人nthESC向HSC诱导分化和胚胎造血标志物表达研究

发布时间：2018-06-03 19:44

本文选题：HSC + nthESC　；参考：《西北农林科技大学》2005年博士论文

【摘要】：造血干细胞(hematopoietic stem cell,HSC)是近年来研究较多的一类组织干细胞,目前发现卵黄囊、胎肝、主动脉-性腺-中肾区(AGM)、胎儿血液以及胎盘中均存在具有高度扩增能力的HSC。本论文主要进行了具有课题组专利的人自体基因胚胎干细胞(nthESC)向造血干细胞的诱导分化、人胚胎发育过程中卵黄囊、胎肝和胎盘中的造血发育以及胎盘贴壁细胞的分离培养、向神经细胞诱导分化的研究,为nthESC 和HSC的临床应用提供了理论依据和技术方法。 1. 探讨了人胚骨髓间充质干细胞(MSC)的体外分离、扩增、冻存、复苏、转GFP基因和向成骨和神经细胞的诱导分化条件,结果发现MSC有60%可转染GFP基因,表达CD29、CD13、CD59,而CD34、CD45和CD38表达阴性。Vimentin染色阳性,mallory染色显示胶原存在,分离的细胞可在神经诱导体系和成骨诱导体系下进一步向神经细胞和成骨细胞分化。表明MSC在体外能迅速扩增,具有向成骨和神经细胞分化潜能,可以作为稳定饲养层来源。 2. 利用人MSC 与人nthESC 共培养,研究人MSC 对胚胎干细胞诱导造血的作用并建立一种有效诱导nthESC 为HSC 的方法。经过20 d 培养,获得大量的CD133+和CD34+绿色荧光标记的HSC。来源于nthESC 的KDR+细胞首先出现在第5 天,CD133 随后出现,CD34+细胞出现在第7 天,随时间延长三者表达增强,第11 天左右表达最强,随后减弱,20 d 仍有表达。培养中发现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,可促进造血细胞形成。结果显示由nthES 与MSC 共培养可产生CD34+细胞,nthESC 的造血分化程序为先形成KDR+细胞,其次是CD133+,再形成CD34+细胞。表明nthESC 可在人胚骨髓MSC 饲养层上有效诱导成HSC。 3. 采用分阶段悬浮培养法,首先诱导nthESC 形成拟胚体(EB),在此过程中添加BMP4 和FGF-1。第二阶段,打散EB,再添加VEGF 和其他细胞因子,对HSC 进行扩增。结果发现,采用低黏附板可有效促进EB 形成,BMP4 和FGF-1 可使EB 形成数量明显增加,可使KDR+和CD133+荧光细胞数量明显增多。添加VEGF 等细胞因子,可有效促进CD34+细胞数量增加。结果表明,联合使用BMP4、FGF-1、VEGF 和SCF 等细胞因子,可使悬浮培养形成EB 数量增加,产生更多的造血前体细胞,继而产生大量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.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R329.2

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