论文标题:绵羊耳成纤维细胞系建立和卵母细胞体外成熟体外受精的研究
Study on the Establishment of the Ear Fibroblast Cell Line and IVM、IFV of the Sheep
论文作者
论文导师 张利平；刘丑生,论文学位 硕士,论文专业 动物遗传育种与繁殖
论文单位 甘肃农业大学,点击次数 144,论文页数 77页File Size2802K
2007-05-28论文网 http://www.lw23.com/lunwen_3428122/
sheep;;fibroblast cell line;;Oocytes ;;IMC;;IVF ;;EFG;;IGF-1
我国是世界上畜禽种质资源最丰害的国家之一。近些年由于高产专门化品种的培育与推广,导致很多畜禽品种受到一定程度的威胁。因此保护动物遗传资源迫在眉睫。 本论文选取了青海贵德裘皮羊,内蒙古乌珠穆沁,陕西同羊为研究对象,采用组织块贴壁培养法和细胞冷冻技术成功构建了成纤维细胞系,并对培养细胞进行了形态学、生长动力学观察、细胞活力测定、核型分析和微生物检测。该细胞系的建立,使得这三个国家重要种质资源在细胞水平上保存下来,也为体细胞克隆等研究提供了理想的生物材料。每个品种的冻存样本含量为40-50之间,每个品种的冻存管数在240-300支之间,每个个体的冻存管数为6支,每支冻存管的细胞数量为3-5×106个/ml。与此同时研究了EGF,IGF-1对绵羊卵母细胞体外成熟和卵裂的影响,半胱氨酸和不同胚胎培养液对体外受精胚胎早期发育的影响,为体外胚胎冷冻保存提供优质高效的胚胎。结果表明: Ⅰ绵羊耳成纤维细胞系的建立 1细胞形态为典型的成纤维型细胞,绝大部分呈梭形或不规则三角形,细胞生长时呈放射状、火焰状走势,细胞增殖快,生长旺盛。细胞生长曲线显示复苏细胞生长状况很好,三个绵羊品种生长曲线均呈“S”型,每代细胞生长均经历了潜伏期、指数生长期和停滞期,符合体外细胞生长规律。 2细胞冻存前后存活率计算结果表明,冻存前细胞活力为96.2%以上,以DMEM/F-12+20%FBS中添加10%DMSO冻存成纤维细胞,解冻后细胞活力为93.6%以上,24h后的贴壁率平均为85%。传代后生长状况与冻存前基本一致,细胞形态和生长速度没有明显变化,冻存细胞生物学特征稳定。 3微生物污染检测呈阴性,表明细胞未受到细菌、真菌、病毒及支原体污染,冻存细胞为健康单一的成纤维细胞。 4在传代10次之前,细胞染色体中二倍体(2n=54)占主体,约为96-98%之间,为稳定的二倍体细胞系。 ⅡEGF ,IGF-1对绵羊卵母细胞体外成熟及不同培养液对体外培养胚胎发育效果影响的研究 1 EGF对卵母细胞体外成熟和卵裂的影响,EGF浓度为50ng/ml时成熟率和卵裂率为71.2%,45.5%,显著高于对照组和10ng/ml,20ng/ml,30ng/ml,40ng/ml组,差异显著(P<0.05) ,当EGF浓度达到100ng/ml时,成熟率和卵裂率最高,分别为72.9%和45.7%。 2 IGF-1对卵母细胞体外成熟和卵裂的影响,IGF-1浓度为40ng/ml时成熟率和卵裂率分别为70.7%,58.5%,显著高于对照组和浓度为10ng/ml,20ng/ml,60ng/ml,80ng/ml,100ng/ml组,差异显著(P<0.05) ,当IGF-I的浓度为100ng/ml时,成熟率和卵裂率最低,分别为38.8%和20.0%,与对照组和其它各实验组差异显著(P<0.05); 3 EGF和IGF-1联合对对卵母细胞体外成熟和卵裂的影响,50ng/ml EGF和40ng/ml IGF-1联合使用,成熟率和卵裂率分别为85.6%和61.0%,同对照组和单独运用50ng/ml EGF,40ng/ml IGF-1组之间差异显著(P<0.05)。 4在mSOFaa液+10%FCS中添加100umol/L半胱氨酸其桑囊胚率为32.7%,高于对照组、50umol/L组和200umol/L组,差异显著(P<0.05), 50umol/L组和200umol/L组无显著差异(P>0.05)。 5在胚胎培养液mSOFaa液+3mg/mL BSA和胚胎培养液mSOFaa液+10%ESS的卵裂率分别为61.2%,和62.5%,差异不显著(P>0.05),桑囊率分别为13.5%和30.0%,差异显著(P<0.05),表明在培养绵羊胚胎时BSA不如ESS的培养效果好。
China is one of the counties that have the richest on resources of specific of the domestic animals .However,in recent years,because of indiscriminate introduction of exotic high-productive breeds,a large number of breeds of the domestic animals face to extinct. So it is imperative to conserve national domestic animal genetic resources . Taking Guide Black fur sheep、Shanxi Tong sheep and Nei Menggu Wu Zhu Mu Qin sheep ear margin as the study materials. we succeeded in establishing a fibroblast cell line by the method of explant adherence culture and cell cryoconservation technology. Observations on morphology,dynamic growth ,determination of viability , analysis of karyotype, test of microorganism were carried out. The newly established cell line not only preserved the gemlplasm resources of this important Guide Black fur sheep on the cell level ,but also provided valuable materials for the research of somatic cloning. The cell freezen amount is about 3×106 cell/ml, sample contents of each breed were between 40 and 50, cryopreservation ampules were 240-360 each breed. And at the same time studied the effects of EGF, IGF-I separation or combination