人类原始生殖细胞的鉴定

发布时间：2019-01-26 22:01

【摘要】：【背景和目的】人类原始生殖细胞(Primordial GermCell, PGC)是人卵母细胞和精子的祖细胞，是人类生存及繁衍后代的重要细胞。PGCs起源于性腺外，经迁移到达生殖嵴与体细胞共同组成生殖腺；PGCs在性腺内经增殖、凋亡、分化等一系列复杂的变化最终形成成熟的卵子和精子，而卵子又是妇女体内最珍贵的细胞，出生后在体内的数量是一定的，并随着年龄增长逐步消耗，不可再生。随着妇女平均生育年龄的推迟，不孕患者日趋增多，，认识PGCs的起源、迁移、增殖、分化等过程对生殖医学发展具有重要意义。本研究主要是用合适的方法制备完整的胚胎切片，根据文献报道人原始生殖细胞的形态、大小及其表面标志物，利用新鲜胚胎组织冰冻切片进行碱性磷酸酶染色，石蜡切片行免疫组化染色的方法初步鉴定早期胚胎人原始生殖细胞。为以后研究hPGCs的迁移、增殖、分化及最终的hPGCs培养等奠定基础。【方法】经我院伦理委员会批准，征得孕妇同意,经签署知情同意书。收集胎龄约为21-35d药物流产胚胎。按孕妇末次月经日减去14d，并结合胚胎发育的外部特征计算胎龄。药流胚胎经多聚甲醛固定、石蜡包埋，进行HE染色，碱性磷酸酶染色和相关抗原免疫组化染色等，免疫组化染色时以小鼠肾组织作为阳性对照，用PBS液代替一抗作为阴性对照，检测未分化多能干细胞标志物Oct-4的表达情况。【结果】1、借助解剖显微镜我们可以成功制备出比较完整的人类早孕期胚胎切片，胎芽头部、上下肢芽、心脏、脊柱和卵黄囊等主要结构显示清楚；2、冰冻新鲜胚胎组织切片碱性磷酸酶染色及石蜡切片碱性磷酸酶染色：在胚胎的后肠部位可见有碱性磷酸酶染色阳性、体积大而圆的细胞，初步鉴定为原始生殖细胞；3、新鲜胚胎石蜡切片免疫组化染色结果：34-35天大小人类胚胎尾部可见Oct-4阳性的细胞，较周围细胞形态大而圆。【结论】1、能够成功制备出人类孕早期胚胎的完整切片；2、人类早期胚胎冰冻切片和新鲜胚胎石蜡切片的后肠部位有碱性磷酸酶染色阳性的细胞；3、人类早期新鲜胚胎石蜡切片尾部可见Oct-4阳性的细胞。Oct-4阳性细胞较其周围细胞形态大而圆，初步鉴定为原始生殖细胞。
[Abstract]:Background and objective: human primordial germ cell (Primordial GermCell, PGC) is the progenitor of human oocyte and sperm, and is an important cell for human survival and reproduction. PGCs originated outside the gonad. After migration to the reproductive crest and somatic cells to form the gonad; A series of complex changes such as proliferation, apoptosis and differentiation of PGCs in the gonad eventually result in mature eggs and sperm, which are the most precious cells in a woman's body, and the number of cells in the body after birth is certain. And with the age of gradually consumption, non-renewable. With the delay of average childbearing age of women, the number of infertile patients is increasing. It is important to understand the origin, migration, proliferation and differentiation of PGCs for the development of reproductive medicine. In this study, complete embryo sections were prepared by suitable methods. The morphology, size and surface markers of human primordial germ cells were reported in literature. Frozen sections of fresh embryonic tissues were used for alkaline phosphatase staining. Human primordial germ cells were identified by immunohistochemical staining in paraffin sections. To lay a foundation for the study of hPGCs migration, proliferation, differentiation and final hPGCs culture. Methods: informed consent was signed with the approval of the ethics committee of our hospital and the consent of pregnant women. The gestational age was about 21-35 days after drug abortion. The gestational age was calculated by subtracting 14 days from the last menstrual day of pregnant women and combining with the external characteristics of embryonic development. Drug flow embryos were fixed by paraformaldehyde, embedded in paraffin, stained with HE, alkaline phosphatase and related antigen immunohistochemical staining. The renal tissues of mice were used as positive control and the first antibody was replaced by PBS solution as negative control. The expression of undifferentiated pluripotent stem cell marker (Oct-4) was detected. [results] 1. With the aid of anatomical microscope, we can successfully produce relatively complete human embryo sections during early pregnancy. The main structures of fetal bud head, upper and lower limb buds, heart, spine and yolk sac can be clearly displayed. 2, alkaline phosphatase staining in frozen fresh embryo tissue and paraffin section alkaline phosphatase staining: alkaline phosphatase positive cells were found in the hindgut of the embryo, which were identified as primordial germ cells. 3The results of immunohistochemical staining on paraffin sections of fresh embryos showed that the Oct-4 positive cells were found in the tail of human embryos from 34 to 35 days, which were larger and more round than the surrounding cells. [conclusion] 1, the complete sections of human early pregnancy embryos can be successfully prepared, 2, the cells in the hindgut of frozen sections and paraffin sections of fresh embryos have alkaline phosphatase staining positive cells. 3. Oct-4 positive cells were found in the tail of paraffin sections of human early fresh embryos. The Oct-4 positive cells were larger and more round than the surrounding cells, and were identified as primordial germ cells.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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