【摘要】：【背景和目的】人類(lèi)原始生殖細(xì)胞(Primordial GermCell, PGC)是人卵母細(xì)胞和精子的祖細(xì)胞，是人類(lèi)生存及繁衍后代的重要細(xì)胞。PGCs起源于性腺外，經(jīng)遷移到達(dá)生殖嵴與體細(xì)胞共同組成生殖腺；PGCs在性腺內(nèi)經(jīng)增殖、凋亡、分化等一系列復(fù)雜的變化最終形成成熟的卵子和精子，而卵子又是婦女體內(nèi)最珍貴的細(xì)胞，出生后在體內(nèi)的數(shù)量是一定的，并隨著年齡增長(zhǎng)逐步消耗，不可再生。隨著婦女平均生育年齡的推遲，不孕患者日趨增多，，認(rèn)識(shí)PGCs的起源、遷移、增殖、分化等過(guò)程對(duì)生殖醫(yī)學(xué)發(fā)展具有重要意義。本研究主要是用合適的方法制備完整的胚胎切片，根據(jù)文獻(xiàn)報(bào)道人原始生殖細(xì)胞的形態(tài)、大小及其表面標(biāo)志物，利用新鮮胚胎組織冰凍切片進(jìn)行堿性磷酸酶染色，石蠟切片行免疫組化染色的方法初步鑒定早期胚胎人原始生殖細(xì)胞。為以后研究hPGCs的遷移、增殖、分化及最終的hPGCs培養(yǎng)等奠定基礎(chǔ)。 【方法】經(jīng)我院倫理委員會(huì)批準(zhǔn)，征得孕婦同意,經(jīng)簽署知情同意書(shū)。收集胎齡約為21-35d藥物流產(chǎn)胚胎。按孕婦末次月經(jīng)日減去14d，并結(jié)合胚胎發(fā)育的外部特征計(jì)算胎齡。藥流胚胎經(jīng)多聚甲醛固定、石蠟包埋，進(jìn)行HE染色，堿性磷酸酶染色和相關(guān)抗原免疫組化染色等，免疫組化染色時(shí)以小鼠腎組織作為陽(yáng)性對(duì)照，用PBS液代替一抗作為陰性對(duì)照，檢測(cè)未分化多能干細(xì)胞標(biāo)志物Oct-4的表達(dá)情況。 【結(jié)果】1、借助解剖顯微鏡我們可以成功制備出比較完整的人類(lèi)早孕期胚胎切片，胎芽頭部、上下肢芽、心臟、脊柱和卵黃囊等主要結(jié)構(gòu)顯示清楚；2、冰凍新鮮胚胎組織切片堿性磷酸酶染色及石蠟切片堿性磷酸酶染色：在胚胎的后腸部位可見(jiàn)有堿性磷酸酶染色陽(yáng)性、體積大而圓的細(xì)胞，初步鑒定為原始生殖細(xì)胞；3、新鮮胚胎石蠟切片免疫組化染色結(jié)果：34-35天大小人類(lèi)胚胎尾部可見(jiàn)Oct-4陽(yáng)性的細(xì)胞，較周?chē)?xì)胞形態(tài)大而圓。 【結(jié)論】1、能夠成功制備出人類(lèi)孕早期胚胎的完整切片；2、人類(lèi)早期胚胎冰凍切片和新鮮胚胎石蠟切片的后腸部位有堿性磷酸酶染色陽(yáng)性的細(xì)胞；3、人類(lèi)早期新鮮胚胎石蠟切片尾部可見(jiàn)Oct-4陽(yáng)性的細(xì)胞。Oct-4陽(yáng)性細(xì)胞較其周?chē)?xì)胞形態(tài)大而圓，初步鑒定為原始生殖細(xì)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.
【學(xué)位授予單位】：中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R329

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