【作者】 路国正；

【导师】 仲跻峰；

【作者基本信息】 山东师范大学 ， 细胞生物学， 2003， 硕士

【摘要】 核移植技术不仅是研究细胞核与细胞质相互作用的重要手段，而且是在医学和农业等领域都将有着广泛应用前景的一项重要胚胎工程技术。本实验用波尔山羊耳皮肤成纤维细胞进行核移植研究，建立了相应的核移植技术程序，如固定针和注射针的制作，卵母细胞的体外培养，成熟卵母细胞的去核，波尔山羊耳皮肤成纤维细胞移入去核卵母细胞的卵周隙，去核卵母细胞-波尔山羊耳皮肤成纤维细胞复合体的电融合，重组胚的激活和培养等。 结果表明： 1．用切剖法采集不同季节屠宰山羊卵巢卵母细胞，结果繁殖季节卵巢采集到可用卵母细胞1.01枚／卵巢（4011枚／3980卵巢），非繁殖季节卵巢可采集到可用卵母细胞0.49枚／卵巢（198枚／405卵巢）。不同季节采集的卵母细胞数差异显著。结果显示，繁殖季节屠宰山羊的卵巢可采集到更多的可用卵母细胞。 2．对不同卵母细胞成熟培养系统的效果进行比较分析，表明在系统ⅠM199+1IUhCG+1ug／ml 17β-E₂+10mg／ml BSA中卵母细胞成熟率达88.8％，在系统ⅡH199+1IU hCG+1ug／ml 17β-E₂+10％HGS中卵母细胞成熟率为75％，在系统ⅢM199+1IU hCG+1ug／ml 17β-E₂+10％FBS中卵母细胞成熟率为27.9％，可见系统Ⅰ具有较高而稳定的成熟率，是一种较理想的无血清培养系统。 3．卵泡的直径大小对卵母细胞的成熟培养有一定的影响，实验表明直径在2-5mm的卵泡内的卵母细胞体外成熟率达88％（142／160），而直径大于6mm的卵泡内的卵母细胞的成熟率只有3.5％（3／80）。 4．卵丘细胞的存在对卵母细胞的发育成熟有重要的影响，有三层以上卵丘细胞包裹的卵母细胞成熟率达89.4％（161／180），半裸卵和裸卵的成熟率分别为46.7％（28／60）和0（0／30）。 5．将繁殖季节采集的2140枚卵丘-卵母细胞复合体用于体外成熟培养，成熟率为85.9％（1818／2140）。非繁殖季节采集的200枚卵丘-卵母细胞复合体，成熟率为35.5％（71／200）。结果表明，不同季节采集的卵母细胞体外成熟率有显著差异。 6．在体细胞的培养中，建立了波尔山羊耳皮肤成纤维细胞的培养程序，获得了波尔山羊耳皮肤成纤维细胞的传代培养物，以此作为山羊体细胞核移植的供体细胞。用作核供体的体细胞在进行核移植前，培养液的血清浓度从10％降至0.5％， 波尔山羊耳成纤维细胞核移植的研究 从而诱导波尔山羊耳皮肤成纤维细胞处于细胞周期的G。期。7．在培养M中用斜口拔尖的针对卵母细胞进行去核，并用同一根针将波尔山羊耳 皮肤成纤维细胞移入卵母细胞的卵周隙，会节省体外操作的时间，有益于胚胎 的发育。8．场强为1．SKV／cm，脉冲宽度为40us，两次脉冲，间隔0．ss是一合适的融合参 数。9．用离子霉素处理 4min，卵裂率是 18％（54／301），桑甚胚发育率是 0（0／54）；用 离子霉素处理4min，然后再用6－DMAP化B激活lh，最后用6－DMAP处理4h卵 裂率是19．15％（91八75），桑甚胚发育率是8．8％（见引）：用肥31所激活4mh， CHX＋CD处理 lh，最后 Cgy处理 4h，卵裂率是 21．16％（111／525），桑甚胚发育率 是 6．3％（7／1 11）。结果表明后两种方法都能用来激活重组胚。本实验研究表明， 山羊一兔异种核移植胚、山羊一山羊同种核移植胚卵裂率分别是24．9％（41 ／165） 和27．l％（52／192），虽然山羊一免异种核移植胚能发育到桑意胚，但发育率 ［4．9％（2／41 ）1显著低于山羊一山羊同种核移植胚［9．6％（5／52 ）j。10．激活的重组胚在M199＋10％FBS、M199＋10％FBS十卵丘细胞两种培养系统中培养， 结果表明在加入卵丘细胞的系统中桑喜胚发育率高。更多还原

