【作者】 李煜；

【导师】 张涌；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2001， 博士

【摘要】 本项研究首先用山羊和牛的皮肤组织进行了细胞培养预备试验，对其细胞系建立方法和基本特性进行了探讨。在此基础上，成功建立了大熊猫皮肤成纤维细胞有限和无限细胞系，获得体外发育至囊胚的大熊猫-牛种间核移植胚胎。结果如下： 一.用含有10％NCS、100IU/ml青霉素、10OIU/ml链霉素的M199 4℃保存皮肤组织，8小时内组织块均能存活。添加10％NCS、100IU/ml青霉素、100IU/ml链霉素的M199、DMEM、HLH培养液，均可用于山羊和牛的皮肤细胞的体外培养，其中青山羊皮肤组织块在上述培养液中原代培养的成活率分别为75.0％、71.4％和80.0％。用添加D-Hanks’配制的0.25％胰蛋白酶（trypsin），0.02％EDTA，胰蛋白酶-EDTA（0.25％胰蛋白酶，0.02％EDTA，简称TE）消化液控时控温消化，重新接种后24小时的细胞贴壁率分别为86.4％、88.5％、95.8％。根据上皮细胞与成纤维细胞对TE消化液的敏感度差别，经1-3次传代，可得到纯化的成纤维细胞和纯化的上皮细胞。体外传代培养的安哥拉山羊、布尔山羊、黑白花奶牛和济宁青山羊皮肤成纤维细胞，以DMSO为冻存保护剂进行冷冻保存，解冻培养后，24小时细胞贴壁率分别为84.2％、86.4％、89.5％和91.76％。解冻细胞经体外3—5次传代培养，生长状态良好，细胞形态保持初始状况。 济宁青山羊皮肤成纤维细胞解冻后作为供核体，用于本实验室克隆山羊的研究，得到世界首例成年体细胞克隆山羊，表明冻存的细胞保持了良好的遗传特性。 二.在山羊和牛皮肤细胞体外培养的基础上，对大熊猫皮肤细胞体外培养的取材、原代培养方法、成纤维细胞纯化、培养液筛选、细胞冻存及解冻、体外生长的生物学特性等方面进行了初步探讨，得到如下结果： 1.将大熊猫皮肤组织置含10％新生牛血清（NCS）、100IU/ml青霉素、100IU/ml链霉素的保存液M199中，4℃、4小时内带回实验室，用DMEM、添加水解乳蛋白的Hanks’液（HLH）和M199基础培养基中添加10％新生牛血清、100IU/ml青霉素和链霉素，气象和温度分别为5％CO2、95％空气、100％湿度，37.5℃进行培养，皮肤组织的成活率分别为83.3％、60.0％和77.7％。体外培养的组织块在培养3-8天开始逸出成纤维细胞，较牛羊原代细胞迁移力强，细胞形态亦有明显区别，成纤维细胞为多角形，细胞核呈圆形或椭圆形，核仁1-3枚，大而明显。 2.0.25％胰酶-0.02％EDTA消化液控温控时消化原代细胞，经传代后可分离纯化皮肤成纤维细胞。传代后8h细胞贴壁率为86％，24h细胞贴壁率为93％。 3.选用M199E、M199H、DMEMH、DMEML、HLH、F12为基础培养液，添加10％NCS、100IU/ml青霉素、链霉素及5mM Hepes培养第三代皮肤成纤维细胞，在相同条件下，DMEML的效果最好。以DMEM为基础培养液，分别添加不同浓度的血清，培养第四代大熊猫皮肤成纤维细胞，培养后摘要 大熊猫成纤维细胞体外培养与异质克隆胚构建第六天进行细胞计数，结果表明，添加 10％、15％、20％NCS对细胞生长无显著差别，无血清的DMLM中，只能维持细胞存活，不能进行细胞增殖。以低糖DMall为基础培养液，在添加血清、双抗和Hepes的同时分别添加不同剂量的TGF、EGP、insulin分组培养，以细胞生长群体倍增值为准，评估生物活性因子对大熊猫皮肤成纤维细胞生长的影响。结果表明，EGF和胰岛素对细胞生长均有较明显的促进作用。 4。用添加血清和双抗的问199为基础培养液，分别添加 fat油、10％乙二橄 10％DMSO冻存大熊猫皮肤成纤维细胞，解冻后24小时细胞贴壁率分别为67．96％、83．33％、87．09％。添加DMSO的冻存液效果最佳，可以满足其细胞传代和长期保存的需要。 5．以低糖DMEM为基础培养液，添加血清、HePes、双抗和sps／ml胰岛素，5＄C02、饱和湿度，37．5”C培养第三代大熊猫皮肤成纤维细胞（PF3），每天进行细胞计数，以其平均值为准绘制生长曲线，用盖玻片培养法测定分裂指数。第六天群体倍增值为4．09。对数生长期细胞群体倍增时间为30．28小时．六天之后，细胞增长减缓，细胞分裂指数急剧下降。生长曲线表明第三代大熊猫皮肤成纤维细胞体外培养条件下存在延缓期和接触抑制，属正常体细胞。 6．第四代大熊猫皮肤成纤维细胞（PF4）染色体组型：Zn＝42，根据着丝粒指数，总体可将其分为两种类型，1—17号常染色体和X染色体为中部或亚中着丝粒，18－－－－0号常染色体为小型近端着丝粒。染色体组型分析表明体外地养的大熊猫皮肤成纤维细胞，－在传代初期保持了哀好一的生活状态；取材、材料的保存、以及原代培养、传代培养过程中的一系列操作，不造成染色体组型的变化。 三．用DMEM体外培养大熊猫皮肤成纤维细胞，传代到第22代时发生群体性细胞衰亡，培养细胞增殖缓慢，传代细胞贴壁数量减少。透射电镜观察，细胞核分叶不明显，核内核仁极少并变小，细胞质中含有较多的溶酶体及其残留物，细胞表面的微绒毛数量急剧减少，并且含有大量髓鞘样结构。上述情况表明，此时的细胞已经发生超微结构的变化，形成更多还原

