本文關(guān)鍵詞： 胚胎干細(xì)胞建系 OG2小鼠 人孤雌胚胎干細(xì)胞 STR 胚胎干細(xì)胞 核移植 擬胚體 造血分化 BMP4 人類(lèi)胚胎干細(xì)胞 造血分化　出處：《廣州醫(yī)學(xué)院》2009年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】： 第一部分小鼠胚胎干細(xì)胞建系及人孤雌胚胎干細(xì)胞的培養(yǎng)鑒定 第一章小鼠胚胎干細(xì)胞建系 研究目的 建立小鼠胚胎干細(xì)胞系,為下一步造血分化提供原材料。 材料與方法 1.使用129/sv雌鼠和OG2公鼠進(jìn)行建系。OG2小鼠的特點(diǎn)是基因Oct4后溶合了基因GFP,所以在ES細(xì)胞在未分化的情況下能夠發(fā)出綠色熒光。 2.建系方法與常規(guī)建系過(guò)程一樣:取第3.5天孕鼠子宮,沖洗子宮獲得囊胚,并接種到飼養(yǎng)層上;第4-6天后挑取克隆消化傳代。 結(jié)果 我們將見(jiàn)栓后第3.5天的3只129/sv小鼠處死,取出子宮,沖洗后獲得16個(gè)胚胎,其中囊胚有9個(gè),桑椹胚有5個(gè),質(zhì)量比較差的胚胎(出現(xiàn)較多的碎片)有2個(gè)。由囊胚發(fā)育而來(lái)的成功建起2株細(xì)胞系,建系率為22%;桑椹胚的沒(méi)有成功建起細(xì)胞系。最后對(duì)其中一株細(xì)胞系進(jìn)行鑒定,結(jié)果都符合ES細(xì)胞的特性 結(jié)論 我們建立的小鼠胚胎干細(xì)胞系符合ES細(xì)胞的特性,具有自我更新以及多向分化的潛能。 第二章人孤雌胚胎干細(xì)胞的培養(yǎng)以及鑒定 研究目的 通過(guò)對(duì)人孤雌胚胎干細(xì)胞(phES cell)進(jìn)行培養(yǎng)鑒定,掌握人胚胎干細(xì)胞培養(yǎng)技能,同時(shí)了解人孤雌胚胎干細(xì)胞的一些生物學(xué)特性。 材料與方法 1.由廣州醫(yī)學(xué)院第三附屬醫(yī)院婦研所提供的人孤雌胚胎干細(xì)胞系。 2.傳代方法:使用膠原酶消化法和機(jī)械法。膠原酶消化法適合培養(yǎng)皿中克隆生長(zhǎng)比較旺盛,狀態(tài)比較好的情況下;機(jī)械法適合于培養(yǎng)皿中分化細(xì)胞比較多,或克隆比較少的情況。 3.我們對(duì)phES細(xì)胞進(jìn)行了Oct4、SSEA-2、SSEA-4、TRA-1-60、TRA-1-81;EB形成試驗(yàn);核型分析;STR以及畸胎瘤進(jìn)行檢測(cè)。 結(jié)果 細(xì)胞能表達(dá)Oct4、SSEA-2、SSEA-4、TRA-1-60、TRA-1-81等指標(biāo);能夠形成EB;核型為45,X0;STR檢測(cè)顯示與供者一致;畸胎瘤到目前還沒(méi)有檢測(cè)出來(lái)。 結(jié)論 phES細(xì)胞的培養(yǎng)條件與正常人的ES細(xì)胞沒(méi)有明顯區(qū)別;通過(guò)檢測(cè)其生物學(xué)特性,發(fā)現(xiàn)這株細(xì)胞的核型異常,而且致瘤性不強(qiáng)。 第二部分胚胎干細(xì)胞的造血分化 第一章核移植小鼠胚胎干細(xì)胞的造血分化潛能 研究目的 對(duì)核移植來(lái)源的小鼠胚胎干細(xì)胞(NT-ES)進(jìn)行造血分化,探討它的造血分化潛能。 材料與方法 1. F-ES細(xì)胞來(lái)源于129/sv雌鼠與OG2雄鼠交配見(jiàn)栓后第3.5天的囊胚,建系后穩(wěn)定傳代到第18代時(shí)進(jìn)行實(shí)驗(yàn);NT-ES細(xì)胞來(lái)源于129/OG2小鼠睪丸的一種滋養(yǎng)細(xì)胞(Sertoli Cell)進(jìn)行核移植后建系獲得,同樣取第18代細(xì)胞進(jìn)行實(shí)驗(yàn)。 