【作者】 王士勇；

【导师】 杨福合；

【作者基本信息】 中国农业科学院 ， 特种经济动物饲养， 2014， 博士

【摘要】 为了探索梅花鹿-牛异种体细胞核移植技术，为梅花鹿胚胎工程研究奠定技术基础，本论文对其供核细胞的培养和冷冻保存、受体卵母细胞的体外成熟、显微操作、重组胚胎外培养等技术环节进行了探索研究，并在水貂、蓝狐和貉等毛皮动物上验证了建立的异种体细胞核移植技术体系。主要内容如下：（1）梅花鹿、水貂、蓝狐和貉耳皮肤成纤维细胞（Ear Skin Fibriblasts，ESF）经组织块法原代培养，利用酶消化时间差与贴壁时间差法处理3～5次纯化，结果显示细胞生长特点为贴壁生长、呈梭形、具伪足，F5代细胞生长曲线呈“潜伏期-对数生长期-停滞期”的模式。DMSO作为抗冻保护剂时，梅花鹿、水貂、蓝狐和貉ESF解冻复苏后复苏率分别为89.64%、92.44、91.24和91.04，显著高于甘油作为抗冻保护剂。结论：梅花鹿、水貂、蓝狐和貉ESF具有典型的皮肤成纤维细胞生长特点，通过酶消化时间与贴壁时间差的方法对其纯化可行，冷冻保存时可采用10%DMSO作为抗冻保护剂。（2）牛卵丘-卵母细胞复合体（Cumulus Oocyte Complexes，COCs）经22h体外成熟培养之后，LH+FSH和PMSG+HCG组成熟率分别为79.1%和75.2%，差异不显著，但是两组成熟率均显著高于对照组；牛COCs经（Brilliant Cresyl Blue, BCB）染色染色90min后卵母细胞着色率为68.1%，显著高于30、60min组，辨识难度为“中”；牛COCs经39μM的BCB染色后，着色率为64.5%，显著高于13μM组的29.3%；BCB+组和BCB-组的成熟率与对照组比较差异均不显著；在体外成熟培养液中添加50ng/mL表皮生长因子（Epidermal Growth Factor，EGF）后培养牛的COCs，22h后成熟率为77.4%，显著高于0、10、20ng/mL组。结论：在牛卵母细胞体外成熟培养液中添加国产激素10IU/mL PMSG、15IU/mL HCG和50ng/mL的EGF能显著提高其体外成熟率；BCB法筛选优质卵母细胞在本试验体系下不适用。（3）牛COCs经体外成熟20、21、22h后，0.4μg/mL脱羰秋水仙碱（Demecolcine，DEME）诱导显核率分别为61.0%、65.3%和66.7%，差异不显著；经0.4μg/mL DEME处理60、120min后，显核率分别为70.0%和71.9%，显著高于30min组；经0.5μg/mL DEME处理60min之后，显核率为78.9%，显著高于0.3、0.4μg/mL组；DEME辅助法的去核率为100.0%，显著高于挤压法和盲吸法，三种去核方法的去核时间差异不显著；带下注核与全细胞胞质内注核的成功率分别为100%和94.8%，注核时间分别为31.0s和35.1s，与破膜后胞质内注核比较差异均显著；一步操作和两步操作的相同注核方法处理组之间在激活率、卵裂率和囊胚率方面均无显著差异；0、0.25、0.50和1.00μM白藜芦醇对梅花鹿-牛ISCNT胚胎卵裂率没有显著影响，0.50和1.00μM组囊胚发育率分别为38.0%和36.7%，显著高于0、0.25μM组；梅花鹿-牛ISCNT胚胎用体细胞共培养时，水貂ESF组的卵裂率为55.7%，显著高于对照组，牛卵丘细胞组囊胚发育率为59.4%，显著高于对照组和其他两组。结论：梅花鹿-牛ISCNT研究可采用下面的技术程序：牛卵母细胞经IVM20～22h后，用0.5μg/mL DEME处理60min诱导显核，Pizeo辅助破膜胞质内注核的一步显微操作核移植，激活后的重组胚用牛卵丘细胞共培养。（4）采用Pizeo辅助全细胞胞质内注核的方法构建毛皮动物-牛ISCNT胚胎时，水貂、蓝狐和貉ESF作为供核细胞，在注核成功率、卵裂率和囊胚率之间差异不显著；牛卵丘细胞作为共培养细胞时，水貂、蓝狐、貉-牛ISCNT胚胎的囊胚率分别为43.6%、40.1%和42.1%，均显著高于对照组。结论：梅花鹿-牛ISCNT技术程序同样适用于水貂、蓝狐和貉等三种毛皮动物与牛的ISCNT。更多还原

