胚胎干细胞建系及造血分化潜能的研究

发布时间：2018-01-26 05:15

本文关键词： 胚胎干细胞建系 OG2小鼠人孤雌胚胎干细胞 STR 胚胎干细胞核移植拟胚体造血分化 BMP4 人类胚胎干细胞造血分化　出处：《广州医学院》2009年硕士论文　论文类型：学位论文

【摘要】： 第一部分小鼠胚胎干细胞建系及人孤雌胚胎干细胞的培养鉴定第一章小鼠胚胎干细胞建系研究目的建立小鼠胚胎干细胞系,为下一步造血分化提供原材料。材料与方法 1.使用129/sv雌鼠和OG2公鼠进行建系。OG2小鼠的特点是基因Oct4后溶合了基因GFP,所以在ES细胞在未分化的情况下能够发出绿色荧光。 2.建系方法与常规建系过程一样:取第3.5天孕鼠子宫,冲洗子宫获得囊胚,并接种到饲养层上;第4-6天后挑取克隆消化传代。结果我们将见栓后第3.5天的3只129/sv小鼠处死,取出子宫,冲洗后获得16个胚胎,其中囊胚有9个,桑椹胚有5个,质量比较差的胚胎(出现较多的碎片)有2个。由囊胚发育而来的成功建起2株细胞系,建系率为22%;桑椹胚的没有成功建起细胞系。最后对其中一株细胞系进行鉴定,结果都符合ES细胞的特性结论我们建立的小鼠胚胎干细胞系符合ES细胞的特性,具有自我更新以及多向分化的潜能。第二章人孤雌胚胎干细胞的培养以及鉴定研究目的通过对人孤雌胚胎干细胞(phES cell)进行培养鉴定,掌握人胚胎干细胞培养技能,同时了解人孤雌胚胎干细胞的一些生物学特性。材料与方法 1.由广州医学院第三附属医院妇研所提供的人孤雌胚胎干细胞系。 2.传代方法:使用胶原酶消化法和机械法。胶原酶消化法适合培养皿中克隆生长比较旺盛,状态比较好的情况下;机械法适合于培养皿中分化细胞比较多,或克隆比较少的情况。 3.我们对phES细胞进行了Oct4、SSEA-2、SSEA-4、TRA-1-60、TRA-1-81;EB形成试验;核型分析;STR以及畸胎瘤进行检测。结果细胞能表达Oct4、SSEA-2、SSEA-4、TRA-1-60、TRA-1-81等指标;能够形成EB;核型为45,X0;STR检测显示与供者一致;畸胎瘤到目前还没有检测出来。结论 phES细胞的培养条件与正常人的ES细胞没有明显区别;通过检测其生物学特性,发现这株细胞的核型异常,而且致瘤性不强。第二部分胚胎干细胞的造血分化第一章核移植小鼠胚胎干细胞的造血分化潜能研究目的对核移植来源的小鼠胚胎干细胞(NT-ES)进行造血分化,探讨它的造血分化潜能。材料与方法 1. F-ES细胞来源于129/sv雌鼠与OG2雄鼠交配见栓后第3.5天的囊胚,建系后稳定传代到第18代时进行实验;NT-ES细胞来源于129/OG2小鼠睾丸的一种滋养细胞(Sertoli Cell)进行核移植后建系获得,同样取第18代细胞进行实验。 2.分化过程中,采用预分化→初步分化→进一步分化的步骤进行造血分化。通过对分化过程中各项指标进行比较,来说明两者的异同。 3.造血移植:检测由这两株细胞分化而来的造血干细胞在体内是否具有造血潜能。结果 1. EB形成能力:NT-ES和F-ES在总EB和造血EB形成的数目分别是:129、134和73、78。 2.流式细胞仪分析结果:在分化的第5、7、10天,NT-ES细胞分化成CD34和Sca-1双阳性细胞的比例分别为:6.58%、32.03%、23.44% ;F-ES细胞的比例分别为:10.35%、32.88%、16.54%。 3. Realtime PCR检测显示,多个造血相关基因在两者分化过程中的变化趋势基本一致。 4. BL-CFC试验显示,NT-ES细胞能够形成爆发集落。 5.造血移植检测结果显示:两者在体内都能够发生造血。结论核移植来源的与受精来源的小鼠胚胎干细胞在体内外几乎具有一样的造血分化潜能。第二章初步探讨BMP4对人类胚胎干细胞造血分化的影响研究目的初步探讨Bmp4在人类胚胎干细胞造血分化过程中的作用,为造血移植提供参考信息。材料与方法 1.由广州医学院第三附属医院妇研所提供的hES1细胞株。 2.实验分成3组:control组(只添加分化基础培养基)、cytokine组(添加常规细胞因子)、Bmp4组(常规细胞因子+Bmp4),采用机械法进行接种。将切成块状的克隆切片接种到超低吸附培养皿中(6孔板),每孔大概接种15-20块克隆切片。 3.同时采用酶消化法与机械法进行形态学的比较,看两者在EB的发育上是否有区别。结果 1.流式细胞仪分析显示,control组、cytokine组和Bmp4组分化至第12天时,CD34+CD38-细胞的比例分别为1.63%、4.77%、9.83%;CD34+CD38+细胞的比例分别为1.48%、2.13%、2.80%;CD34+CD117+细胞的比例分别为3.09%、4.40%和9.91%。 2. control组、cytokine组和Bmp4组的造血集落形成数目分别为8、26、35. 3.用机械法处理后接种形成的EB,形态典型而且发育良好;酶消化法处理克隆后,不易于形成EB。结论 1、Bmp4能够显示促进hES细胞的造血分化活性。 2、在hES细胞形成EB的过程中,机械法比酶消化法更有利于EB的形成。
[Abstract]:The first part of mouse embryonic stem cell line construction and human parthenogenetic embryonic stem cell culture identification
