【摘要】：背景:視網(wǎng)膜色素變性(retinitis pigmentosa,RP)是一類以感光細(xì)胞凋亡為特征的遺傳性致盲性眼病,目前尚無治愈方法,通過移植干細(xì)胞來源的感光細(xì)胞或視網(wǎng)膜色素上皮細(xì)胞成為RP治療最有希望的新方法之一。視網(wǎng)膜是免疫豁免器官,加之玻璃體視網(wǎng)膜手術(shù)進(jìn)行細(xì)胞移植是成熟技術(shù),細(xì)胞移植后的視網(wǎng)膜功能和植入細(xì)胞的情況可以通過無創(chuàng)方法進(jìn)行檢測(cè),使得眼睛成為細(xì)胞移植研究的理想器官。受制于倫理等因素,目前涉及從直接從人體組織獲得干細(xì)胞進(jìn)行臨床研究的進(jìn)展較緩慢。人胚胎干細(xì)胞(human embryonic stem cells,hESCs)具有向內(nèi)中外三胚層分化的潛能,是一類理想的種子細(xì)胞。體外三維誘導(dǎo)hESCs形成類器官的方法較好地解決了種子細(xì)胞來源問題,同時(shí),由于三維誘導(dǎo)hESCs形成類器官的過程很好地重演了胚胎發(fā)育發(fā)育過程中器官發(fā)生的過程,已經(jīng)使其成為發(fā)育學(xué)研究的良好模型。目前,利用三維培養(yǎng)離體誘導(dǎo)hESCs已經(jīng)可以形成包括視杯在內(nèi)的多種器官組織,但三維培養(yǎng)形成神經(jīng)視網(wǎng)膜(neural retina,NR)仍存在有以下幾點(diǎn)問題亟待解決:1、hESCs三維誘導(dǎo)形成的NR與在體發(fā)育過程中形成的NR仍存在結(jié)構(gòu)和功能的差異;2、影響三維培養(yǎng)hESCs所形成的NR的因素不明,誘導(dǎo)效率偏低。此外,由于誘導(dǎo)形成的NR尚處發(fā)育階段,其內(nèi)細(xì)胞組分混雜,缺乏特異膜標(biāo)記物直接定位和分選視網(wǎng)膜前體細(xì)胞(retinal progenitor cells,RPCs)�；诖�,選擇合適的手段改善三維培養(yǎng)方法并選擇恰當(dāng)?shù)哪?biāo)記物分離細(xì)胞是當(dāng)下研究的關(guān)鍵點(diǎn)。氧對(duì)生命的維系至關(guān)重要,一方面,生命體通過氧化磷酸化的有氧呼吸方式大量產(chǎn)生ATP保障了生命活動(dòng)的高效運(yùn)轉(zhuǎn);另一方面,機(jī)體發(fā)育伴隨了外環(huán)境氧濃度的變化,而干細(xì)胞的命運(yùn)與氧濃度的高低密切相關(guān)。通常認(rèn)為:低氧是決定干細(xì)胞調(diào)節(jié)自我更新以及維持干性的重要因素;高氧則會(huì)促進(jìn)干細(xì)胞的分化。三維誘導(dǎo)hESCs形成NR的過程通常都在常氧條件下進(jìn)行,但胚胎發(fā)育過程中,中樞神經(jīng)系統(tǒng)發(fā)育先于心血管系統(tǒng),因此在血液循環(huán)建立前后,視網(wǎng)膜的發(fā)育會(huì)經(jīng)歷一個(gè)氧濃度由低升高的過程。目前,大多數(shù)三維培養(yǎng)沒有涉及氧濃度的轉(zhuǎn)變過程。此外,理想的可移植細(xì)胞應(yīng)具備以下條件:1、處于前體細(xì)胞階段,便于移植前離體擴(kuò)增以及移植后分化為目的細(xì)胞;2、無胚胎源性,不會(huì)在移植入器官后無限增殖形成畸胎瘤。