【Abstract】 abstract Ph.D. Dissertation FIBROBLAST CELL CULTURE AND HETEROGENEOUS EMBRYO RECONSTRUCTION IN GAINT PANDA Doctoral Candidate: LI YU Advisor: Prof.: Zhang Yong The College of Animal Science and Veterinary Medicine, Northwest Science and Technology University of Agriculture and Forestry, Yangling, Shaanxi, P. R. China 712100 Abstract Experiments of generating the skin fibroblast cell line from the tissues of goat and bovine had been made in advance in order to investigate the methods of the cell line establishment and characterization. Then the finite and infinite skin fibroblast cell line from the tissue of giant panda was successfully established, and the interspecies nuclear transplant blastocysts between giant panda and bovine were obtained. Following is the results: 1. All block of skin tissue from goat can be survived in M199 supplemented with 10%NCS. 100 IU/ml penicillin. 100 lU/mi streptomycin at 4XD in 8 h. The survival rate of the skin tissue pieces in M199. DMEM. HLH supplemented with I0%NCS. 100 ILT/ml penicillin. 100 lU/mI streptomycin was 75.0%. 71.4%. 80.0% respectively. When the fibroblast cells were trypsined by D-hanks?with 0.25% trypsin . 0.02 EDTA. 0.25% trypsin桬DTA (TE) at controlled temperature and time, Attachment rate of the cells after 24 h was 86.4%. 88.5%. 95.8% respectively. According to the different sensitivity to TE, and through 1-3 passages, epithelia and fibroblast cells can be separated and purified. Cultured fibroblast cell line of Angora goat. Boar goat. Milk cow and Ji-ning goat was preserved at low temperature with the cryogen of DMSO and when they were thawed, the seeding efficiencies were 84.2%. 86.4%. 89.5% and 91.8% respectively. Thawed cells were passaged 3-5 times in culture with the natural characteristics. After thawed, the fibroblast cells of Ji-ning goat were used to study animal cloning in our laboratory, and the first adult somatic cloned goat were obtained in the world. This demonstrates that preserved cells maintain the natural genetic pattern. 2. Following the cell culture experiments of goats and bovines, we investigate the capture of skin tissue, primary culture, cell purifying, medium selection, cell preservation and the characterization of giant panda: - 97 - 2.1. The skin tissue from giant panda was brought to laboratory in M199 supplemented with 10%NCS, 100 lU/mi penicillin, 100 lU/mi streptomycin at 4C in 4 h. When cultured in the incubator at 5%C02, 95%air, 100%humidity, 37.5X2, the survival rate of the skin tissue pieces in DMEM, HLH, M199 supplemented with 10/oNCS, 100 lU/mi penicillin, 100 lU/mi streptomycin, was 75.0%, 7 1.4%, 80.0% respectively. Outgrowth of fibroblast cells started at 3-8 day from the culture beginning. The migratory efficiency of the fibrobiast cells is higher than that of the goat and cow. The configuration of the fibrobiast cells of giant panda is either different from those of goats and cows. 2.2 The fibroblast cells of giant panda can be separated and purified when trypsined with TE at p controlled tem更多还原