小鼠8-细胞胚胎单一卵裂球的体外培养及其囊胚类胚胎干细胞的初步分离与鉴定

发布时间：2018-01-21 22:22

本文关键词： 昆明小鼠 8-细胞体外受精胚胎抗氧化体外受精胚胎培养基单个卵裂球类ES细胞　出处：《西南大学》2011年硕士论文　论文类型：学位论文

【摘要】：分离培养单个卵裂球生产同卵双胎或一卵多胎动物已有成功报道；提取体外受精胚胎单个卵裂球活体检测着床前胚胎的基因遗传问题是人医临床诊断的热门课题(PGD诊断)；利用小鼠不同发育阶段的早期胚胎分离培养卵裂球获取胚胎干细胞(embryonic stem cells,ES细胞)的实验研究是近期研究胚胎干细胞来源的热点之一。以上研究内容是近十年来以早期胚胎卵裂球的分离培养为研究手段,为寻求动物快速扩繁新途径、探索人类生前遗传病诊断方式和寻求无伦理争议的人体自身Es细胞生前储备库的有效方法的三个不同目的所开展的研究。以上三方面的研究报道很多,在羊和牛已经获得了来源于2-细胞和4-细胞胚胎的单个卵裂球的活体后代,在兔获得了来源于4-细胞胚胎的单个卵裂球后代,在猪获得了8-细胞单个卵裂球后代。通过活组织检查卵裂球途径建立ES细胞系的成功率很低,而且不是所有的姐妹卵裂球都能建立ES细胞系,由此引发了姐妹卵裂球是否都具有相同的衍生成Es细胞的能力或是否具有不同的命运,卵裂球的选取可能减少供体细胞的发育能力,因此确定随机选取单卵裂球产生ES细胞是否破坏胚胎的完整性以及是否危害胚胎的后续发育,是-个十分重要的问题。小鼠单个卵裂球培养后,ES细胞的建系成功率与胚胎的细胞分裂次数或胚胎细胞的发育阶段具有成反比趋势,这些结果显示出每一个胚胎阶段并不是所有卵裂球,而是只有一个或两个卵裂球有衍生成ES细胞的能力。以上三方面的研究报道均表明各自方向都有成功潜力,成功率很低,影响成功率的焦点问题主要集中在单一卵裂球的质量上,这种质量问题是由于早期胚胎在分裂增殖过程中因时序所致的决定因子分布不均所引起,或是众多研究者沿用的早期胚胎培养基不能满足单一卵裂球更高的无分化性增殖的培养条件所致,前者是遗传性的,后者是后天获得性的问题。本试验针对后者,以昆明小鼠为实验材料,以培养体外受精8-细胞胚胎卵裂球生成囊胚后分离培养出类胚胎干细胞的效果为主要检测目标,从改善体外受精胚胎的早期培养条件入手,探索体外受精8-细胞胚胎单个卵裂球的体外培养体系,因此设计以下3个试验,旨在观察单个卵裂球体外培养条件对其干细胞生产的非遗传因素作用,为ES细胞的研究提供实验依据。试验总结如下：试验一：8-细胞体外受精胚胎单一卵裂球培养基的选择与优化本试验在分析和选择小鼠早期体外受精胚胎培养液的基础上,自制不含抗氧化剂的CZB’培养液,以此为基础培养液,通过添加不同浓度维生素E、半胱氨酸或L-谷氨酰胺等抗氧化剂,观察抗氧化培养对体外受精胚胎2-细胞、4-细胞、8-细胞和桑囊胚发育率的作用,旨在改进小鼠早期体外受精胚胎培养体系的基础上,为选取小鼠体外受精8-细胞胚胎单个卵裂球的培养基奠定基础。结果显示0.15 mg·ml-1L-谷氨酰胺处理组、0.05 mg·ml-1半胱氨酸处理组、100μmol·L-1VE处理组的抗氧化培育效果均优于对照组,能显著提高体外受精胚胎的2-细胞(68.68%、61.35%、67.88%VS 53.14%)、4-细胞(51.77%、49.72%、50.71%VS 27.83%)、8-细胞率(42.18%、41.32%、40.52% VS 17.70%)和桑囊胚发育率(33.75%、32.37%、30.89% VS 12.70%)。