【摘要】： 背景： 人類植入前胚胎的染色體異常是很普遍的，常導致胚胎發(fā)育的停滯或引起自發(fā)流產(chǎn)。正常受精卵具有兩個原核，而異常原核數(shù)目的受精卵包括單原核受精卵、多原核受精卵及無原核受精卵，這些受精卵一般都被丟棄，可一些研究顯示這些原核形態(tài)異常的胚胎有一定比率的染色體正常率，有一定的移植價值。 目前對人類植入前胚胎染色體異常的研究多采用免疫熒光原位雜交(FISH)的方法。然而，F(xiàn)ISH只能檢測有限數(shù)目的染色體，并且檢測每一種染色體異常都需要相應的特定探針，局限了FISH技術的應用。目前，有研究者試圖將胚胎單卵裂球的染色體誘導至中期相，可以在不需要特定探針的情況下，對卵裂球進行核型分析，彌補FISH的不足。 目的： 1．檢測原核數(shù)目異常胚胎中的染色體正常率，分析這些胚胎是否具有移植價值。 2．探索單卵裂球核型分析技術，以期為胚胎植入前遺傳學診斷提供一種新的方法。 方法： 1．將IVF及ICSI周期中原核評分為1PN的受精卵繼續(xù)培養(yǎng)，第五天如胚胎發(fā)育為囊胚則分離出內細胞團后將余下滋養(yǎng)層細胞行雙色FISH檢測，如未發(fā)育至囊胚則將胚胎的全部卵裂球活檢后分別行FISH； 2．對卵裂球進行體外操作以期得到染色體中期相，方法包括： 1)直接用秋水仙素阻斷卵裂球至中期； 2)與體外成熟的人去核MⅡ卵母細胞融合，誘發(fā)PCC(染色體提前凝集)； 3)與小鼠MⅡ卵母細胞融合，誘發(fā)PCC； 4)與小鼠受精卵融合，誘導至中期。 結果： 1．對138枚1PN胚胎行雙色FISH檢測，得到可分析的結果共87枚，其中來源于IVF的48枚，單體信號占18.8％、二體信號54.2％、嵌合體20.8％、多體信號6.2％；來源于ICSI的39枚，單體信號46.2％、二體信號25.6％、嵌合體23.1％、多體信號5.1％。87枚1PN胚胎中，有16枚發(fā)育到囊胚，其中12枚為二體信號(75.0％)，4枚為嵌合體(25.0％)； 2．對胚胎單卵裂球核型分析的結果為： 1)采用秋水仙素直接阻滯卵裂球57枚，得到中期染色體9枚，成功率15.8％； 2)采用體外成熟后人卵誘導卵裂球26枚，得到中期染色體4枚，成功率15.4％； 3)采用小鼠成熟卵母細胞誘導卵裂球13枚，電融合時小鼠卵母細胞皆被激活，成功率為0； 4)采用小鼠受精卵誘導卵裂球160枚，得到中期染色體25枚，成功率15.6％。 結論： 1．ICSI來源的1PN多為單體信號，可能是由于精子未解聚而卵母細胞激活所致；IVF來源的1PN則多為正常二體信號，可能其主要的原因是原核形成不同步或原核融合。第五天發(fā)育到囊胚的胚胎則皆顯示二體信號或嵌合體，可能是由于單倍體的胚胎缺乏發(fā)育至囊胚能力。 2．本實驗采用四種方法所得到的中期染色體數(shù)都約為15.5％，成功率較低，，目前采用核轉換技術來將卵裂球染色體誘導至中期相的效率還偏低，尚需要進一步改進。
[Abstract]:Background:
Chromosomal abnormalities in human preimplantation embryos are common, often leading to stagnation of embryonic development or spontaneous abortion. Normal fertilized eggs have two prokaryotes, while abnormal prokaryotic eggs include mono prokaryotic ovum, multiple prokaryotic and non prokaryotic fertilized eggs, and these fertilized eggs are generally discarded. Some studies show this Some embryos with abnormal nuclear morphology have a certain percentage of normal chromosomes and have certain transplantation value.
At present, the study of chromosomal abnormalities in human preimplantation embryos uses immunofluorescent in situ hybridization (FISH). However, FISH can only detect a limited number of chromosomes, and the detection of each chromosome abnormality requires a specific probe, limiting the use of FISH technology. At present, researchers are trying to dye the single blastomere of the embryo. Karyotype analysis of blastomeres can be made without the need for specific probes when the chromophore is induced to the metaphase phase, thus making up for the deficiency of FISH.
Objective:
1. detect the normal rate of chromosomes in the abnormal number of embryos and analyze whether these embryos have the value of transplantation.
2. explore the single blastomere karyotype analysis technology, in order to provide a new method for preimplantation genetic diagnosis.
Method:
1. the fertilized eggs of 1PN in IVF and ICSI cycle were continued to be cultured. On the fifth day, if the embryo developed into blastocyst, the remainder trophoblast cells were detected by double color FISH. If the blastocyst was not developed to the blastocyst, the whole blastomere of the embryo was performed FISH respectively.
2. in vitro operation of blastomere to obtain metaphase chromosomes.
1) direct colchicine to block the blastomere to the mid-term;
2) fusion with human mature M (II) oocytes and induce PCC (chromosome agglutination).
3) fusion with mouse M II oocytes and induced PCC;
4) fusion with mouse fertilized eggs, induced to mid-term.
Result:
1. pairs of 138 1PN embryos were detected by double color FISH. The results were 87, of which 48 were derived from IVF, 18.8% of the monomer, 54.2% for two body signals, 20.8% for chimerism and 6.2% in multibody signals, and from 39 of ICSI, two signal 25.6%, chimerism 23.1%, 5.1%.87 1PN embryos of multibody signals, 16 of them developed into blastocysts, of which 12 were two body signals (75%) and 4 were chimeras (25%).
2. the results of karyotype analysis of embryo single blastomere are as follows:
1) using colchicine to block 57 blastomere directly and get 9 metaphase chromosomes, the success rate was 15.8%.
2) 26 eggs from blastomere were induced by human egg maturation in vitro, and 4 metaphase chromosomes were obtained. The success rate was 15.4%.
3) 13 mature blastomere cells were induced by mouse oocytes, and the oocytes were activated by electrofusion, and the success rate was 0.
4) 160 fertilized eggs were used to induce blastomere, and 25 metaphase chromosomes were obtained, with a success rate of 15.6%.
Conclusion:
1.ICSI source 1PN is mostly mono signal, possibly due to sperm undisaggregation and oocyte activation; IVF source 1PN is mostly normal two body signal, which may be mainly due to the formation of prokaryotic asynchronous or prokaryotic fusion. The fifth day embryos developed to blastocyst were all two body signals or chimeras, probably due to unfolding. The body's embryo lacks the ability to develop to the blastocyst.
2. the number of metaphase chromosomes obtained by four methods is about 15.5%, and the success rate is low. The efficiency of using nuclear conversion technology to induce the blastomere chromosomes to medium-term phase is still low, and further improvement is needed.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R321-33

