人胚胎干細(xì)胞向造血細(xì)胞誘導(dǎo)分化的研究
發(fā)布時(shí)間：2018-07-13 14:45
【摘要】： 血細(xì)胞輸注和造血干細(xì)胞(hematopoietic stem cells ,HSCs)移植是細(xì)胞治療的常用手段,可用來治療惡性血液疾病、感染性疾病、遺傳性疾病、重癥免疫缺陷、AIDS等多種疾病。然而目前血細(xì)胞來源緊張及污染問題給其臨床使用的安全性和廣泛性帶來了極大挑戰(zhàn)。因此人們期望更為安全、有效和經(jīng)濟(jì)的血細(xì)胞資源。隨著干細(xì)胞研究及其相關(guān)領(lǐng)域的生物學(xué)技術(shù)的快速發(fā)展,以人胚胎干細(xì)胞(human embryonic stem cells,hESCs)為啟動細(xì)胞的造血干細(xì)胞工程為臨床輸血和干細(xì)胞的移植帶來了新希望。 目前,研究者主要使用造血發(fā)育相關(guān)因子或造血微環(huán)境的基質(zhì)細(xì)胞促進(jìn)人胚胎干細(xì)胞向造血細(xì)胞分化。雖然上述誘導(dǎo)方法在誘導(dǎo)胚胎干細(xì)胞向造血細(xì)胞分化上取得了良好的效果,但仍然存在著一些難以克服的局限性:鼠源飼養(yǎng)層是目前常用的較為高效的誘導(dǎo)方法;然而鼠源飼養(yǎng)層所帶來的異源污染極大地限制了該研究的臨床應(yīng)用前景。誘導(dǎo)中使用的各種細(xì)胞因子多為基因工程產(chǎn)品,價(jià)格昂貴,可在研究中使用卻不能大量推廣。因此我們希望建立一種新的誘導(dǎo)方案既可以高效誘導(dǎo)人胚胎干細(xì)胞向造血細(xì)胞分化,又可以避免異源污染,同時(shí)可降低實(shí)驗(yàn)成本。 造血發(fā)生經(jīng)歷3個(gè)階段:卵黃囊造血、胎肝造血和骨髓造血。人卵黃囊造血時(shí)期為4～6周;胎肝造血時(shí)期為6～22周;骨髓造血從22周至出生。在胚胎發(fā)育的不同階段,不同的造血微環(huán)境對造血發(fā)生發(fā)揮著重要作用。在利用ESCs體外定向分化為造血細(xì)胞的模型中,誘導(dǎo)條件(微環(huán)境)的選擇策略是研究造血發(fā)生和分化調(diào)控機(jī)制的重要環(huán)節(jié)。胎肝是造血發(fā)育的主要位點(diǎn),該組織的微環(huán)境為造血細(xì)胞的生成提供了必要的條件。理論上來說胎肝造血微環(huán)境對胚胎干細(xì)胞向造血細(xì)胞的分化應(yīng)該會有很好的誘導(dǎo)支持作用。因此在本實(shí)驗(yàn)中我們模擬胎肝造血微環(huán)境,聯(lián)合使用15周人胎肝基質(zhì)細(xì)胞和人胎肝組織提取物誘導(dǎo)人胚胎干細(xì)胞分化為造血細(xì)胞,并比較該方法與人胎肝基質(zhì)細(xì)胞誘導(dǎo)法及人胎肝基質(zhì)細(xì)胞/細(xì)胞因子誘導(dǎo)法之間的差別,旨在建立一種高效安全的造血細(xì)胞誘導(dǎo)體系。 首先,我們分離15周人流產(chǎn)胎兒胎肝基質(zhì)細(xì)胞并制備胎肝組織細(xì)胞提取物,使用半定量RT-PCR和流式細(xì)胞技術(shù)分析了胎肝基質(zhì)細(xì)胞表面標(biāo)志及基因表達(dá)情況。結(jié)果表明人胎肝基質(zhì)細(xì)胞表達(dá)間質(zhì)細(xì)胞表面標(biāo)志CD90、CD29,不表達(dá)造血細(xì)胞表面標(biāo)志CD34和CD45;此外,人胎肝基質(zhì)細(xì)胞表達(dá)造血發(fā)育支持因子EPO、Flt-3和SCF,但隨著傳代時(shí)間的延長這些因子的表達(dá)水平逐漸降低。由此我們選擇前3代的胎肝基質(zhì)細(xì)胞作為胚胎干細(xì)胞造血誘導(dǎo)的飼養(yǎng)層。 