本文關(guān)鍵詞： 昆明小鼠 8-細(xì)胞體外受精胚胎 抗氧化體外受精胚胎培養(yǎng)基 單個卵裂球 類ES細(xì)胞　出處：《西南大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：分離培養(yǎng)單個卵裂球生產(chǎn)同卵雙胎或一卵多胎動物已有成功報道；提取體外受精胚胎單個卵裂球活體檢測著床前胚胎的基因遺傳問題是人醫(yī)臨床診斷的熱門課題(PGD診斷)；利用小鼠不同發(fā)育階段的早期胚胎分離培養(yǎng)卵裂球獲取胚胎干細(xì)胞(embryonic stem cells,ES細(xì)胞)的實驗研究是近期研究胚胎干細(xì)胞來源的熱點之一。以上研究內(nèi)容是近十年來以早期胚胎卵裂球的分離培養(yǎng)為研究手段,為尋求動物快速擴繁新途徑、探索人類生前遺傳病診斷方式和尋求無倫理爭議的人體自身Es細(xì)胞生前儲備庫的有效方法的三個不同目的所開展的研究。以上三方面的研究報道很多,在羊和牛已經(jīng)獲得了來源于2-細(xì)胞和4-細(xì)胞胚胎的單個卵裂球的活體后代,在兔獲得了來源于4-細(xì)胞胚胎的單個卵裂球后代,在豬獲得了8-細(xì)胞單個卵裂球后代。通過活組織檢查卵裂球途徑建立ES細(xì)胞系的成功率很低,而且不是所有的姐妹卵裂球都能建立ES細(xì)胞系,由此引發(fā)了姐妹卵裂球是否都具有相同的衍生成Es細(xì)胞的能力或是否具有不同的命運,卵裂球的選取可能減少供體細(xì)胞的發(fā)育能力,因此確定隨機選取單卵裂球產(chǎn)生ES細(xì)胞是否破壞胚胎的完整性以及是否危害胚胎的后續(xù)發(fā)育,是-個十分重要的問題。小鼠單個卵裂球培養(yǎng)后,ES細(xì)胞的建系成功率與胚胎的細(xì)胞分裂次數(shù)或胚胎細(xì)胞的發(fā)育階段具有成反比趨勢,這些結(jié)果顯示出每一個胚胎階段并不是所有卵裂球,而是只有一個或兩個卵裂球有衍生成ES細(xì)胞的能力。以上三方面的研究報道均表明各自方向都有成功潛力,成功率很低,影響成功率的焦點問題主要集中在單一卵裂球的質(zhì)量上,這種質(zhì)量問題是由于早期胚胎在分裂增殖過程中因時序所致的決定因子分布不均所引起,或是眾多研究者沿用的早期胚胎培養(yǎng)基不能滿足單一卵裂球更高的無分化性增殖的培養(yǎng)條件所致,前者是遺傳性的,后者是后天獲得性的問題。本試驗針對后者,以昆明小鼠為實驗材料,以培養(yǎng)體外受精8-細(xì)胞胚胎卵裂球生成囊胚后分離培養(yǎng)出類胚胎干細(xì)胞的效果為主要檢測目標(biāo),從改善體外受精胚胎的早期培養(yǎng)條件入手,探索體外受精8-細(xì)胞胚胎單個卵裂球的體外培養(yǎng)體系,因此設(shè)計以下3個試驗,旨在觀察單個卵裂球體外培養(yǎng)條件對其干細(xì)胞生產(chǎn)的非遺傳因素作用,為ES細(xì)胞的研究提供實驗依據(jù)。試驗總結(jié)如下： 試驗一：8-細(xì)胞體外受精胚胎單一卵裂球培養(yǎng)基的選擇與優(yōu)化 本試驗在分析和選擇小鼠早期體外受精胚胎培養(yǎng)液的基礎(chǔ)上,自制不含抗氧化劑的CZB’培養(yǎng)液,以此為基礎(chǔ)培養(yǎng)液,通過添加不同濃度維生素E、半胱氨酸或L-谷氨酰胺等抗氧化劑,觀察抗氧化培養(yǎng)對體外受精胚胎2-細(xì)胞、4-細(xì)胞、8-細(xì)胞和桑囊胚發(fā)育率的作用,旨在改進(jìn)小鼠早期體外受精胚胎培養(yǎng)體系的基礎(chǔ)上,為選取小鼠體外受精8-細(xì)胞胚胎單個卵裂球的培養(yǎng)基奠定基礎(chǔ)。結(jié)果顯示0.15 mg·ml-1L-谷氨酰胺處理組、0.05 mg·ml-1半胱氨酸處理組、100μmol·L-1VE處理組的抗氧化培育效果均優(yōu)于對照組,能顯著提高體外受精胚胎的2-細(xì)胞(68.68%、61.35%、67.88%VS 53.14%)、4-細(xì)胞(51.77%、49.72%、50.71%VS 27.83%)、8-細(xì)胞率(42.18%、41.32%、40.52% VS 17.70%)和桑囊胚發(fā)育率(33.75%、32.37%、30.89% VS 12.70%)。結(jié)果表明：在實驗室自配的CZB'培養(yǎng)液中添加適宜濃度的抗氧化劑對比明小鼠體外受精胚胎進(jìn)行抗氧化培養(yǎng)效果較好,使用這種抗氧化培養(yǎng)基可以作為體外受精8-細(xì)胞單個卵裂球的基礎(chǔ)培養(yǎng)液。 試驗二：8-細(xì)胞體外受精胚胎單一卵裂球的培養(yǎng)方法研究 在試驗一的基礎(chǔ)上,本試驗選取抗氧化性早期胚胎培養(yǎng)基CZB培養(yǎng)液作為8-細(xì)胞胚胎單一卵裂球的培養(yǎng)液,通過改進(jìn)細(xì)胞的共培養(yǎng)體系、比較人工透明帶使用效果和觀察有限培養(yǎng)液內(nèi)卵裂球培養(yǎng)密度對其培養(yǎng)效果的影響,從這三個途徑入手,研究體外培養(yǎng)單一卵裂球的有效培養(yǎng)方法。結(jié)果顯示：比較小鼠胚胎成纖維細(xì)胞(Mouse embryonic fibroblast, MEF)原代(7.33%)、MEF 3代(15.90%)、MEF 6代(0.68%)共培養(yǎng)體系,小鼠子宮內(nèi)膜細(xì)胞共培養(yǎng)體系(16.06%),小鼠顆粒細(xì)胞共培養(yǎng)體系(10.53%)等對單個卵裂球發(fā)育的影響,MEF 3代共培養(yǎng)體系和子宮內(nèi)膜細(xì)胞共培養(yǎng)體系更有助于提高8-細(xì)胞單個卵裂球發(fā)育能力和囊胚率,與對照組相比差異顯著(15.90%、16.06% VS 3.48%);將單一卵裂球放入人工透明帶內(nèi)培養(yǎng)其囊胚發(fā)育率沒有得到明顯改善(16.15% VS 15.87%),但有透明帶培養(yǎng)的多腔囊胚率要顯著低于無透明帶培養(yǎng)(1.09% VS 4.63%)；在培養(yǎng)微滴中采取10個·20μl-1培養(yǎng)密度培養(yǎng)能顯著提高囊胚率(18.44% VS 14.33%)。結(jié)果表明：選取MEF 3代共培養(yǎng)體系,用包裹透明帶的方法,以10個·20μl-1培養(yǎng)密度培養(yǎng)能顯著增強單一卵裂球的體外發(fā)育能力和囊胚出現(xiàn)比率。 