【共引文獻】

相關期刊論文 前8條

1 徐艷文,莊廣倫;植入前遺傳學診斷技術研究的新進展[J];北京大學學報(醫(yī)學版);2004年06期

2 陳瑛,張玉蘭,郝彥娟,魏明竟;遺傳病與植入前遺傳學診斷技術[J];國外醫(yī)學.臨床生物化學與檢驗學分冊;2001年02期

3 陳欣潔;胚胎細胞種植前遺傳學診斷的研究進展[J];廣州醫(yī)學院學報;2001年02期

4 李秀蓉,陸長富,范立青,朱文斌,盧光t;小鼠單卵裂球體外培養(yǎng)及染色體制備[J];生命科學研究;2000年01期

5 劉建兵;馮云;;胚胎植入前非整倍體篩查的研究進展[J];醫(yī)學綜述;2007年23期

6 徐艷文;周燦權;;輔助生育技術的新進展[J];中國實用婦科與產(chǎn)科雜志;2006年01期

7 徐艷文,莊廣倫,周燦權,張敏芳,李莉琳;應用熒光原位雜交技術進行人類早期胚胎性染色體嵌合型的初步研究[J];中華婦產(chǎn)科雜志;1999年10期

8 陳欣潔,孫筱放;熒光原位雜交在種植前遺傳學診斷中的應用[J];中國優(yōu)生與遺傳雜志;2001年01期

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2 廖宏慶;人類不同原核數(shù)目受精卵中篩選正常優(yōu)質胚胎的策略研究[D];中南大學;2010年

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4 張敏敏;利用熒光原位雜交對人類植入前胚胎行性別診斷的研究[D];安徽醫(yī)科大學;2008年

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