本文選題：原始生殖細胞 + 增殖率��； 參考：《南方醫(yī)科大學》2010年碩士論文

【摘要】： C57BL/6J小鼠是標準的近交系小鼠,其胚胎期生殖發(fā)育的過程與人的有許多相同之處,而且遺傳背景清楚,便于研究,是胚胎生殖發(fā)育研究較理想的生物模式材料。胚胎組織切片標本的制作與觀察是研究生殖發(fā)育的一個必不可少的重要手段,為形態(tài)學研究與免疫熒光技術(shù)等研究提供技術(shù)平臺。目前小鼠胚胎的生殖發(fā)育仍有很多難題未被解決,已有的資料大多是通過體外分離培養(yǎng)研究所得,很多體內(nèi)的發(fā)育問題仍困惑著科學家,需要進行更多的研究來解答。C57BL/6J小鼠妊娠期19-21天,在胚胎第11天至13.5天之間生殖嵴與原始生殖細胞的發(fā)育變化明顯可見。8.5天-10.5天時原始生殖細胞向生殖嵴移動并增殖。11.5天時多半進入生殖嵴,失去其運動能力,并大量增殖。13.5天之后原始生殖細胞在生殖嵴內(nèi)的增殖完成。接著雌性生殖細胞即開始進入減數(shù)分裂,在15.5天大多數(shù)進入第一次減數(shù)分裂并最終停留在前期的雙線期。而雄性生殖細胞則處于靜止狀態(tài),出生后5天再開始有絲分裂。目前國內(nèi)外關(guān)于小鼠生殖嵴胚胎發(fā)育形態(tài)學研究的全面報道較少,而且很少有研究關(guān)注不同性別之間胚胎期生殖細胞發(fā)育的區(qū)別。本研究以C57BL/6J小鼠胚胎生殖嵴為實驗對象,采用石蠟切片蘇木精-伊紅染色、免疫熒光組織化學法以及蛋白印跡分析等方法,研究C57BL/6J小鼠在11天至13.5天之間生殖嵴發(fā)育的形態(tài)學改變以及雌性和雄性之間原始生殖細胞在數(shù)量與增殖率的區(qū)別,初步探討正常C57BL/6J小鼠雌性與雄性原始生殖細胞發(fā)育的區(qū)別,為生殖胚胎發(fā)育的分子水平調(diào)控的深入研究提供了依據(jù)。 一、鼠整胚組織切片標本的制作與觀察 分別取不同天數(shù)的小鼠胚胎(11天,11.5天,12天,12.5天,13天,13.5天),每個胚胎取少量組織PC R法鑒定胚胎性別。整胚組織采用4%多聚甲醛固定液固定,接著從低濃度乙醇開始梯度脫水,二甲苯透明,浸蠟后包埋,5um連續(xù)切片。石蠟塊切片后做HE染色。選擇含有生殖嵴的切片,光學顯微鏡下觀察11天至13.5天胚胎生殖嵴的組織形態(tài)學變化以及性別分化特征。組織切片觀察發(fā)現(xiàn),小鼠胚胎的細胞與組織的形態(tài)被很好的展現(xiàn)出來,我們能很輕松的觀察到從11天至13.5天生殖嵴形態(tài)結(jié)構(gòu)上的改變、胚胎性別分化的特征以及原始生殖細胞的形態(tài)。 結(jié)果發(fā)現(xiàn),小鼠胚胎11-13.5天生殖嵴的發(fā)育迅速。11天的鼠胚生殖嵴已經(jīng)形成,且其中可見遷移來的原始生殖細胞。原始生殖細胞體積大,核大且呈圓形或卵圓形。11.5天鼠胚,生殖嵴變厚變長。12天的鼠胚生殖嵴性別尚未分化。12.5天的鼠胚開始出現(xiàn)性別分化的跡象。13天的鼠胚性別明顯分化,雄性的原始性索開始分化為實心原始生精小管。13.5天的鼠胚,雄性原始生精小管結(jié)構(gòu)明顯,雌性皮質(zhì)索孤立細胞團形成原始卵泡。生殖嵴石蠟切片的制作,可以為生殖嵴免疫熒光研究提供材料,為更進一步研究小鼠胚胎生殖發(fā)育以及原始生殖細胞的發(fā)育提供了實驗依據(jù)。 二、生殖嵴中原始生殖細胞的免疫熒光組織化學檢測 雌雄性生殖細胞特異性表達小鼠脈管同系物(Mouse vasa homolog, Mvh)。采用免疫熒光雙染的方法用Mvh抗體標記原始生殖細胞,用增殖細胞核抗原(Proliferating Cell Nuclear Antigen, PCNA)標記處于增殖中的細胞。從每個胚胎的連續(xù)切片中間隔選取3-4張切片進行免疫熒光雙染,拍照后截取相同大小的面積,計數(shù)Mvh陽性與Mvh與PCNA共陽性的細胞數(shù),即原始生殖細胞數(shù)與正處于增殖中的原始生殖細胞數(shù)。根據(jù)后者與前者的比值計算出原始生殖細胞的增殖率。 結(jié)果發(fā)現(xiàn),11、天生殖嵴面積小,未參與計數(shù)。對于原始生殖細胞的數(shù)量與增殖率,11.5天-12.5天的雌性和雄性胚胎之間都沒有顯著差別(P0.01),但其數(shù)量在雄性13天(135.83±4.57)和13.5天(152.23±5.54)顯著多于雌性13天(97.08±4.72)和13.5天(121.22±9.27)(P=0.000),增殖率在13天雄性(20.23±0.34%)較雌性(16.95±0.35%)高(P=0.000),13.5天沒有顯著區(qū)別(P0.01)。雌性和雄性胚胎原始生殖細胞的增殖率都在13天最高,數(shù)量在13.5天最多。 三、蛋白印跡法檢測生殖嵴中Mvh和PCNA蛋白的表達 取13天、13.5天胚胎的生殖嵴,提取蛋白做蛋白印跡檢測,分析比較雌性與雄性Mvh與PCNA蛋白的表達。結(jié)果發(fā)現(xiàn)：以β-tubulin為內(nèi)參,Mvh蛋白的表達在13天與13.5天雄性顯著高于雌性,PCNA表達在13天雄性也高于雌性,13.5天沒有顯著區(qū)別。說明13天、13.5天雄性原始生殖細胞數(shù)量較雌性多,13天時雄性生殖嵴內(nèi)的細胞增殖率較雌性高,與免疫熒光結(jié)果大致相同。 本實驗結(jié)果表明C57BL/6小鼠雌性和雄性胚胎的原始生殖細胞發(fā)育是有顯著區(qū)別的。在原始生殖細胞進入生殖嵴且性別分化明顯之后、停止增殖之前,即主要在胚胎期13天雄性的原始生殖細胞較雌性增殖迅速,且數(shù)量上顯著高于雌性,這為更進一步研究小鼠胚胎發(fā)育過程中原始生殖細胞的基因表達變化以及某些基因敲除后對不同性別小鼠生殖的影響提供了實驗依據(jù)。
[Abstract]:In this study , the reproductive development of C57BL / 6J mice was studied by means of morphological study and immunofluorescence technique .



