification but also for medicine screening. It also could offer target cells for producing gene knockout animals.
In this study, the effects of different basic medium, addition of small moleculars, LIF and bFGF and different feeder cells on the establishment of porcine haploid parthenogenetic embryonic stem cells and IVF embryonic stem cells were investigated. This study consisted of four parts.1. We compared different methods for activating porcine oocyte, in order to obtain high efficient method to produce porcine haploid parthenogenetic embyos.2. The method which could result in high percentage haploid parthenogenetic embyos was used to produce parthenogenetic embyos, and these embryos were used for culture of haploid parthenogenetic embryonic stem cell.3. The IVF embryos were produced with conventional method and used for culture of porcine embryonic stem cell.4. The development rate and quality of parthenogenetic and IVF blastocysts were compared, in order to elucidate the potential reasons resulted in different competences of these two kinds of embryos to attach the dish and outgrowth cell clone. The results were as follows:
1. The different physical and chemical methods were applied to activate MII porcine oocytes. In experiment 1, electrical activation, a combination of electrical activation and cytochalasin B (CB), and ethanol activation at different concentrations and durations were used to activate porcine oocytes. In experiment 2, electrical activation followed by MG-132 or Thimerosal/DTT treatments were investigated for their effects on porcine oocytes activation. The results of experiment 1 indicated that the development rates of blastocysts in CB group (27.34%) were significantly higher than that of electrical activation alone (16.92%) (P<0.05), and both of them were significantly higher than that of all ethanol groups (P<0.05). In all ethanol groups, 9% ethanol treatment for 11min got the highest blastocyst rate (10.20%). The results of experiment 2 showed that the rate of blastocysts formation in MG-132 group (24.69%) was not significantly different with that of CB group (27.50%), but they were significantly higher than that of Thimerosal/DTT (14.10%) and electrical activation (17.5%) groups (P< 0.05). The ploidy analysis of blastocysts derived from different groups indicated that the percentages of haploid blastocysts in electrical activation,9% ethanol and Thimerosal/DTT group were 41.7%,40% and 35.3% respectively, they were significantly higher than that of CB (0) and MG-132 (6.7%) groups (P<0.05).Considering both blastocysts formation rate (out of oocytes) and haploid rate (out of total blastocysts), the final haploid blastocyst percentages (out of oocytes) in electrical activation,9% ethanol, Thimerosal/DTT and MG-132 groups were 7.3%(17.5%*41.7%)、4.1%(10.2%*40%)、5.0%(14.1%*35.3%) and 1.7% (24.69%*6.7%), respectively. The electrical activation might be the best activation way to obtain haploid embryos.
2. The parthenogenetic embryos were removed of zona pellucida and cultured in different media with different feeder cells. The attachment rates of embryos cultured in mediumⅠ(DMEM+bFGF+LIF+nucleoside mix+FBS) with MEF as feeder cells were 0. The attachment rate of embryos cultured in mediumⅡ(bafflo rat liver cell conditioned TCM-199) without feeder cells was 21.7%, but the attached cells differentiated apparently and they did not form clone. Adding LIF in this medium reduced the attachment rate to 9.3%, and no clone was formed. Embryos did not attach when cultured in this medium with MEF as feeder cells. When the embryos were cultured in mediumⅢ(DMEM/F12+Neurobasal medium+N2B27+ PD0325901+CHIR99021) with MEF or L-cell as feeder cells, the attachment rates were 0. When the mixation of 1/2MEF+1/2L-cell was used as feeder cells, attachment rate was 23.1%. The attached cells did not form clone, though they did not differentiate. The attachment rate of embryos in mediumⅣ(DMEM/F12 +KSR+bFGF) with MEF as feeder cells was 0. The attachment rate of embryos in mediumⅤ(DMEM+HAM'S-F10+NUTRIENT MIX+LIF+bFGF+KSR+FBS) with MEF as feeder cells was 10%, but the attached cells did not form clone.
3. Four kinds of different media (Ⅰ,Ⅱ,ⅢandⅣmentioned above) were used to culture IVF embryos. The attachment rate of IVF embryos cultured in mediumⅠwith MEF as feeder cells were 0. The attachment rate of IVF embryos cultured in BRL conditioned medium without feeder cells was 37.5%, but the attached cells differentiated apparently and they did not form clone. The attachment rate of embryos cultured in mediumⅢwith 1/2MEF+1/2L-cell as feeder cells was 27.8%, the attached cells did not form clone. The IVF embryos were cultured in mediumⅣwith MEF or STO as feeder cells. The attachment rate of embryos cultured were 22% and 25% respectively. The rate of embryos formed primary clone was 63.6% and 75.0% respectively. Three attached embryos in MEF group passaged for 3 times. One attached embryo in STO group passaged for 1 time. The cells attached on both MEF and STO were detected positive by AKP staining. The attached cells on MEF were detected positive by Oct-4 and SSEA-1 staining.The results shown that the attached cells were porcine embryonic stem cell-like cells.
4. The parthenogenetic and IVF embryos were compared for their development rates, quality and immuno fluorescence staining of blastocyst. The blastocyst formation percentages were not significantly different between parthenogenetic and IVF embryos, they were 28.7% and 25.9% respectively. The cell number of blastocysts did not show significant difference either, they were 51 and 49.Both parthenogenetic and IVF embryos shown positive Oct-4 staining.The results shown that neither poor quality nor the lack of pluripotence could result in the poor attachment and outgrowth competence of parthenogenetic embryos.
引文
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