試驗三：來源于8-細(xì)胞胚胎單一卵裂球囊胚類干細(xì)胞的分離、培養(yǎng)與鑒定 本試驗在獲得由單一卵裂球發(fā)育而來的小鼠囊胚后,從共培養(yǎng)體系中通過全胚飼養(yǎng)層法培養(yǎng),再經(jīng)過顯微外科手段挑選出增殖的囊胚內(nèi)細(xì)胞團(tuán)(Inner cell mass, ICM)集落,進(jìn)一步通過對內(nèi)細(xì)胞團(tuán)的離散、分離、培養(yǎng),最后通過形態(tài)學(xué)鑒定、堿性磷酸酶(alkaline phosphatase,AKP)染色和體外分化試驗,初步鑒定來源于小鼠8-細(xì)胞體外受精胚胎單一卵裂球的類胚胎干細(xì)胞。結(jié)果顯示：分離所得細(xì)胞集落與飼養(yǎng)層界線清楚,表面光滑,細(xì)胞致密,集落呈典型的鳥巢狀,顯微鏡下觀察,細(xì)胞體積小但核很大,符合ES細(xì)胞的形態(tài)學(xué)特征；AKP試劑盒檢測,發(fā)現(xiàn)該種細(xì)胞被染成紅色,且周圍飼養(yǎng)層細(xì)胞不著色；將該種細(xì)胞集落挑出,接種于不加白血病抑制因子(Leukemia Inhibitory Factor, LIF)的ES細(xì)胞培養(yǎng)液中,發(fā)現(xiàn)該種細(xì)胞開始分化。結(jié)果表明：這種來源于小鼠體外受精8-細(xì)胞胚胎單一卵裂球的細(xì)胞集落,經(jīng)過初步鑒定可以判定為類ES細(xì)胞。 結(jié)論：在分析和選擇幾種小鼠常用體外受精胚胎培養(yǎng)基的基礎(chǔ)上,本試驗白制的抗氧化培養(yǎng)基能夠有效改進(jìn)體外受精胚胎的培養(yǎng)效果；以此培養(yǎng)基作為小鼠8-細(xì)胞體外受精胚胎單一卵裂球的基礎(chǔ)培養(yǎng)液,對單一卵裂球的培養(yǎng)方法進(jìn)行研究表明,采用MEF 3代共培養(yǎng)體系,將單一卵裂球放入人工透明帶內(nèi)培養(yǎng)和在培養(yǎng)微滴中采取10個·20μl-1培養(yǎng)密度培養(yǎng)的方法能夠顯著提高小鼠8細(xì)胞體外受精胚胎單一卵裂球發(fā)育至囊胚的比率：從這種來源于小鼠體外受精8-細(xì)胞胚胎單一卵裂球的細(xì)胞集落中能夠分離培養(yǎng)出界線清楚、表面光滑、細(xì)胞致密、呈典型鳥巢狀的集落,通過形態(tài)學(xué)鑒定、AKP染色和體外分化試驗初步鑒定為類胚胎干細(xì)胞。
[Abstract]:Isolation of single blastomere produce identical twins or polyembryony animal has been successfully reported; extraction of genetic problem of in vitro fertilization embryo in vivo detection of single blastomere embryos is a hot topic in clinical diagnosis of human medicine (PGD diagnosis); early embryos in different developmental stages of the isolation and culture of blastomeres derived embryonic stem cells (embryonic stem cells, ES cells) experimental research is one of the recent research focus from embryonic stem cells. The above research content is nearly ten years for the separation of embryo blastomeres culture as research means, to seek new ways of animal fast propagation, to carry out exploration and research of the three different human genetic disease diagnosis and effective way of living no method of seeking the ethical controversy of human body's Es cell pool. The above three studies reported in many sheep and cattle have been Live offspring got source of single blastomere from embryonic 2- cells and 4- cells, single blastomere in Rabbit Offspring obtained from embryonic 4- cells, 8- cells were obtained in single blastomere offspring pigs. ES cell lines by examining the way to establish the blastomere live success rate is very low, and not all sister blastomeres can ES cell line was established, which led to whether have the same sister blastomeres derived Es cell or with a different fate, the selection of blastomeres may reduce viability of donor cells, thus determine the integrity of the randomly selected single blastomere ES cell damage of the embryo and the subsequent development is harmful to the embryo, is a very important problem. The mouse single blastomere culture, developmental stage construction of ES cell line of the success rate and the number of cell division or embryo embryo cells Is inversely proportional to the