2.分化過(guò)程中,采用預(yù)分化→初步分化→進(jìn)一步分化的步驟進(jìn)行造血分化。通過(guò)對(duì)分化過(guò)程中各項(xiàng)指標(biāo)進(jìn)行比較,來(lái)說(shuō)明兩者的異同。 3.造血移植:檢測(cè)由這兩株細(xì)胞分化而來(lái)的造血干細(xì)胞在體內(nèi)是否具有造血潛能。 結(jié)果 1. EB形成能力:NT-ES和F-ES在總EB和造血EB形成的數(shù)目分別是:129、134和73、78。 2.流式細(xì)胞儀分析結(jié)果:在分化的第5、7、10天,NT-ES細(xì)胞分化成CD34和Sca-1雙陽(yáng)性細(xì)胞的比例分別為:6.58%、32.03%、23.44% ;F-ES細(xì)胞的比例分別為:10.35%、32.88%、16.54%。 3. Realtime PCR檢測(cè)顯示,多個(gè)造血相關(guān)基因在兩者分化過(guò)程中的變化趨勢(shì)基本一致。 4. BL-CFC試驗(yàn)顯示,NT-ES細(xì)胞能夠形成爆發(fā)集落。 5.造血移植檢測(cè)結(jié)果顯示:兩者在體內(nèi)都能夠發(fā)生造血。 結(jié)論 核移植來(lái)源的與受精來(lái)源的小鼠胚胎干細(xì)胞在體內(nèi)外幾乎具有一樣的造血分化潛能。 第二章初步探討B(tài)MP4對(duì)人類(lèi)胚胎干細(xì)胞造血分化的影響 研究目的 初步探討B(tài)mp4在人類(lèi)胚胎干細(xì)胞造血分化過(guò)程中的作用,為造血移植提供參考信息。 材料與方法 1.由廣州醫(yī)學(xué)院第三附屬醫(yī)院婦研所提供的hES1細(xì)胞株。 2.實(shí)驗(yàn)分成3組:control組(只添加分化基礎(chǔ)培養(yǎng)基)、cytokine組(添加常規(guī)細(xì)胞因子)、Bmp4組(常規(guī)細(xì)胞因子+Bmp4),采用機(jī)械法進(jìn)行接種。將切成塊狀的克隆切片接種到超低吸附培養(yǎng)皿中(6孔板),每孔大概接種15-20塊克隆切片。 3.同時(shí)采用酶消化法與機(jī)械法進(jìn)行形態(tài)學(xué)的比較,看兩者在EB的發(fā)育上是否有區(qū)別。 結(jié)果 1.流式細(xì)胞儀分析顯示,control組、cytokine組和Bmp4組分化至第12天時(shí),CD34+CD38-細(xì)胞的比例分別為1.63%、4.77%、9.83%;CD34+CD38+細(xì)胞的比例分別為1.48%、2.13%、2.80%;CD34+CD117+細(xì)胞的比例分別為3.09%、4.40%和9.91%。 2. control組、cytokine組和Bmp4組的造血集落形成數(shù)目分別為8、26、35. 3.用機(jī)械法處理后接種形成的EB,形態(tài)典型而且發(fā)育良好;酶消化法處理克隆后,不易于形成EB。 結(jié)論 1、Bmp4能夠顯示促進(jìn)hES細(xì)胞的造血分化活性。 2、在hES細(xì)胞形成EB的過(guò)程中,機(jī)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.

【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R329

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相關(guān)期刊論文 前1條

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本文編號(hào)：1464775