【作者】 蔡中涛；

【导师】 李碧春； 丁家桐；

【作者基本信息】 扬州大学 ， 养殖， 2006， 硕士

【摘要】 随着鸡胚胎和遗传工程的快速发展，迫切需要鸡胚胎发育规律的统一判断依据和标准，以及对鸡早期胚胎在体内的生长发育规律和早期胚胎细胞体外生长能力的掌握。本实验通过对鸡早期胚胎发育研究，以探索鸡胚胎生长发育的规律性；采用不同方法分离鸡胚胎干(ES)细胞，同时观察鸡ES细胞对不同酶消化的耐受性、ES细胞体外培养和传代的能力；采用显微注射法将人生长激素(hGH)基因和人组织激肽释放酶(KLK1)基因直接注入发育鸡胚的胚盘中，研究ES细胞转染外源基因的能力，为禽类胚胎发育生物学研究以及转基因鸡生产提供素材和新途径。研究结果如下：1、鸡早期胚胎体内发育规律的研究：在鸡产蛋后的不同时间段内分别将鸡处死，从输卵管或子宫内取出发育的受精卵，显微镜下观察并记录。结果显示：受精卵的第一次分裂发生在产蛋后5h(0-0.5h子宫龄)，桑椹期形成在产蛋后11.5h(6-6.5h子宫龄)，囊胚期形成在产蛋后15.5h(10-10.5子宫龄)。根据胚盘形态发育的变化，可将鸡胚在体内发育过程分为两个时期：一是在距前一个蛋产出后5.5h(0-1h子宫龄)至15.5h(10-10.5h子宫龄)为鸡胚的卵裂期，卵裂的结果形成厚约6-7层细胞的胚层；二是在距前一个蛋产出后17.5h(12-12.5h子宫龄)至明区形成期，即鸟类的囊胚期，结果在胚盘中央形成透明的周围不透明的明区和暗区。对鸡ES细胞培养或制作嵌合体鸡，根据需要可在不同的时间获取所需卵裂球或胚胎，早期囊胚阶段即在分裂期的10.5h子宫龄前可获取较大卵裂球，在12.5-13.5h子宫龄后可获取较小的卵裂球。2、对鸡ES细胞分离与培养的研究：采用药勺获取、单独酶解离法分离得到的鸡第X期(24-26h子宫龄)胚盘细胞，接种到四种不同的培养体系中，比较不同的培养体系中各种因素对ES细胞培养传代的影响。实验结果表明：在无饲养层，培养液中不添加外源性细胞因子的培养体系1中，无ES细胞的AKP阳性克隆形成；在无饲养层，培养液中添加细胞因子培养体系2中，培养72 h后有一代ES细胞的AKP阳性克隆形成，克隆形成率为33.33％，但ES细胞在传代后不能重新聚集形成集落；在以鸡胚胎成纤维细胞为饲养层，培养液中不添加外源性细胞因子的培养体系3中，培养48 h后可见细胞成集落状隆起生长，但传至三代后未观察到ES细胞集落形成，第一代和二代ES细胞的AKP阳性克隆形成率分别为40％和20％。与其它三个培养体系相比，在以鸡成纤维细胞为饲养层，用添加了10％的胎牛血清、2％的鸡血清、2mmol/L L-谷氨酰胺、1mmol/L丙酮酸钠、5.5×10-5mol/Lβ-巯基乙醇、10μl/ml非必需氨基酸、以及1000U/ml白血病抑制因子(Leukemia inhibitory factor，LIF)、10ng/ml碱性成纤维生长因子(Fibroblast growth factor-basic，bFGF)和5ng/ml干细胞生长因子(Stem cell factor，SCF)的高糖DMEM培养体系4中，培养约2～3d时开始出现鸟巢状的ES细胞集落，以多次离散法进行传代获得了传至5～6代的鸡ES细胞克隆。AKP阳性克隆形成率与其它三个培养体系相比，存在着极显著的差异(P<0.01)。通过对传代培养后的鸡ES细胞进行AKP染色鉴定和SSEA-1的鉴定，证实细胞未发生分化，保持了ES细胞的特征。3、对鸡胚盘内转染外源基因的研究：采用显微注射法，分别将人生长激素(hGH)基因和人组织激肽释放酶(KLK1)基因直接注射到孵化24h的鸡胚中继续孵化。在注射的320枚鸡胚中，孵化前7d死亡194枚，8～18d死亡108枚，孵出雏鸡18只，孵出率为5.6％。8只雏鸡分别于出壳后的第一、二、三、五天死亡。7只于3月龄死亡。3只鸡存活并正常生长。死亡雏鸡具有脚瘫、体弱、斜颈、站立不稳等异常表现，存活鸡无外观异常。从孵化4d后死亡的150枚鸡胚和18只出壳雏鸡的组织中提取基因组DNA，并用DNA杂交试验进行基因整合检测，结果发现其中的3枚鸡胚和3只雏鸡为人生长激素基因整合阳性，基因整合率为12.5％；18枚鸡胚和6只雏鸡为KLK1整合阳性，整合率为22.2％。结果表明：鸡胚盘内细胞通过显微注射法可以转染外源基因，是转基因鸡制备的一条有效途径。更多还原