研究發(fā)現(xiàn),器官來源的絡(luò)氨酸蛋白激酶受體(tyrosine-protein kinase Kit,C-Kit)陽性細(xì)胞是一類具有自我更新和分化潛能的干細(xì)胞,將其移植入對(duì)應(yīng)的器官可發(fā)揮組織修復(fù)功能。而階段特異的胚胎抗原4 (stage-specific embryonic antigen 4, SSEA4)為人器官組織在胚胎階段表達(dá)的表面標(biāo)記物,SSEA4將有利于鑒別胚胎源性的細(xì)胞。目的:hESCs三維培養(yǎng)以探究其形成的NR的細(xì)胞和組織構(gòu)筑特點(diǎn);研究氧濃度升高對(duì)三維誘導(dǎo)hESCs形成NR效率的影響,從而通過調(diào)控氧濃度改良三維誘導(dǎo)方法;研究從三維誘導(dǎo)hESCs形成NR中分離的C-Kit+/SSEA4-細(xì)胞的生物學(xué)特點(diǎn)以及低氧對(duì)其干性維持的影響,進(jìn)而獲取狀態(tài)良好和足夠數(shù)量的RPCs;利用全基因譜掃描分析從三維誘導(dǎo)hESCs形成NR分離的C-Kit+/SSEA4-細(xì)胞的基因表達(dá)譜,全面深入了解該細(xì)胞的特點(diǎn)。方法與結(jié)果:本研究分為三個(gè)部分:第一部分:hESCs的三維培養(yǎng)及神經(jīng)視網(wǎng)膜的自發(fā)形成1、利用細(xì)胞免疫熒光、流式細(xì)胞分析研究hESCs細(xì)胞系H1的細(xì)胞特性。發(fā)現(xiàn)H1-hESCs成克隆聚集樣生長,核分裂相明顯。高達(dá)95%以上的細(xì)胞表達(dá)胚胎干細(xì)胞標(biāo)志物 OCT4, SOX2 和 SSEA4。2、通過免疫熒光染色鑒定三維誘導(dǎo)hESCs形成的視泡、視杯樣結(jié)構(gòu)�？梢姅M胚體(embryonic bodies, EBs)在8天左右即可形成視泡樣結(jié)構(gòu),繼續(xù)培養(yǎng)24天后,視泡的形態(tài)更加清晰,并可觀察到雙層凹陷的視杯樣結(jié)構(gòu),三維誘導(dǎo)38D的hESCs來源NR可表達(dá)增殖標(biāo)記物Ki67; RPCs標(biāo)記物:RAX、PAX6、CHX10;神經(jīng)前體細(xì)胞標(biāo)記物:SOX2、NESTIN;神經(jīng)節(jié)細(xì)胞(retinal ganglion cells, RGCs)標(biāo)記物 Tuj1及感光前體細(xì)胞標(biāo)記物Crx,其中RAX在全神經(jīng)視網(wǎng)膜均有表達(dá)。第二部分:氧濃度對(duì)三維誘導(dǎo)hESCs形成神經(jīng)視網(wǎng)膜發(fā)育的影響1、利用免疫熒光染色分析高氧對(duì)三維誘導(dǎo)hESCs自發(fā)形成NR的影響,發(fā)現(xiàn)40%O2的高氧可以明顯促進(jìn)神經(jīng)視網(wǎng)膜內(nèi)細(xì)胞的增殖,且40%O2組中NR內(nèi)增殖細(xì)胞在尖-底(apical-basal)端兩側(cè)間遷移增加,高氧可促進(jìn)細(xì)胞核動(dòng)態(tài)遷移(Interkinetic nuclear migration)的發(fā)生。2、利用免疫熒光染色研究高氧對(duì)三維誘導(dǎo)hESCs自發(fā)形成NR中神經(jīng)玫瑰花節(jié)(neural rossetes)尖-底端極性的影響,發(fā)現(xiàn)在20%O2組中,神經(jīng)玫瑰花結(jié)內(nèi)側(cè)尖端面會(huì)翻轉(zhuǎn)至外側(cè),而40%O2組中神經(jīng)玫瑰花節(jié)可按照正常生理的尖-底端極性發(fā)育。