Chapter 1 the establishment of mouse embryonic stem cell line
research objective
The mouse embryonic stem cell line was established to provide the raw material for the next step of hematopoietic differentiation.
Materials and methods
1., using 129/sv female mice and OG2 male mice to build.OG2 mice, the characteristic is that Oct4 gene dissolves the gene GFP, so ES cells can send out green fluorescence when they are undifferentiated.
2. line method and the conventional construction system: the 3.5 day pregnant rat uterus, uterine flushing obtained blastocysts, and inoculated into the feeder layer; the positive clones were digested in 4-6 days.
Result
We will see the death, 3 129/sv mice 3.5 days after the removal of the thrombus after flushing the uterus, 16 embryos, blastocysts which have 9, 5 morulas, relatively poor quality embryos (more debris) 2. By the blastocyst to successfully build 2 cell lines. The Department of construction, the rate is 22%; morula cells built without success. At the end of one cell lines were identified, the results are consistent with the characteristics of ES cells
conclusion
The mouse embryonic stem cell line has been established in accordance with the characteristics of ES cells and has the potential of self renewal and multidifferentiation.
Culture and identification of human parthenogenetic embryonic stem cells in the second chapter
research objective
Through the culture and identification of human embryonic stem cells (phES cell), we can master the culture skills of human embryonic stem cells and understand some biological characteristics of human parthenogenetic embryonic stem cells.
Materials and methods
1. by the Guangzhou Medical College Third Affiliated Hospital and Institute of parthenogenetic embryonic stem cell lines.
2. passage method: collagenase digestion and mechanical method. Collagenase digestion is suitable for Petri dishes, clone growth is relatively vigorous, and the condition is relatively good. Mechanical method is suitable for culture dishes with more differentiated cells or less cloning.
3. we carried out Oct4, SSEA-2, SSEA-4, TRA-1-60, TRA-1-81; EB formation test on phES cells; karyotype analysis; STR and teratoma.