之后,我們將人胚胎干細(xì)胞培養(yǎng)于低黏附性培養(yǎng)皿中誘導(dǎo)形成擬胚體(human embryoid bodies ,hEBs),并用骨形成蛋白4(Bone morphogenetic proteins 4, BMP4)對擬胚體進(jìn)行了處理,通過檢測擬胚體向中胚層發(fā)育的情況,選擇了適當(dāng)培養(yǎng)天數(shù)的擬胚體,采用三種不同的誘導(dǎo)方法進(jìn)行誘導(dǎo)分化。在誘導(dǎo)的過程中,我們收集不同培養(yǎng)天數(shù)的細(xì)胞,應(yīng)用流式細(xì)胞技術(shù)檢測了分化體系中CD34、CD45等造血表面抗原的表達(dá)情況,以確定各組的造血分化情況。結(jié)果表明三種不同的誘導(dǎo)方法均能促進(jìn)人胚胎干細(xì)胞向造血細(xì)胞定向分化。但在不添加細(xì)胞因子和細(xì)胞提取物的情況下,胎肝基質(zhì)細(xì)胞誘導(dǎo)人胚胎干細(xì)胞向造血細(xì)胞分化的能力較弱,在誘導(dǎo)的10天中CD34+細(xì)胞比率與CD45+細(xì)胞比率均低于10%。在添加細(xì)胞因子之后,人胚胎干細(xì)胞向造血細(xì)胞分化的能力明顯增強(qiáng),其中CD34+細(xì)胞最高可達(dá)24.68%, CD45+細(xì)胞最高可達(dá)13.57%。聯(lián)合使用胎肝基質(zhì)細(xì)胞與胎肝細(xì)胞提取物則能更為高效的誘導(dǎo)人胚胎干向造血細(xì)胞發(fā)育,其中CD34+細(xì)胞最高可達(dá)32.73%, CD45+細(xì)胞最高可達(dá)27.96%。此外,我們收集誘導(dǎo)10天的細(xì)胞,應(yīng)用RT-PCR檢測了細(xì)胞造血相關(guān)基因的表達(dá)變化情況;應(yīng)用克隆形成實(shí)驗(yàn)檢測了細(xì)胞的造血克隆形成能力;應(yīng)用Wright—Giemsa染色觀測了細(xì)胞的形態(tài);應(yīng)用免疫熒光染色技術(shù)檢測了誘導(dǎo)集落造血表面抗原的表達(dá)情況。結(jié)果發(fā)現(xiàn),誘導(dǎo)產(chǎn)生的細(xì)胞具有造血細(xì)胞的一般特性,表達(dá)造血相關(guān)基因AML、SCL、GATA-1;在半固體培養(yǎng)基中可分化為不同種類的造血克隆。但不同誘導(dǎo)體系中產(chǎn)生的細(xì)胞造血基因的表達(dá)量與造血克隆的生成能力不同,胎肝基質(zhì)細(xì)胞/胎肝細(xì)胞提取物體系產(chǎn)生細(xì)胞AML、SCL、GATA-1的表達(dá)量及形成的造血克隆最多,且以紅系克隆為主。在上述研究的基礎(chǔ)上,我們進(jìn)一步探討造血祖細(xì)胞向紅系發(fā)育的機(jī)制,并建立了高效的造血祖細(xì)胞向紅細(xì)胞的分化體系,為以胚胎干細(xì)胞為啟動細(xì)胞誘導(dǎo)分化規(guī)�；漠a(chǎn)生紅細(xì)胞奠定一定的基礎(chǔ)。紅細(xì)胞的產(chǎn)生受到造血因子和造血干細(xì)胞自身內(nèi)在基因的共同調(diào)控,其中促紅細(xì)胞生成素(erythropoietin,EPO)是其產(chǎn)生的最重要因子。細(xì)胞因子信號轉(zhuǎn)導(dǎo)抑制因子-3(suppressor of cytokine signaling-3 ,SOCS-3)最早發(fā)現(xiàn)于1997年,有研究證明EPO受體上有SOCS-3的高親和結(jié)合位點(diǎn),SOCS-3對EPO具有負(fù)調(diào)控作用。因此,我們推測降低胚胎干細(xì)胞或造血祖細(xì)胞中SOCS-3基因的表達(dá)水平有利于它們向紅系的發(fā)育。