山羊原始生殖细胞分离与培养的影响因素  

Factors Influencing the Isolation and Cultivation of Primordial Germ Cell in Goat

作　　者：熊浩[1] 崔雯[1] 王杏龙[1] 刘翔[1] 

机构地区：[1]扬州大学动物科学与技术学院,江苏225009

出　　处：《中国草食动物》2010年第5期12-15,共4页China Herbivores

基　　金：家畜胚胎干细胞技术研究(2008AA101005)

摘　　要：以本地白山羊胎儿的生殖嵴为试验材料,从原始生殖细胞(primordial germ cells,PGCs)中分离培养得到胚胎生殖细胞(embryonic germ cells,EGCs),对其进行体外培养和鉴定等。胎儿生殖嵴在室温(25℃)下消化15 min,使用0.125%胰酶+0.02%EDTA比0.25%胰酶+0.04%EDTA效果好,差异显著(P<0.05);"挑取集落法"和"胰酶消化法"均能将PGCs传至第2代,两种方法差异不显著(P>0.05);以GEF为饲养层,在培养液中添加不同浓度和种类的细胞因子:①LIF:10 ng/mL;②LIF:20 ng/mL;③LIF(10 ng/mL)+SCF(10 ng/mL);④LIF(20 ng/mL)+SCF(20 ng/mL)。4种情况对于原代集落形成率的影响差异不显著(P>0.05),均能得到传至第2代的EGCs;研究不同胎龄胎儿分离培养PGCs的效果,结果发现4例冠臀长小于15 mm的胎儿仅观察到一个原代集落,6例大于30 mm的胎儿没有观察到原代集落。In this study,the fetal genital ridge from local White goats were used to isolate and culture EGCs from PGCs.The digestion of the fetal genital ridge at 25℃ for 15 min with 0.125% Tryosin＋0.02%EDTA was better（P0.05）than that with 0.250% Trypsin ＋0.04% EDTA;PGCs could be transmitted to the second generation in both ＂picked colony method＂and ＂Trypsin digestion method＂,and there was no significant difference（P0.05） in two methods;By adding different cytokines in different concentrations,the effects on the transmission of PGCs with GEF as the feeder layer were studied under four conditions（①LIF 10 ng/mL;② LIF 20 ng/mL;③LIF 10 ng/mL＋SCF 10 ng/mL;④LIF 20 ng/mL＋ SCF 20 ng/mL）,and no differences（P0.05） in the primary colony rates and the transmission to the second generation were found in the four conditions.Effects of different gestational ages on isolation and passage of PGCs were studied,only a primary colon was observed in 4 fetus with crown-rump length less than 15 mm,and no primary colon was observed in 6 fetus with crown-rump length more than 30 mm.

关 键 词：白山羊 胚胎干细胞 原始生殖细胞 分离培养 

分 类 号：S814.8[农业科学—畜牧学]