【作者】 张林；

【导师】 张涌；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2007， 硕士

【摘要】 本实验旨在对牛克隆胚胎以及山羊-牛核移植胚胎的体外培养基进行优化,并且研究了在培养过程中不同时间添加不同大分子物质对于胚胎发育的影响。从而建立一套较为系统、稳定、高效的胚胎培养体系,提高胚胎体外囊胚发育率,为提高制作转基因动物、克隆动物、试管动物等的效率提供技术平台,为该技术走出实验室,在实践中得以应用提供理论依据。试验设计结果为:1.以DMEM为基础培养液,对供体牛以及山羊胎儿耳朵组织块进行组织块法培养,又以0.2%胶原酶II和0.25%胰蛋白酶(Trypsin)按2:3的比例配成混合消化液消化山羊胎儿供体组织,以2.5 g/L胰蛋白酶+1 mmol/L EDTA(Gibco BRL)消化液消化牛胎儿供体组织,结果两种方法都获得了体外培养的胎儿耳朵成纤维细胞。可在含10%DMSO(二甲亚讽)的冷冻液中于-70℃条件下短期冻存,也可在-196℃条件下长期保存。2.通过细胞质内注射法将牛和山羊胎儿耳朵成纤维细胞移入去核的牛卵母细胞中构建重组胚胎以及山羊-牛异种胚胎,分别采用mCR2aa以及mSOF进行培养,结果表明:mSOF液中培养同种胚胎和异种胚胎的卵裂率、8/16-cell发育率,囊胚发育率以及囊胚细胞数均明显高于mCR2aa中的培养结果。(同种依次为74.4±6.5 % Vs54.2±4.3%、45.1±6.2% Vs31.5±2.9 %、18.5±2. 3% Vs9.3±1.8%以及98.2±4.3 Vs78.4±4.1,异种依次为46.6±3.7% Vs36.4±5.1%、30.4±3.8% Vs 20.3±3.2%、8.8±2.1% Vs 3.7±1.5%以及81.1±3.2 Vs68.0±2.5)(P<0.05)。3.在mSOF中按不同培养时间添加8mg/mlBSA或者10%FBS,培养前三天和培养三天后添加的补充物质及次序为:⑴B SA+ FBS;⑵BSA+BSA;⑶F BS+BSA;⑷FBS+FBS。结果表明:添加BSA+FBS和BSA+BSA的培养体系培养的同种胚胎和异种胚胎卵裂率(同种为79.8±7.1%及74.0±3.6%,异种为40.1±6.3%及36.2±2.7%)均比添加FBS+BSA,FBS+FBS的培养体系结果明显高(同种为49.8±5.1%及49.5±2.4%,异种为26.1±3.2%及23.4±2.8%)(P<0.05)。添加BSA+FBS培养的同种胚胎及异种胚胎的8/16-cell发育率,囊胚发育率和囊胚细胞数均比其他组高(同种8/16-cell发育率为49.7±3.5% Vs37.5±2.6% Vs20.4±2.4% Vs19.0±2.1%;同种囊胚发育率为:21.5±1.8% Vs10.9±2.0% Vs5.0±2.3% Vs4.3±2.0%;同种囊胚细胞数依次为115.2±4.3 Vs 103.4±4.4 Vs 101.1±5.2 Vs 102.9±7.7,异种8/16-cell发育率为29.2±2.0% Vs18.1±2.2% Vs19.9±2.8% Vs18.2±2.6%;异种囊胚发育率为13.4±2.1% Vs6.9±2.1% Vs3.3±1.6% Vs3.1±1.7%;异种囊胚细胞数为100.1±3.0 Vs 99.0±2.2 Vs 79.7±5.3 Vs 77.7±4.6)(P<0.05)。更多还原

【Abstract】 This study is conducted to explore an effective cultivated method for supporting the embryo development and to make these technologies going out of laboratory to be used in the fields of embryonic engineering. We studied the effect of different culture medium and the different order of supple macromolecules to mSOF on the culture development of the bovine and goat-bovine cloned embryo. The test and results is as following:1. Bovine ear Fetal Fibroblasts and Goat ear Fetal Fibroblasts were obtained either by the basic culture media DMEM or Goat Fetal tissues were digested by a mixture of 0.2% collagenase II and 0.25% trypsin at the ratio of 2/3 and Bovine Fetal tissues were digested by a mixture of 2.5 g/L trypsin +1 mmol/L EDTA. The cells could be stored both at-196℃for a long time and at -70℃for a short time under the condition that 10% DMSO was added into freezing media.2. The cattle ear fetal fibroblasts and the goat ear fetal fibroblasts are transplanted into the enucleated cattle oocytes separately by using oocyte intraplasmic nuclear injection method to develop bovine-bovine cloned embryos and goat-bovine cloned embryos. The embryos are cultivated in modified charles rosenkrans 2 amino acid medium (mCR2aa) and modified synthetic oviduct fluid medium (mSOF) separately. Result show: cleavage rate, 8/16-cell rate, blastocysts rate and total cell number of blastocysts, cultivated in mSOF solution are all higher than those cultivated in mCR2aa (p<0.05).3. BSA(8mg/ml) or FBS(10%) can be added to mSOF according to the different periods of cultivation. The supplements and orders, added during the first three days and after three days are as follow: BSA and BSA, BSA and FBS, FBS and BSA, FBS and FBS. Result: the cleavage rate, 8/16-cell rate, blastocysts rate and total cell number of blastocysts, adding BSA and FBS into mSOF, are in turn 79.8±7.1%, 49.7±3.5%, 21.5±1.8%, and 115.2±4.3 in bovine-bovine cloned embryo, and 40.1±6.3%、29.2±2.0%、13.4±2.1% and 100.1±3.0 in goat-bovine cloned embryo, which are evidently higher than other cultivation groups(p<0.05).更多还原