Result
Cells can express Oct4, SSEA-2, SSEA-4, TRA-1-60, TRA-1-81 and other indicators, form EB, the karyotype is 45, X0, STR detection is consistent with donors, teratoma has not yet been detected.
conclusion
The culture conditions of phES cells were not significantly different from those of ES cells. By detecting their biological characteristics, we found that the karyotype of these cells was abnormal and their tumorigenicity was not strong.
The hematopoietic differentiation of second parts of embryonic stem cells
Chapter 1 the hematopoietic differentiation potential of nuclear transplanted mouse embryonic stem cells
research objective
The hematopoietic differentiation of mouse embryonic stem cells (NT-ES) from nuclear transplantation was carried out to explore the potential of its hematopoietic differentiation.
Materials and methods
1. F-ES cells derived from 129/sv female mice were mated with male OG2 in 3.5 days after the bolt blastocyst, built stable passage experiment was carried out by the eighteenth generation; a NT-ES on trophoblast cells derived from mouse testis 129/OG2 (Sertoli Cell) after the construction of nuclear transfer system, also take the eighteenth generation cells were experiment.
2. in differentiation, hematopoietic differentiation is achieved by pre differentiation, primary differentiation and further differentiation.
3. hematopoietic transplantation: to detect the hematopoietic potential of hematopoietic stem cells derived from these two cells in the body.
Result
1. EB formation ability: the number of NT-ES and F-ES in total EB and hematopoietic EB are: 129134 and 73,78., respectively.
2. flow cytometry analysis results: on the first day of differentiation, the proportion of NT-ES cells differentiated into CD34 and Sca-1 double positive cells were 6.58%, 32.03% and 23.44%, respectively, and the proportion of F-ES cells was 10.35%, 32.88%, 16.54%., respectively. The ratio of F-ES cells to CD34 cells was 10.35%.
3. Realtime PCR detection showed that the trend of multiple hematopoietic related genes in the process of differentiation was basically the same.
The 4. BL-CFC test showed that NT-ES cells were able to form an outbreak colony.
The results of 5. hematopoietic transplantation showed that both of them could have hematopoiesis in the body.
conclusion
The mouse embryonic stem cells from the source of nuclear transplantation and the source of fertilization almost have the same hematopoietic differentiation potential in the body and in vivo.
The second chapter discusses the effect of BMP4 on the hematopoietic differentiation of human embryonic stem cells
research objective
The role of Bmp4 in the hematopoietic differentiation of human embryonic stem cells was preliminarily discussed in order to provide reference information for hematopoietic transplantation.
Materials and methods
1. hES1 cell lines by the Institute of Guangzhou Medical College Third Affiliated Hospital and provide.
2. experiments were divided into 3 groups: group control (add only differentiation culture medium), cytokine group (adding conventional cytokines), group Bmp4 (conventional cytokine +Bmp4), using the mechanical method of inoculation. The cut slices inoculated to clone massive ultra-low attachment culture dish (plate 6), probably every hole inoculation of 15-20 block cloning sections.
3. at the same time, the morphological comparison between the enzyme digestion method and the mechanical method was used to see whether there was a difference between the two in the development of EB.
Result
1. flow cytometry analysis showed that in group control, group cytokine and group Bmp4 differentiated to twelfth days, the proportion of CD34+CD38- cells was 1.63%, 4.77%, 9.83%, the proportion of CD34+CD38+ cells was 1.48%, 2.13% and 2.80%, respectively, and the proportion of CD34+CD117+ cells was 3.09%, 4.40% and 9.91%. respectively.
The number of hematopoietic colonies in the 2. control group, the cytokine group and the Bmp4 group was 8,26,35., respectively.
3. the EB formed by inoculation after mechanical treatment is typical and well developed, and the enzyme digestion method is not easy to form EB. after cloning.
conclusion
1, Bmp4 can show the hematopoietic differentiation activity of hES cells.
2, in the process of the formation of EB in hES cells, the mechanical method is more beneficial to the formation of EB than the enzyme digestion.

【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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