在本實(shí)驗(yàn)中我們選擇具有造血干細(xì)胞特性的人紅白血病細(xì)胞株K562作為研究對象,構(gòu)建了SOCS-3慢病毒siRNA干涉載體,轉(zhuǎn)染K562細(xì)胞。根據(jù)綠色熒光蛋白的表達(dá)進(jìn)行流式分選后,我們獲得了高表達(dá)慢病毒干涉載體的細(xì)胞。實(shí)時(shí)熒光定量PCR和Western-blot檢測了轉(zhuǎn)染細(xì)胞中SOCS-3基因的干涉效率,結(jié)果顯示與對照組相比,siRNA干涉后K562細(xì)胞SOCS-3基因的表達(dá)量僅為其相對表達(dá)量的22.1%,干涉效率77.9%;Western -blot結(jié)果顯示SOCS-3在蛋白質(zhì)水平表達(dá)也明顯受抑制。我們進(jìn)一步對SOCS-3基因沉默后的K562細(xì)胞進(jìn)行了誘導(dǎo)分化,并采用聯(lián)苯胺染色法檢測K562細(xì)胞向紅系分化比例變化,免疫熒光染色檢測細(xì)胞表面抗原的變化,RT-PCR檢測造血相關(guān)基因的變化,發(fā)現(xiàn)SOCS-3沉默后K562細(xì)胞向紅系的發(fā)育能力顯著提高。 綜上所述,在本研究中我們采用三種不同方法誘導(dǎo)人胚胎干細(xì)胞分化為造血細(xì)胞,并證明聯(lián)合使用胎肝基質(zhì)細(xì)胞和胎肝組織細(xì)胞提取物是較好的誘導(dǎo)方法,該方法可高效誘導(dǎo)胚胎干細(xì)胞向造血細(xì)胞分化,且避免了鼠源飼養(yǎng)層所帶來的異源污染,降低了實(shí)驗(yàn)成本。在此基礎(chǔ)上我們通過構(gòu)建慢病毒干涉載體的方法,穩(wěn)定干涉了K562細(xì)胞中SOCS-3表達(dá),建立了高效的造血祖細(xì)胞向紅系細(xì)胞誘導(dǎo)體系,證明了SOCS-3基因沉默有利于造血祖細(xì)胞向紅系的發(fā)育。上述研究為深入探討造血細(xì)胞發(fā)育調(diào)控機(jī)制以及以胚胎干細(xì)胞或造血干細(xì)胞為啟動細(xì)胞大規(guī)模的誘導(dǎo)產(chǎn)生紅細(xì)胞體系的建立奠定了基礎(chǔ)。
[Abstract]:Blood cell infusion and hematopoietic stem cells (HSCs) transplantation are the common means of cell therapy, which can be used to treat many diseases, such as malignant blood diseases, infectious diseases, hereditary diseases, severe immunodeficiency, AIDS and so on. However, the problem of blood cell source tension and pollution to the clinical use is safe and extensive. It brings great challenges. So people expect more safe, effective and economical blood cells. With the rapid development of stem cell research and its related fields, human embryonic stem cells (human embryonic stem cells, hESCs) for the hematopoietic stem cell engineering that start the cells for clinical transfusions and stem cell transplantation New hope.