结果表明：在实验室自配的CZB'培养液中添加适宜浓度的抗氧化剂对比明小鼠体外受精胚胎进行抗氧化培养效果较好,使用这种抗氧化培养基可以作为体外受精8-细胞单个卵裂球的基础培养液。试验二：8-细胞体外受精胚胎单一卵裂球的培养方法研究在试验一的基础上,本试验选取抗氧化性早期胚胎培养基CZB培养液作为8-细胞胚胎单一卵裂球的培养液,通过改进细胞的共培养体系、比较人工透明带使用效果和观察有限培养液内卵裂球培养密度对其培养效果的影响,从这三个途径入手,研究体外培养单一卵裂球的有效培养方法。结果显示：比较小鼠胚胎成纤维细胞(Mouse embryonic fibroblast, MEF)原代(7.33%)、MEF 3代(15.90%)、MEF 6代(0.68%)共培养体系,小鼠子宫内膜细胞共培养体系(16.06%),小鼠颗粒细胞共培养体系(10.53%)等对单个卵裂球发育的影响,MEF 3代共培养体系和子宫内膜细胞共培养体系更有助于提高8-细胞单个卵裂球发育能力和囊胚率,与对照组相比差异显著(15.90%、16.06% VS 3.48%);将单一卵裂球放入人工透明带内培养其囊胚发育率没有得到明显改善(16.15% VS 15.87%),但有透明带培养的多腔囊胚率要显著低于无透明带培养(1.09% VS 4.63%)；在培养微滴中采取10个·20μl-1培养密度培养能显著提高囊胚率(18.44% VS 14.33%)。结果表明：选取MEF 3代共培养体系,用包裹透明带的方法,以10个·20μl-1培养密度培养能显著增强单一卵裂球的体外发育能力和囊胚出现比率。试验三：来源于8-细胞胚胎单一卵裂球囊胚类干细胞的分离、培养与鉴定本试验在获得由单一卵裂球发育而来的小鼠囊胚后,从共培养体系中通过全胚饲养层法培养,再经过显微外科手段挑选出增殖的囊胚内细胞团(Inner cell mass, ICM)集落,进一步通过对内细胞团的离散、分离、培养,最后通过形态学鉴定、碱性磷酸酶(alkaline phosphatase,AKP)染色和体外分化试验,初步鉴定来源于小鼠8-细胞体外受精胚胎单一卵裂球的类胚胎干细胞。结果显示：分离所得细胞集落与饲养层界线清楚,表面光滑,细胞致密,集落呈典型的鸟巢状,显微镜下观察,细胞体积小但核很大,符合ES细胞的形态学特征；AKP试剂盒检测,发现该种细胞被染成红色,且周围饲养层细胞不着色；将该种细胞集落挑出,接种于不加白血病抑制因子(Leukemia Inhibitory Factor, LIF)的ES细胞培养液中,发现该种细胞开始分化。结果表明：这种来源于小鼠体外受精8-细胞胚胎单一卵裂球的细胞集落,经过初步鉴定可以判定为类ES细胞。结论：在分析和选择几种小鼠常用体外受精胚胎培养基的基础上,本试验白制的抗氧化培养基能够有效改进体外受精胚胎的培养效果；以此培养基作为小鼠8-细胞体外受精胚胎单一卵裂球的基础培养液,对单一卵裂球的培养方法进行研究表明,采用MEF 3代共培养体系,将单一卵裂球放入人工透明带内培养和在培养微滴中采取10个·20μl-1培养密度培养的方法能够显著提高小鼠8细胞体外受精胚胎单一卵裂球发育至囊胚的比率：从这种来源于小鼠体外受精8-细胞胚胎单一卵裂球的细胞集落中能够分离培养出界线清楚、表面光滑、细胞致密、呈典型鸟巢状的集落,通过形态学鉴定、AKP染色和体外分化试验初步鉴定为类胚胎干细胞。
[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.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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