發(fā)布時(shí)間：2018-06-03 19:44

  本文選題：HSC + nthESC�。� 參考：《西北農(nóng)林科技大學(xué)》2005年博士論文

【摘要】：造血干細(xì)胞(hematopoietic stem cell,HSC)是近年來研究較多的一類組織干細(xì)胞,目前發(fā)現(xiàn)卵黃囊、胎肝、主動(dòng)脈-性腺-中腎區(qū)(AGM)、胎兒血液以及胎盤中均存在具有高度擴(kuò)增能力的HSC。本論文主要進(jìn)行了具有課題組專利的人自體基因胚胎干細(xì)胞(nthESC)向造血干細(xì)胞的誘導(dǎo)分化、人胚胎發(fā)育過程中卵黃囊、胎肝和胎盤中的造血發(fā)育以及胎盤貼壁細(xì)胞的分離培養(yǎng)、向神經(jīng)細(xì)胞誘導(dǎo)分化的研究,為nthESC 和HSC的臨床應(yīng)用提供了理論依據(jù)和技術(shù)方法。 1. 探討了人胚骨髓間充質(zhì)干細(xì)胞(MSC)的體外分離、擴(kuò)增、凍存、復(fù)蘇、轉(zhuǎn)GFP基因和向成骨和神經(jīng)細(xì)胞的誘導(dǎo)分化條件,結(jié)果發(fā)現(xiàn)MSC有60%可轉(zhuǎn)染GFP基因,表達(dá)CD29、CD13、CD59,而CD34、CD45和CD38表達(dá)陰性。Vimentin染色陽性,mallory染色顯示膠原存在,分離的細(xì)胞可在神經(jīng)誘導(dǎo)體系和成骨誘導(dǎo)體系下進(jìn)一步向神經(jīng)細(xì)胞和成骨細(xì)胞分化。表明MSC在體外能迅速擴(kuò)增,具有向成骨和神經(jīng)細(xì)胞分化潛能,可以作為穩(wěn)定飼養(yǎng)層來源。 2. 利用人MSC 與人nthESC 共培養(yǎng),研究人MSC 對(duì)胚胎干細(xì)胞誘導(dǎo)造血的作用并建立一種有效誘導(dǎo)nthESC 為HSC 的方法。經(jīng)過20 d 培養(yǎng),獲得大量的CD133+和CD34+綠色熒光標(biāo)記的HSC。來源于nthESC 的KDR+細(xì)胞首先出現(xiàn)在第5 天,CD133 隨后出現(xiàn),CD34+細(xì)胞出現(xiàn)在第7 天,隨時(shí)間延長(zhǎng)三者表達(dá)增強(qiáng),第11 天左右表達(dá)最強(qiáng),隨后減弱,20 d 仍有表達(dá)。培養(yǎng)中發(fā)現(xiàn)11 d 可形成小造血集落,14 d 形成大集落。在BMP4、FGF-1和VEGF 等因子作用下,可促進(jìn)造血細(xì)胞形成。結(jié)果顯示由nthES 與MSC 共培養(yǎng)可產(chǎn)生CD34+細(xì)胞,nthESC 的造血分化程序?yàn)橄刃纬蒏DR+細(xì)胞,其次是CD133+,再形成CD34+細(xì)胞。表明nthESC 可在人胚骨髓MSC 飼養(yǎng)層上有效誘導(dǎo)成HSC。 3. 采用分階段懸浮培養(yǎng)法,首先誘導(dǎo)nthESC 形成擬胚體(EB),在此過程中添加BMP4 和FGF-1。第二階段,打散EB,再添加VEGF 和其他細(xì)胞因子,對(duì)HSC 進(jìn)行擴(kuò)增。結(jié)果發(fā)現(xiàn),采用低黏附板可有效促進(jìn)EB 形成,BMP4 和FGF-1 可使EB 形成數(shù)量明顯增加,可使KDR+和CD133+熒光細(xì)胞數(shù)量明顯增多。添加VEGF 等細(xì)胞因子,可有效促進(jìn)CD34+細(xì)胞數(shù)量增加。結(jié)果表明,聯(lián)合使用BMP4、FGF-1、VEGF 和SCF 等細(xì)胞因子,可使懸浮培養(yǎng)形成EB 數(shù)量增加,產(chǎn)生更多的造血前體細(xì)胞,繼而產(chǎn)生大量HSC。
[Abstract]:Hematopoietic stem cell (stem) is a kind of tissue stem cells studied in recent years. Yolk sac, fetal liver, aorta-gonadal-mesonephric area (AGM), fetal blood and placenta all have high amplification ability. In this paper, the differentiation of human autologous embryonic stem cells from human autologous embryonic stem cells into hematopoietic stem cells, hematopoietic development in yolk sac, fetal liver and placenta, and isolation and culture of placental adherent cells were studied. The study of neuronal differentiation provides a theoretical basis and a technical method for the clinical application of nthESC and HSC. 1. The conditions of isolation, expansion, cryopreservation, resuscitation, transfer of GFP gene and induction of differentiation into osteoblasts and neural cells of human embryonic bone marrow mesenchymal stem cells (MSCs) in vitro were studied. The results showed that 60% of MSC could be transfected into GFP gene. The expression of CD29, CD13, CD59, and negative expression of CD34, CD45 and CD38 showed that collagen was present. The isolated cells could further differentiate into neural cells and osteoblasts in neuro-induced and osteogenic systems. The results showed that MSC could be amplified rapidly in vitro and had the potential to differentiate into osteoblasts and nerve cells and could be used as a source of stable feeder layer. 2. The effect of human MSC on hematopoietic induction of embryonic stem cells was studied by co-culture of human MSC and human nthESC, and an effective method of inducing nthESC to HSC was established. After 20 days of culture, a large number of CD133 and CD34 labeled HSCs were obtained. The KDR cells derived from nthESC first appeared on the 5th day, then on the 7th day, and then on the 7th day. The expression of CD34 cells increased with time, and the expression was the strongest on the 11th day, and then the expression was still present on the 20th day of abatement. It was found in culture that small hematopoietic colonies could be formed at 11 days and large colonies could be formed at 14 days. BMP4 FGF-1 and VEGF can promote hematopoietic cell formation. The results showed that the hematopoietic differentiation of CD34 cells induced by co-culture of nthES and MSC was followed by KDR cells, CD133 and CD34 cells. The results showed that nthESC could be effectively induced on the feeder layer of human embryonic bone marrow MSC. 3. In this paper, nthESC was induced to form embryoid EBV by the method of suspension culture, and BMP4 and FGF-1 were added in the process. In the second stage, HSC was amplified by adding VEGF and other cytokines. The results showed that the low adhesion plate could effectively promote the formation of EB, BMP4 and FGF-1 could significantly increase the number of EB formation, and increase the number of KDR and CD133 fluorescence cells. The addition of cytokines such as VEGF can effectively increase the number of CD34 cells. The results showed that combined use of BMP4, FGF-1, VEGF and SCF could increase the number of EB and produce more hematopoietic precursor cells in suspension culture, and then produce a large number of HSCs.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類號(hào)】：R329.2

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