At present, researchers mainly use hematopoietic growth related factors or stromal cells in hematopoietic microenvironment to promote human embryonic stem cells to differentiate into hematopoietic cells. Although the afore-mentioned induction methods have achieved good results in inducing embryonic stem cells to differentiate into hematopoietic cells, there are still some insuperable limitations: the rat feeder layer is the eye. However, the heterologous pollution caused by the rat feeder layer greatly restricts the prospect of the clinical application. The various cytokines used in the induction are mostly genetic engineering products, which are expensive and can not be widely used in the study. Therefore, we hope to establish a new induction scheme. It can effectively induce the differentiation of human embryonic stem cells into hematopoietic cells and avoid heterogenous pollution, and at the same time, it can reduce the cost of experiments.
Hematopoiesis occurs in 3 stages: yolk sac hematopoiesis, fetal liver hematopoiesis and marrow hematopoiesis. Human yolk sac hematopoiesis period is 4~6 weeks; fetal liver hematopoiesis period is 6~22 weeks; bone marrow hematopoiesis is from 22 weeks to birth. In different stages of embryo development, different hematopoietic microenvironment plays an important role in hematopoiesis. The differentiation of hematopoiesis in vitro by ESCs is made in vitro. In the blood cell model, the selection strategy of the induction condition (microenvironment) is an important link in the study of the regulation mechanism of hematopoiesis and differentiation. Fetal liver is the main locus of hematopoiesis, and the microenvironment of this tissue provides the necessary conditions for the generation of hematopoietic cells. In this experiment, we simulated fetal liver hematopoietic microenvironment, combined with 15 weeks human fetal liver stromal cells and human fetal liver tissues to induce human embryonic stem cells to differentiate into hematopoietic cells, and compare this method with human fetal liver stromal cell induction and human fetal liver stromal cells / cell causes. The difference between sub induction methods is to establish an efficient and safe induction system for hematopoietic cells.
First, we isolated fetal liver stromal cells and prepared fetal liver tissue extracts for 15 weeks. The surface markers and gene expressions of fetal liver stromal cells were analyzed by semi quantitative RT-PCR and flow cytometry. The results showed that human fetal liver stromal cells expressed CD90, CD29, and no expression of hematopoietic cell surface. In addition, human fetal liver stromal cells express hematopoietic growth support factor EPO, Flt-3 and SCF, but the expression level of these factors gradually decreases with the prolongation of passage time. Therefore, we choose fetal liver stromal cells in the first 3 generations as the feeding layer induced by embryonic stem cell hematopoiesis.