【Abstract】 In order to explore Somatic Cell Nuclear Transfer (ISCNT) technology using sika deer-bovineInterspecies and to lay a foundation for embryonic engineering of sika deer, we investigatedbiocytoculture and cryopreservation of donor cells, in vitro maturation (IVM) of receptor oocyte,micro-manipulation and in vitro culture of reconstructed embryos, etc. In addition, a technology systemof somatic cell nuclear transfer was validated by experimenting on mink, blue fox and recoon dog.Maincontents were as follow:1. The ear skin fibroblasts of sika deer, mink, blue foxand recoon dog were initially cultured byexplant culture, then purified based on the differences in both enzymatic digestion time and adherencetime (for3~5times). The results showed that the growth of the cells characterized as adherent to thesurface, spindle shaped and with pseudopods. The Cell growth curve of F5generation followed by thepattern of “incubation-logarithmic growth-stagnation”. When DMSO was used as cryoprotectant, thedefrost recovery rate of ESF of sika deer, mink, blue fox and recoon dog was89.64%,92.44%,91.24%and91.04%respectively, which were significantly higher than those when glycerin glycerol was used.Conclusion: the ear skin fibroblasts of sika deer, mink, blue fox and recoon dog possess the typicalgrowth characteristics of skin fibroblast, they can be purified based on the difference inboth enzymaticdigestion time and adherence time.10%DMSO can be used as cryoprotectant during cryopreservation.2. The maturation rate of bovine cumulus oocyte complexes (COCs) after22h IVM was79.1%and75.2%when LH+FSH and PMSG+HCG were used respectively, showed no significant difference(P>0.05), but were significantly higher than the control group (P<0.05). The tinctorial yield of oocyte ofbovine COCs after stained for90min with Brilliant Cresyl Blue (BCB) was68.1%, significantly higherthan the groups of30min and60min (P<0.05) respectively, the identification difficulty classified as“normal”. The tinctorial yield of bovine COCs with BCB of39μM was64.5%, significantly higher thanthat of13μM (P<0.05). The differences between BCB+and control group had no significance, as wellas that between BCB-group and control group (P>0.05). After maturated in vitro for22h, thematuration rate of bovine COCs cultured in medium supplemented with Epidermal Growth Factor (EGF)50ng/mL had been remarkably higher than that with0,10and20ng/mL (P<0.05).Conclusion: the in vitro maturation rate of bovine oocytes can be significantly increased whenhormone such as10IU/mL PMSG,15IU/mL HCG and50ng/mL EGF were added into IVM medium.BCB method is not applicable to identify bovine oocytes of good quality in this study.3. The rate of extrusion of bovine COCs after treatment with0.4μg/mL Demecolcine (DEME) wasnot significantly different after cultured in vitro for20h (61.0%),21h (65.3%), and22h (66.7%)(P>0.05), but was significantly higher after treatment with0.4μg/mL DEME for60min (70.0%) and120min (71.9%) than for30min (P<0.05). The rate of extrusion in the group of0.5μg/mL DEME was78.9%, which was significantly higher than that of0.3and0.4μg/mL groups (P<0.05). The enucleationrate with DEME method (100%) was significantly higher than that with McGrath’s method andextrusion method (P<0.05), but there was no significantly difference in enucleation time (P>0.05).Therate of nuclear injection and the duration of nuclear injection by Perivitelline Microinjection (PM,100%and31.0s) and Whole-Cell Intracytoplasmic Microinjection (WCIM,94.8%and35.1s) were significantly better than by Break-Membrane-Cell Intracytoplasmic Microinjection (BMCIM, P<0.05).There was no significantly difference among the rate of activation, cleavage and blastocyst between theone-step and the two-step methods (P>0.05). The cleavage rate of Sika deer-Bovine embryo treatedwith resveratrol had no significantly difference among the groups of0,0.25,0.50and1.00μM. Theblastocyst rate in group of0.50μM (38.0%) and1.00μM (36.7%) was significantly higher than in thegroup of0μM and0.25μM (P<0.05). The cleavage rate of Sika deer-Bovine embryo in mink ESFco-culture system (55.7%) was significantly higher than the control group (P<0.05). The blastocyst ratein bovine culumus cells co-culture system (59.4%) was significantly higher than that in the two othersystemand control group (P<0.05).Conclusion: this procedures is applicable for the technical sika deer-bovine ISCNT: IVM bovineoocytes for20~22h; process with0.5μg/mL DEME for60min; BMCIM for nuclear transfer;co-culture of activated reconstructed embryo with bovine oocytes.4. When Pizeo was used for cell intracytoplasmic microinjection to construct fur animal-bovineISCNT embryo, there were no significant difference in success rate of nuclear injection, cleavage rateand blastocyst rate, no matter the donor cell was from mink, blue fox or recoon dog (P>0.05).Blastocyst rate of mink-bovine, blue fox-bovine and recoon dog-bovine ISCNT embryo was43.6%,40.1%and42.1%respectively when co-cultured with bovine cumulus cell, all significantly higher thanthose of control group (P<0.05).Conclusion: the procedure of sika deer-bovine ISCNT was also applicable to mink-bovine, bluefox-bovine and recoon dog-bovine ISCNT.更多还原