【作者】 曹鸿国；

【导师】 张涌；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2005， 博士

【摘要】 体细胞核移植同ES 细胞研究与组织工程相结合,诞生治疗性克隆的新疗法,修复人体病变的组织或器官,不仅可以解决移植物与受体间的免疫排斥反应问题,还可以解决移植物的来源问题。本次研究通过对体细胞核移植克隆胚适宜构建条件、激活方法、不同供体细胞在体细胞核移植中的应用、ES 细胞的适宜分离方法、克隆胚ES 细胞的分离培养、诱导分化等进行系列研究,为人类治疗性克隆的临床应用积累知识和经验。1、小鼠卵母细胞经乙醇和氯化锶激活同小鼠输卵管上皮细胞饲养层共培养,胚胎发育较为一致,卵裂均匀;小鼠孤雌胚的激活率乙醇加饲养层组最高97.15 %;小鼠孤雌胚的2-细胞率乙醇加饲养层组最高93.14 %;小鼠孤雌胚的桑椹胚率乙醇加饲养层组最高80.38 %;小鼠孤雌胚的囊胚率氯化锶加饲养层组最高76.21 %;孤雌胚的ICM 孵出率氯化锶加饲养层组最高75.28 %;孤雌胚ES 细胞孵出率氯化锶加饲养层组最高29.21 %,同乙醇加饲养层组、氯化锶组之间差异极显著(p<0.01)。表明氯化锶激活的昆明小鼠孤雌胚体外具有较强的发育能力。2、LIF 和大鼠心肌条件培养基在小鼠ES 细胞分离培养过程中,昆明小鼠ICM 孵出率心肌条件培养基为76.30 %,LIF 条件液为59.35 %,两者差异极显著(p<0.01); LIF条件液比心肌条件培养基能较早地分离出小鼠ICM,平均时间差为11.2 h;ES 细胞集落培养48 h时周边出现分化现象的集落所占比例在心肌条件培养基中为51.55 %, LIF条件液中为31.69 %, 两者差异极显著(p<0.01);第5 代小鼠ES 细胞核型正常率在心肌细胞条件培养基中为78.60 %,稍高于LIF 条件液中的76.00 %。表明大鼠心肌条件培养基较LIF 条件液更能成功分离培养昆明小鼠ES 细胞。3、小鼠在注射hCG 后14～16 h 间进行去核,去核成功率最高80.09 %;注射hCG后的20～22 h 间去核的卵母细胞,重构胚激活率最高85.14 %;注射hCG 后14～16 h去核重构胚囊胚率最高13.94 %,同16～18 h 间的差异显著(p< 0.05),同18～20 h、20～22 h 间的差异极显著(p< 0.01)。表明小鼠卵母细胞适宜在注射hCG 后14～16 h 间进行去核。4、小鼠卵丘细胞核移植重构胚氯化锶加饲养层组325 枚,195 枚发育到2-细胞阶段,71 枚发育到桑椹胚阶段,38 枚发育到囊胚阶段,9 枚重构囊胚分离出ES 细胞,5枚囊胚分离的ES 细胞集落可稳定传代;分离出ES 细胞集落呈鸟巢状突起,生长旺盛的ES 细胞集落周围可见到分化的上皮样或梭形细胞;ES 细胞碱性磷酸酶染色强阳性;体外形成类胚体。核移植重构胚氯化锶组289 枚,162 枚重构胚发育到2-细胞阶段,10更多还原