【Abstract】 With the rapid development of embryonic and genetic engineering in chick, the reseach in the embryonic development biology demands a uniform criterion for the embryonic development rules in chick, and the reseach on the early-stage embryonic development rules in vivo or in vitro in chick is necessary for fowl breeding and transgenic research. Through the investigation on the early-stage and incubating-stage embryonic development and organ growth in chick, the study explored the growth rules of chick embryo. Moreover, through direct microinjection of human growth hormone(hgh) gene and human tissue kallikein-1(KLK1) gene into the germinal disc of developing embryos in chick, the study explored the new approaches for producing transgenic chickens.1、The early embryo development rule in chicken: By slaughtering the chick on different time periods after laying an egg, the developing fertilized eggs were obtained from the oviduct or uterine to be observed and investigated under the microscope. The results showed that: the first cleavage happened 5 hours(0-0.5 hour of uterine age)after laying an egg, morular stage happened 11.5 hours(6-6.5 hours of uterine age)after laying an egg, blastocyst stage happened 15.5 hours(10-10.5 hours of uterine age)after laying an egg. According to morphological condition of the blastodisc, the embryo development can be divided into two stages: cleavage stage, from 5.5 hours(0-1 hour of uterine age) to 15.5 hours(10-10.5 hours of uterine age) after laying an egg, was characteristic of the formation of a blastodisc by 6-7 layers of cells; and blastocyst stage, from 17.5 hours(12-12.5 hours of uterine age) after laying an egg to pellucida area forming period, was characteristic of the formation of a light area and a dark area at the center of the blastodisc.2、ES were isolated from the stage X of chicken embryos with 0.25% trypsin-0.04% EDTA, then cultured in the culture system 4 with high glucose DMEM supplemented with 10% fetal bovine serum, 2% chicken serum, 2mmol/L L-glutamine, 5.5×10-5β-mercaptoethanol, 1mmol/L sodium pyruvatel, with leukemia inhibitory factor(LIF, 1000U/ml), basic fibroblast growth factor(bFGF, 10ng/ml) and stem cell factor(SCF, 5ng/ml), ES-like colonies were developed 2-5 days later, and there were 1 to 5 passages of ES-derived AKP positive colonies formed, with the values of 80%, 66.67%, 46.67%, 26.67% and 3.33% respectively. However, there was no AKP positive colony observed in culture system 1, the formation of one passage ES-derived AKP positive colonies was 33.33% in this system 2, and in system 3, there were 1st and 2nd passages of ES AKP positive colonies observed, with the value of 40% and 20%, respectively. The 1st to 5th passage of ES were characterized by morphology, colony formation, proliferation capability, karyotype, alkaline phosphatase activity and cell differentiation. Results showed that EScultured in the culture system 4 could be subcultured for five times and still maintained undifferentiated situation with normal chromosome karyotype. It seemed that the cultured chicken ES maintained characters of pluripotent stem cells.3、Transfection of exogenous genes into the germinal disc of chicken: Through direct microinjection of human growth hormone(hgh) gene and human tissue kallikein-1(KLK1) gene into the germinal disc of 320 developing chicken embryos of 24 hours after incubating, 18 chicks(5.6%) were hatched, but 8 died respectively 1, 2, 3 and 5 days after hatch, and 7 died 3 months later, only 3 survived. The dead chicks showed some symptoms like standing unsteadily and wryneck., while the survived exhibited no abnormalties. The genomic DNA were extracted from the tissues of 150 dead embryos and 18 chicks and conducted DNA hybridization test to detect the integration of exogenous genes. The results showed that: 3 embryo samples and 3 chick samples were detected hgh-positive, the integration rate was 12.5%; 18 embryo samples and 6 chick samples were detected KLK1-positive, the integration rate was 22.2%. The findings demonstrated that direct microinjection of foreign genes into the germinal disc of developing embryos in chick was an effective method to make transgenic chicken.更多还原