After that, we induce the human embryonic stem cells to form the human embryoid bodies (hEBs) in the low adhesion culture dish, and use the bone forming protein 4 (Bone morphogenetic proteins 4, BMP4) to deal with the embryoid body. By detecting the development of the mesoembryonic body to the middle embryo layer, the proper culture number of the pseudo embryoid body is selected. Three different induction methods were used to induce differentiation. In the process of induction, we collected the cells with different days of culture and detected the expression of CD34, CD45 and other hematopoietic surface antigens in the differentiation system by flow cytometry to determine the hematopoietic differentiation in each group. The results showed that three different induction methods could promote the human embryo. The fetal stem cells differentiate into hematopoietic cells, but without the addition of cytokines and cell extracts, the ability of fetal liver stromal cells to induce human embryonic stem cells to differentiate into hematopoietic cells is weak. The ratio of CD34+ cells to CD45+ cells in the 10 days of induction is lower than that of 10%. in the addition of cytokines. The ability of the differentiation of blood cells increased obviously, of which the highest CD34+ cells could reach 24.68%, the highest of CD45+ cells could reach 13.57%. and the combination of fetal liver stromal cells and fetal hepatocyte extract could induce the development of human embryonic stem to hematopoietic cells more efficiently, of which the maximum of CD34+ cells was up to 32.73%, and the maximum of CD45+ cells could reach 27.96%.. The cells which were induced for 10 days were collected and the expression of hematopoiesis related genes was detected by RT-PCR. The ability of the hematopoietic clone formation was detected by the cloning and formation test. The morphology of the cells was observed by Wright Giemsa staining, and the expression of the induced colony hematopoietic surface antigen was detected by immunofluorescence staining. The results showed that the induced cells had general characteristics of hematopoietic cells, expressed hematopoietic related genes AML, SCL, GATA-1, and differentiated into different kinds of hematopoietic clones in the semisolid medium, but the expression of hematopoietic genes produced in different induction systems was different from that of hematopoietic clones, and the fetal liver stromal cells / fetal liver were fine. The cell extract system produces AML, SCL, GATA-1 and the most hematopoietic clones, which are mainly red clones. On the basis of the above study, we further explore the mechanism of hematopoietic progenitor cells to the erythroid development, and establish a highly efficient hematopoietic progenitor cell differentiation system for the initiation of embryonic stem cells. The production of erythrocytes is controlled by the co regulation of hematopoietic and hematopoietic stem cells's own intrinsic genes, in which erythropoietin (erythropoietin, EPO) is the most important factor in its production. Cytokine signal transduction inhibitor -3 (suppressor of cytokine signali) NG-3, SOCS-3) was first discovered in 1997. Studies have shown that the EPO receptor has a high affinity binding site for SOCS-3, and SOCS-3 has a negative regulatory effect on EPO. Therefore, we speculate that the reduction of the expression level of the SOCS-3 gene in embryonic stem cells or hematopoietic progenitor cells is beneficial to the development of their erythroid system. In this experiment, we chose to have hematopoietic stem cells. The cell characteristic human erythroleukemia cell line K562 was used as the research object to construct the SOCS-3 lentivirus siRNA interference carrier and transfect the K562 cells. We obtained the cells with high expression of the lentivirus interference carrier based on the expression of the green fluorescent protein. The real-time fluorescence quantitative PCR and Western-blot were used to detect the SOCS-3 base in the transfected cells. The results showed that the expression of SOCS-3 gene in K562 cells after siRNA interference was only 22.1% of the relative expression and 77.9% in the interference efficiency compared with the control group, and the Western -blot results showed that the expression of SOCS-3 at the protein level was also obviously inhibited. We further induced the differentiation of K562 cells after the SOCS-3 gene silencing, The differentiation ratio of K562 cells to erythroid differentiation was detected by diphenyl amine staining, and the changes of cell surface antigen were detected by immunofluorescence staining. The changes of hematopoiesis related genes were detected by RT-PCR. It was found that the development ability of K562 cells to the red system was significantly improved after SOCS-3 silencing.
In summary, in this study, we used three different methods to induce human embryonic stem cells to differentiate into hematopoietic cells, and proved that combined use of fetal liver stromal cells and fetal liver tissue cells extracts is a good induction method. This method can effectively induce embryonic stem cells to differentiate into hematopoietic cells and avoid the result of rat feeder layer. Heterogenous pollution reduces the cost of experiment. On this basis, we establish a highly efficient hematopoietic progenitor cell induction system by interfering with the expression of SOCS-3 in K562 cells by constructing the lentivirus interferometric vector. It is proved that the silence of SOCS-3 gene is beneficial to the development of hematopoietic progenitor cells. The mechanism of the regulation of hematopoietic cell development and the establishment of a large-scale induction of erythrocyte system by embryonic stem cells or hematopoietic stem cells is the basis for the establishment of a red cell system.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R329
[Abstract]:Blood cell infusion and hematopoietic stem cells (HSCs) transplantation are the common means of cell therapy, which can be used to treat many diseases, such as malignant blood diseases, infectious diseases, hereditary diseases, severe immunodeficiency, AIDS and so on. However, the problem of blood cell source tension and pollution to the clinical use is safe and extensive. It brings great challenges. So people expect more safe, effective and economical blood cells. With the rapid development of stem cell research and its related fields, human embryonic stem cells (human embryonic stem cells, hESCs) for the hematopoietic stem cell engineering that start the cells for clinical transfusions and stem cell transplantation New hope.