【Abstract】 Therapeutic cloning can be used restoring human pathological tissue and organ by somatic cell nuclear transfer and ES cell biotechnology, which not only can avoid graft versus host reaction, but also provide lots of seed cells used in cell engineering, tissue engineering and organ transplant. In order to provide knowledge and experience in clinic application of human therapeutic cloning, feasible reconstructed condition, activation method of reconstructed embryos, application of different donor cells in somatic cell nuclear transfer, suitable isolation method of embryonic stem cells, isolation and cultivation of ES cells from reconstruction embryos and others were studied in this paper. 1. Mice embryos had normal image and synchronous development by ethanol or SrCl2 activation and co-culture with mice oviduct epidermal cells; Mice activation rate of parthenogenetic embryos in ethanol plus feeder cells group was highest 97.15%; Mouse 2-cell rate of parthenogenetic embryos in ethanol plus feeder cells group was highest 93.14%,; Mice morula rate of parthenogenetic embryos in ethanol plus feeder cells group was highest 80.38%; Mice blastocyst rate of parthenogenetic embryos in SrCl2 plus feeder cells group was highest 76.21%; Mice hatched inner cell mass rate of parthenogenetic embryos in SrCl2 plus feeder cells group was highest 75.28%; Mice embryonic stem cell hatched rate of parthenogenetic embryos with SrCl2 plus feeder cells group was highest 29.21%, significant difference with ethanol plus feeder cells group, and SrCl2 group (p<0.01). It was indicated that Kunming species mice parthenogenetic embryos activated with SrCl2 have stronger development ability in vitro. 2. Isolation rate of mice inner cell masses with cardiomyocyte media conditioned was 76.30 %, leukemia inhibitor factor media was 59.35 %, and difference between two medias was significant (p<0.01); Isolation of mice inner cell masses with leukemia inhibitor factor media was earlier than that with cardiomyocyte media conditioned, mean time difference was 11.2 h; Differentiation rate of embryonic stem cell masses of 48th h with cardiomyocyte media conditioned was 51.55 %, leukemia inhibitor factor media was 31.69 %, and difference between two medias was significant (p<0.01); Normal karyotype rate of mice embryonic stem cells with cardiomyocyte media conditioned 78.6 % was slightly higher than that with leukemia inhibitor factor media 76 %. It was indicated that rat cardiomyocyte media conditioned is more feasible for isolation and cultivation of Kunming species mouse embryonic stem cells than leukemeia inhibitor factor media conditioned. 3. Encleation rate of mice oocytes 14~16 h after hCG injection was highest 80.09%; Activation rate of embryos reconstructed during 20~22 h was highest 85.14 %; Blastocyst rate of mice oocytes during 14~16 h was highest 13.94%, significant difference with 16~18 h (p<0.05), 18~20 h, 20~22 h (p< 0.01). It was indicated that encleation time of mice oocytes 14-16 h after hCG injection is good. 4. 195 of 325 mice reconstructed embryos with cumulus cells in SrCl2 plus feeder cells group were developed to 2-cell embryo, 71 to morula, 38 to blastocyst, 9 to embryonic stem cells, in which 5 embryonic stem cells could be passaged and cultivated successfully; Embryonic stem cell colonies isolated were with island-like image, strong positive by AKP staining, could become embryoid bodies and spontaneously differentiate into epidermal-like cells around them in vitro; 162 of 289 mice reconstructed embryos in SrCl2 group were developed to 2-cell embryo, 10 to morula, 3 to early blastocyst. It is approved that feeder cells, modified media, and cardiomyocyte media conditioned play a key role to development of reconstructed embryos in vitro, and mice cumulus cells as donor cells of somatic cell nuclear transfer can be applied in therapeutic cloning field. 5. 227 of 420 mice reconstructed embryos with skin fibroblast as donor cells in SrCl2 plus feeder cells group were developed to 2-cell embryo, 72 to morula, 29 to blastocyst, 6 to embryonic stem cell, in which 3 embryonic stem cell-like colonies could be passaged and cultivated successfully; Karyotype analysis to 5th passage embryonic stem cells was researched, normal rate of karyotype was 77.84 %, 75.18 %, 77.20 % respectively; Embryonic stem cells isolated were island-like, positive by AKP staining, and could spontaneously differentiate into epidermal or shuttle-like cells around them in vitro; In addition, after frozen and thawed routinely, embryonic stem cells were with strong proliferation and previous image; 184 of 372 mice reconstructed embryos with skin fibroblasts in SrCl2 group were developed to 2-cell embryo, 5 to morula. It is indicated that feeder cells, modified media, and cardiomyocyte media conditioned are key to development of reconstructed embryos in vitro, and mice skin fibroblasts as donor cells of somatic cell nuclear transfer can be feasible in therapeutic cloning field. 6. 128 of 312 mice reconstructed embryos with blood lymphocytes as donor cells inSrCl2 plus feeder cells group were developed to 2-cell embryo, 69 to morula stage, 6 to blastocyst stage, 3 to inner cell mass, which had similar image with inner cell masses from embryos produced in vivo; 121 of 343 mice reconstructed embryos with blood lymphocytes in SrCl2 group were developed to 2-cell embryo, 2 to morula. It was indicated that feeder cells, modified media, and cardiomyocyte media conditioned have a key role to development of reconstructed embryos in vitro, and it is feasible to construct embryos with peripheral blood lymphocytes in mice, warranting further study in this area. 7. Nuclear transfer embryonic stem cells from reconstructed embryos with cumulus cells and skin fibroblasts were induced and differentiated into epithelial cells, which had high purity and cell proliferation capability, cytokeratin identification of epithelial cells was positive, so epithelial cells can be used as seed cells in clinic application. 8. Cardiomyocytes with rhythmical beat were induced and differentiated from nuclear transfer embryos reconstructed with cumulus cells and fibroblasts as donor cells, which still had the rhythmical synchronization beat after passaged; Immunocytochemistry identification of cardiomyocytes a-sarcmeric actin and cardiac troponin-T was positive, and lots of glucogen were in cardiomyocyte cytoplasma; Cardiomyocytes had very strong division potential in vitro. It was indicated that cardiomyocytes could be induced and differentiated from nuclear transfer embryos reconstructed, and applied in clinic as seed cells.更多还原