Preparation and observation of tissue sections of mouse whole embryo tissue



The histological changes of embryonic germ cells and the characteristics of sex differentiation were observed in the embryonic germ cells from 11 to 13.5 days . The morphological changes and the sex differentiation of the embryonic germ crista were observed from 11 to 13.5 days . The changes of the morphological structure of the genital ridge from 11 to 13.5 days were observed . The characteristics of the sex differentiation and the morphology of the primordial germ cells were observed .



The results showed that the reproductive ridge of mouse embryo in 11 - 13.5 days was developed rapidly . The reproductive ridge of mouse embryo in 11 days had been formed , and the primordial germ cells migrated were observed .



Immunofluorescence histochemical detection of primordial germ cells in the genital ridge



Male and female germ cells specifically express the mouse vascular homologue ( Mouse vasa sarcoma , Mv h ) . The number of cells with the same size was taken from the continuous section of each embryo , and the number of cells with the same size was taken after taking pictures . The number of cells with the same size was counted after taking pictures . The number of primordial germ cells and the number of primordial germ cells in the proliferating cell were counted . The proliferative rate of primordial germ cells was calculated from the ratio of the latter to the former .



The results showed that there was no significant difference in the number of primordial germ cells ( 135.83 鹵 4.57 ) and 13.5 days ( 152.23 鹵 5.54 ) than that in females ( 97.08 鹵 4.72 ) and 13.5 days ( 125.22 鹵 9.27 ) ( P = 0.000 ) , but there was no significant difference between males ( 20.23 鹵 0.34 % ) and females ( 16.95 鹵 0.35 % ) ( P = 0.000 ) .



3 . Expression of PCNA protein in the genital ridge by Western blotting



The results showed that the expression of PCNA in 13 days and 13.5 days was significantly higher than that in females and 13.5 days . The results showed that the number of male primordial germ cells was higher in 13 days and 13.5 days .



The results showed that the primordial germ cell development of the female and male embryos of C57BL / 6 mice was significantly different . After the primordial germ cells entered the genital ridge and the sex differentiation was obvious , the primordial germ cells were more rapidly and significantly higher than that of the female in 13 days of the embryonic stage , which provided an experimental basis for the further study of the gene expression changes of the primordial germ cells during the mouse embryo development and the influence of certain gene knockout mice on the reproduction of different sex mice .

【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R329.1

【共引文獻】

相關(guān)期刊論文 前9條

1 魏燁昕;;哺乳動物生殖細胞的性別決定[J];安徽農(nóng)業(yè)科學;2012年14期

2 傅文玉,樸英杰,路艷蒙,陳英,喬東訪,安連兵;大鼠原生殖細胞的形態(tài)學及分化特性[J];第一軍醫(yī)大學學報;2001年09期

3 秦潔,肖小s，

本文編號：1887602