At present, researchers mainly use hematopoietic growth related factors or stromal cells in hematopoietic microenvironment to promote human embryonic stem cells to differentiate into hematopoietic cells. Although the afore-mentioned induction methods have achieved good results in inducing embryonic stem cells to differentiate into hematopoietic cells, there are still some insuperable limitations: the rat feeder layer is the eye. However, the heterologous pollution caused by the rat feeder layer greatly restricts the prospect of the clinical application. The various cytokines used in the induction are mostly genetic engineering products, which are expensive and can not be widely used in the study. Therefore, we hope to establish a new induction scheme. It can effectively induce the differentiation of human embryonic stem cells into hematopoietic cells and avoid heterogenous pollution, and at the same time, it can reduce the cost of experiments.
Hematopoiesis occurs in 3 stages: yolk sac hematopoiesis, fetal liver hematopoiesis and marrow hematopoiesis. Human yolk sac hematopoiesis period is 4~6 weeks; fetal liver hematopoiesis period is 6~22 weeks; bone marrow hematopoiesis is from 22 weeks to birth. In different stages of embryo development, different hematopoietic microenvironment plays an important role in hematopoiesis. The differentiation of hematopoiesis in vitro by ESCs is made in vitro. In the blood cell model, the selection strategy of the induction condition (microenvironment) is an important link in the study of the regulation mechanism of hematopoiesis and differentiation. Fetal liver is the main locus of hematopoiesis, and the microenvironment of this tissue provides the necessary conditions for the generation of hematopoietic cells. In this experiment, we simulated fetal liver hematopoietic microenvironment, combined with 15 weeks human fetal liver stromal cells and human fetal liver tissues to induce human embryonic stem cells to differentiate into hematopoietic cells, and compare this method with human fetal liver stromal cell induction and human fetal liver stromal cells / cell causes. The difference between sub induction methods is to establish an efficient and safe induction system for hematopoietic cells.
First, we isolated fetal liver stromal cells and prepared fetal liver tissue extracts for 15 weeks. The surface markers and gene expressions of fetal liver stromal cells were analyzed by semi quantitative RT-PCR and flow cytometry. The results showed that human fetal liver stromal cells expressed CD90, CD29, and no expression of hematopoietic cell surface. In addition, human fetal liver stromal cells express hematopoietic growth support factor EPO, Flt-3 and SCF, but the expression level of these factors gradually decreases with the prolongation of passage time. Therefore, we choose fetal liver stromal cells in the first 3 generations as the feeding layer induced by embryonic stem cell hematopoiesis.