【Abstract】 Nuclear transfer technique is important not only for the study of nuclear-cytoplasm interaction, but also has extensive applications in human clinics and animal husbandry. In recent years, the technique of nuclear transfer of somatic cell is developing very rapidly. The fibroblast cell from goats was used for nuclear transfer and a relevant procedure of nuclear transfer was made in this experiment, the procedure includes the production of holding and injection pipette, the culture of oocytes in vitro maturation, the removal of nucleus from matured oocyte, the injection of fibroblast cell into perivitelline space of enucleated oocyte, the fusion of enucleated oocyte-fibroblast cell complexes, the action and culture of reconstructed embryos, etc.Some conclusions were drawn from the experiment as follows:1. The usable cumulus-oocyte (COCs) was collected from slaughtered goat ovaries during the breeding and nonbreeding seasons by surface-follicles cutting. The average total number of COCs recovered per ovary was 4.01(4011/3980) and 0.49(198/405), respectively. There is significant different between the numbers of COCs collected from the two seasons. The results show that more COCs can be recovered by surface-follicles cutting during the breeding season than the nonbreeding seasons.2. To compare effects of different mediums on the maturation rates of oocytes, the COCs were matured in different mediums as follows: 1 M199+lIU/ml hCG+1ug/ml 17β -E2+10mg/ml BSA; II M199+1IU/ml hCG+1ug/ml 17β-E2+10%HGS; III M199+1IU hCG+lug/ml 17 β -E2+1096FBS, the maturation rates of oocytes in group I , group II , group III were 88. 8%, 75% and 27.9%separately, the results indicated that the maturation rate of oocytes in group I was higher than that in group II and group III .3. The diameter of follicles has an effect on the maturation rate of the goat oocyes. The maturation rate of the goat oocyes from the follicleswith diameter 2< <5mm and 6mm< was 88% (142/160) and 3.5% (3/80).4. The cumulus cells have a important effect on the maturation of oocytes that the maturation rates of oocytes having three layers of cumulus, half naked and naked oocyes were 89. 4%(161/180), 46. 7%(28/60) and 0(0/30).5. Usable goat COCs recovered by surface-follicles cutting from breeding seasons and nonbreeding seasons were matured in vitro. The percentage of maturation were 85. 9% (1818/2140) and 35. 5% (71/200), respectively. The results show that COCs recovered from breeding seasons were better than from nonbreeding seasons.6. The system of culturing fibroblast cell was established. Cells of P1-P10 were used as donor cells. The fibroblast cells that were used as donor cells were induced to enter G0 stage of the cell cycle when they were cultured in the medium, the serum of which was decreased from 10% to 0. 5%.7. The enucleation and injection of fibroblast cell into perivitelline space were operated in the culture dish using the point-belved pipette, which would make the time for operation decreased and be helpful to the development of the embryos.8. Two electric pulses (1.5KV/cm, 40usec each, 0. 5sec interval) were applied. It was the optimal parameter of electrofusion for the oocyte-fibroblast cell complexes.9. When reconstructed embryos were activated by ionomycin for 4min, the percentage of cleavage of reconstructed embryos was 18% (54/301). And the proportion of reconstructed embryos developing to the morula stage was 0(0/54). When reconstructed embryos were activated by ionomycin for 4min and then by 6-DMAP for 5h, the percentage of cleavage of reconstructed embryos was 19. 15% (91/475). And the proportion of reconstructed embryos developing to the morula stage was 8.8% (8/91). When reconstructed embryos were cultured in A23187 for 4min following activated by CHX+CD for Ih, then CHX 4h, the percentage of cleavage of reconstructed embryos were 21. 16%(111/525); And the proportion of reconstructed embryos developing to the morula stage were 6. 3%(7/lll). The percentage of cleavage of goat-goat and goat-更多还原