on sheep oocytes maturation in vitro and cleavage, cysteamine and different medium on sheep embryos development. In order to provide the adequate quality and high performance embryo for Cryopreservation. The experiment results were as follows: ⅠThe Establishment of the ear Fibroblast Cell Line 1 The cultured cells were typical fibroblast morpha,which growed as irregular triangle or shuttle,appearing as radioactive blaze in shape.Their growth curve were "S",which showed since the second day, the cells understood latent phase, logarithmic growth phase and stagnate phase. 2 Cell viability before freezing was 96.2%, The fibroblasts were frozen in DMEM/F-12+20%FBS by adding 10% DMSO, After thawing Cell viability was 93.6% , The attachment rates of thawed cells after 24h was 85%. Adherent cell after revival growed as fast as cell before freezing. Meantime the fibroblasts became more purified. 3 The fibroblasts identified with Microbial detection test were negative ,which demonstrated cultured cells were not contaminated by bacteria,yeasts,fungi,virus and mycoplasmas. 4 The number of chromosome is 2n=54 ,Diploid (2n=54)account for 96-98% in cells chromosome. Results of chromosomal karyotypes indicated that cells are stable diploid cell line. ⅡThe effects of the EGF and IGF-1 on IVM、different culture medium on the embryo development in vitro of the sheep 1 The maturation rate and the cleavage rate derived from oocytes matured in medium supplemented with 50ng/ml EGF is 71.2% and 45.5% Seperatately. It′s significantly highter compared with controd and 10ng/ml,20ng/ml,30ng/ml,40ng/ml groups (P<0.05) ,when EGF solution arrive to 100ng/ml,the maturation rate and the cleavage rate is 72.9% and 45.7%. 2 The maturation rate and the cleavage rate derived from oocytes matured in medium supplemented with 40ng/ml IGF-1 is 70.7% and 58.5% Seperatately. It′s significantly highter compared with controd and 10ng/ml,20ng/ml, 60ng/ml,80ng/ml,100ng/ml groups (P<0.05) ,when IGF-1 solution arrive to 100ng/ml,the maturation rate and the cleavage rate is 38.8% 20.0% Seperatately It′s significantly lower than other group(P<0.05). 3 The maturation rate and the cleavage rate derived from oocytes matured in medium supplemented with 50ng/mL EGF and 40ng/mL IGF-1 combation group was significantly higher compared with controd and 50ng/mLEGF and 40ng/mLIGF-1 groups(P<0.05).The maturation rate and the cleavage rate is 85.6%and 61.0% Seperatately. 4 The blastocyst rate derived from matured in medium mSOFaa +10%FCS supplemented with 100umol/L cysteamine is 43.6%,Its significantly highter compared with controd, 50 umol/L and 200umol/L (P<0.05) ,There is no differences between 50 umol/L and 200 umol/L (P>0.05). 5 The Cleavage rate in medium(mSOFaa+3mg/mL BSA) and medium (mSOFaa+10%ESS) is 61.2%,62.5% respectively(P﹥0.05) .the blastocyst rate is 13.5% and 30.0% respectively(P﹤0.05).It is illuminated that the effect of BSA is worse than the effect of ESS in sheep embryo development.
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