trend, the results showed that each embryo stage and not all blastomeres, but only one or two blastomeres derived from ability of ES cells. The above three aspects of research reports indicate that the respective direction have potential for success, the success rate is very low, quality issues affecting the success rate of the focus in a single blastomere, the quality problem is due to the early embryo in the proliferation process because of factors caused by the uneven distribution of the timing caused by, or early embryos of many researchers use the medium can not meet due to culture conditions without undifferentiated proliferation of single blastomere is higher, the former is hereditary, the latter is acquired. The test for the latter, in Kunming mice as experimental materials, in order to cultivate 8- in vitro fertilization embryo blastomeres isolated from the blastocyst formation type embryo The effect of stem cells as the main targets, from the improvement of in vitro fertilization and embryo culture conditions of the early start, explore the culture system of 8- cells in vitro fertilization embryo single blastomere in vitro, so the design of the following 3 experiments to observe single blastomere in vitro conditions on the role of stem cell production of non genetic factors, to provide the experimental basis for the study of ES cell tests are summarized as follows:
Experiment one: the selection and optimization of 8- cells in vitro fertilization embryo single blastomere culture medium
In this experiment the selection and analysis of liquid foundation early in vitro fertilization and embryo culture of mouse, homemade without antioxidants CZB medium as basic culture medium, by adding different concentrations of vitamin E, cysteine or L- glutamine antioxidants, observe the antioxidant culture of in vitro fertilization embryo 2- cells, 8- cells and 4- cells. Mulberry blastocyst development rate, to improve early mouse embryos fertilized in vitro culture system on the basis of lay the foundation for the selection of culture medium of mouse embryos fertilized in vitro 8- cells single blastomere. The results showed that 0.15 mg / ml-1L- glutamine treatment group, ml-1 treatment group 0.05 mg cysteine, 100 mol L-1VE treatment group antioxidant cultivation the effect was better than the control group, can significantly improve the in vitro fertilization embryo 2- cells (68.68%, 61.35%, 53.14% 67.88%VS), 4- cells (51.77%, 49.72%, 27.83% 50.71%VS), 8- cell The rate (42.18%, 41.32%, 40.52% VS 17.70%) and mulberry blastocyst rate (33.75%, 32.37%, 30.89% VS 12.70%). The results showed that: in the laboratory with CZB'medium adding suitable concentration of antioxidants on mice than in in vitro fertilization and embryo culture of antioxidant effect is good, the use of this medium can be cultured as antioxidant based on in vitro fertilization 8- cell single blastomere.