After that, we induce the human embryonic stem cells to form the human embryoid bodies (hEBs) in the low adhesion culture dish, and use the bone forming protein 4 (Bone morphogenetic proteins 4, BMP4) to deal with the embryoid body. By detecting the development of the mesoembryonic body to the middle embryo layer, the proper culture number of the pseudo embryoid body is selected. Three different induction methods were used to induce differentiation. In the process of induction, we collected the cells with different days of culture and detected the expression of CD34, CD45 and other hematopoietic surface antigens in the differentiation system by flow cytometry to determine the hematopoietic differentiation in each group. The results showed that three different induction methods could promote the human embryo. The fetal stem cells differentiate into hematopoietic cells, but without the addition of cytokines and cell extracts, the ability of fetal liver stromal cells to induce human embryonic stem cells to differentiate into hematopoietic cells is weak. The ratio of CD34+ cells to CD45+ cells in the 10 days of induction is lower than that of 10%. in the addition of cytokines. The ability of the differentiation of blood cells increased obviously, of which the highest CD34+ cells could reach 24.68%, the highest of CD45+ cells could reach 13.57%. and the combination of fetal liver stromal cells and fetal hepatocyte extract could induce the development of human embryonic stem to hematopoietic cells more efficiently, of which the maximum of CD34+ cells was up to 32.73%, and the maximum of CD45+ cells could reach 27.96%.. The cells which were induced for 10 days were collected and the expression of hematopoiesis related genes was detected by RT-PCR. The ability of the hematopoietic clone formation was detected by the cloning and formation test. The morphology of the cells was observed by Wright Giemsa staining, and the expression of the induced colony hematopoietic surface antigen was detected by immunofluorescence staining. The results showed that the induced cells had general characteristics of hematopoietic cells, expressed hematopoietic related genes AML, SCL, GATA-1, and differentiated into different kinds of hematopoietic clones in the semisolid medium, but the expression of hematopoietic genes produced in different induction systems was different from that of hematopoietic clones, and the fetal liver stromal cells / fetal liver were fine. The cell extract system produces AML, SCL, GATA-1 and the most hematopoietic clones, which are mainly red clones. On the basis of the above study, we further explore the mechanism of hematopoietic progenitor cells to the erythroid development, and establish a highly efficient hematopoietic progenitor cell differentiation system for the initiation of embryonic stem cells. The production of erythrocytes is controlled by the co regulation of hematopoietic and hematopoietic stem cells's own intrinsic genes, in which erythropoietin (erythropoietin, EPO) is the most important factor in its production. Cytokine signal transduction inhibitor -3 (suppressor of cytokine signali) NG-3, SOCS-3) was first discovered in 1997. Studies have shown that the EPO receptor has a high affinity binding site for SOCS-3, and SOCS-3 has a negative regulatory effect on EPO. Therefore, we speculate that the reduction of the expression level of the SOCS-3 gene in embryonic stem cells or hematopoietic progenitor cells is beneficial to the development of their erythroid system. In this experiment, we chose to have hematopoietic stem cells. The cell characteristic human erythroleukemia cell line K562 was used as the research object to construct the SOCS-3 lentivirus siRNA interference carrier and transfect the K562 cells. We obtained the cells with high expression of the lentivirus interference carrier based on the expression of the green fluorescent protein. The real-time fluorescence quantitative PCR and Western-blot were used to detect the SOCS-3 base in the transfected cells. The results showed that the expression of SOCS-3 gene in K562 cells after siRNA interference was only 22.1% of the relative expression and 77.9% in the interference efficiency compared with the control group, and the Western -blot results showed that the expression of SOCS-3 at the protein level was also obviously inhibited. We further induced the differentiation of K562 cells after the SOCS-3 gene silencing, The differentiation ratio of K562 cells to erythroid differentiation was detected by diphenyl amine staining, and the changes of cell surface antigen were detected by immunofluorescence staining. The changes of hematopoiesis related genes were detected by RT-PCR. It was found that the development ability of K562 cells to the red system was significantly improved after SOCS-3 silencing.
In summary, in this study, we used three different methods to induce human embryonic stem cells to differentiate into hematopoietic cells, and proved that combined use of fetal liver stromal cells and fetal liver tissue cells extracts is a good induction method. This method can effectively induce embryonic stem cells to differentiate into hematopoietic cells and avoid the result of rat feeder layer. Heterogenous pollution reduces the cost of experiment. On this basis, we establish a highly efficient hematopoietic progenitor cell induction system by interfering with the expression of SOCS-3 in K562 cells by constructing the lentivirus interferometric vector. It is proved that the silence of SOCS-3 gene is beneficial to the development of hematopoietic progenitor cells. The mechanism of the regulation of hematopoietic cell development and the establishment of a large-scale induction of erythrocyte system by embryonic stem cells or hematopoietic stem cells is the basis for the establishment of a red cell system.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R329
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相關(guān)期刊論文 前3條
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