Experiment two: the culture of 8- cells in vitro fertilization embryo single blastomere
On the basis of Experiment 1, this experiment selected antioxidant early embryo cultured in CZB culture medium as the medium 8- single cell embryo blastomeres, through improved cell co culture system, compared with artificial transparent effect and observe the medium effect of culture density on the blastomere culture effect, starting from the three in a way, effective training methods of single blastomere culture in vitro. The results showed that compared with mouse embryonic fibroblasts (Mouse embryonic, fibroblast, MEF) primary (7.33%), MEF 3 (15.90%), MEF 6 (0.68%) co culture system, co culture system of mouse endometrial cells (16.06%), system co culture of mouse granulosa cells (10.53%) of single blastomere development effects, the 3 generation of MEF co culture system and endometrial cell co culture system is helpful to improve the 8- cells with single blastomeres and blastocyst development ability The rate, compared with the control group the difference was significant (15.90%, 16.06% VS 3.48%); single blastomere in artificial zona cultured in the blastocyst rate has not been significantly improved (16.15% VS 15.87%), but there are many coeloblastula zona cultured rate was significantly lower than that of zona free culture (1.09% VS 4.63%); in in the droplet culture take 10 - 20 L-1 medium density culture can significantly increase the blastocyst rate (18.44% VS 14.33%). The results show that the selection of the 3 generation of MEF co culture system with the method of wrapping transparent tape, with 10 and 20 L-1 can significantly enhance the culture density cultivation of single blastomere in vitro development ability and blastocyst ratio.
Test three: isolation, culture and identification of a single cleavage stem cell like stem cells from 8- cell embryos
This experiment was obtained from single blastomeres of mouse blastocyst development comes from the system, through the whole embryo culture method of feeder layer were cultured after microsurgical methods selected proliferation of ICM (Inner cell, mass, ICM) colony, further through the discrete, of the inner cell mass separation, culture. Finally, through morphological identification, alkaline phosphatase (alkaline phosphatase, AKP) staining and differentiation in vitro, identified from mouse 8- cells in vitro fertilization embryo single blastomere of embryonic stem cell. The results showed that the separation of cell colony and feeder layer boundaries clear, smooth surface, compact cells, colony showed typical nest under the microscope, the cells are small, but large nuclear, accord with the morphological characteristics of ES cells; AKP kit, found that the cells were dyed red, and the surrounding feeder cells not stained; the Cell colony picking inoculated without leukemia inhibitory factor (Leukemia Inhibitory, Factor, LIF) ES cell culture medium, found that the cells began to differentiate. The results show that the derived from embryos in vitro fertilization of mouse 8- cells single blastomere cell colony, after preliminary identification can be determined as ES cells.
Conclusion: on the basis of the analysis and selection of several commonly used in in vitro fertilization and embryo culture medium on the test system of antioxidant white culture medium effect can be effectively improved in vitro fertilization embryo; the medium liquid as the basis of mouse 8- cells in vitro fertilization embryo single blastomere, cultured method of single blastomere studies showed that the the 3 generation of MEF co culture system, the single blastomere in artificial culture and transparent band ratio in the medium drop in take 10 - 20 L-1 training method can significantly improve the density of culture in vitro of 8 cell mouse fertilized embryo blastocysts: single blastomere colony can be isolated from the clear boundaries, smooth surface. From the compact cells derived from embryos in vitro fertilization of mouse 8- cells single blastomere cells, showed typical nest like colonies, through morphological identification, AKP The staining and in vitro differentiation test was preliminarily identified as a type of embryonic stem cell.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

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