3、利用免疫熒光染色研究高氧對(duì)三維誘導(dǎo)hESCs自發(fā)形成NR中RPCs的影響,發(fā)現(xiàn)氧濃度變化并不會(huì)改變神經(jīng)視網(wǎng)膜標(biāo)記物的表達(dá)模式,但40%O2下PAX6陽性視網(wǎng)膜祖細(xì)胞數(shù)量較20%O2顯著增加,RPCs向神經(jīng)內(nèi)層遷移更為明顯,且40%O2顯著促進(jìn)RGCs的形成。相比20%O2的常氧處理,高氧下三維誘導(dǎo)hESCs形成的RGCs的突起更長,向基底側(cè)遷移增加。第三部分:氧濃度對(duì)hESCs三維誘導(dǎo)NR來源C-Kit+/SSEA4- (hESC-NR-C-Kit+/SSEA4-)細(xì)胞的增殖的影響及其生物學(xué)特性分析1、利用改進(jìn)的三維誘導(dǎo)方法誘導(dǎo)hESCs形成NR,研究氧濃度的影響對(duì)NR標(biāo)記物表達(dá)的影響,發(fā)現(xiàn)改進(jìn)的三維誘導(dǎo)方法所得EBs形成相對(duì)較慢,其形成的NR細(xì)胞與組織構(gòu)筑特點(diǎn)沒有顯著變化,高氧對(duì)EBs生長仍有促進(jìn)作用。2、利用免疫熒光染色研究改進(jìn)的三維誘導(dǎo)hESCs形成NR中C-Kit的表達(dá)的時(shí)空分布特點(diǎn),發(fā)現(xiàn)C-Kit陽性細(xì)胞主要分布于神經(jīng)視網(wǎng)膜的內(nèi)層,C-Kit陽性細(xì)胞同時(shí)表達(dá)干細(xì)胞標(biāo)記Nestin、PAX6、RAX,但不表達(dá)CHX10。隨著誘導(dǎo)時(shí)間的進(jìn)展,NR內(nèi)C-Kit的表達(dá)水平不斷下降。3、利用細(xì)胞生存實(shí)驗(yàn)分析hESC-NR-C-Kit+/SSEA4-細(xì)胞增殖特性,發(fā)現(xiàn)hESC-NR-C-Kit+/SSEA4-細(xì)胞在3%O2的低氧下細(xì)胞增殖明顯增加,在誘導(dǎo)30D、45D及60D分選細(xì)胞,以30D分選的細(xì)胞增殖活性最好。4、利用細(xì)胞免疫熒光染色,研究hESC-NR-C-Kit+/SSEA4的-細(xì)胞的特性,發(fā)現(xiàn)hESC-NR-C-Kit+/SSEA4-細(xì)胞可以表達(dá)包括 Nestin、PAX6、RAX 等多種 RPCs 的標(biāo)記物和增殖標(biāo)記物Ki67,且可以誘導(dǎo)分化形成RGCs、雙極細(xì)胞、感光細(xì)胞及Muller細(xì)胞。5、利用全基因組轉(zhuǎn)錄譜表達(dá)分析,研究hESC-NR-C-Kit+/SSEA4-細(xì)胞的基因表達(dá)譜特點(diǎn),發(fā)現(xiàn)30D、45D及60D分選的hESC-NR-C-Kit+/SSEA4-視網(wǎng)膜前體細(xì)胞與人胚眼分離的RPCs之間的Pearson相關(guān)系數(shù)均在0.88以上,且以30D最高(0.908),hESC-NR-C-Kit+/SSEA4-細(xì)胞較RPCs增殖和遷移相關(guān)基因表達(dá)增加,p53信號(hào)通路激活增加,但細(xì)胞粘附分子通路激活降低。結(jié)論:1、三維誘導(dǎo)hESCs形成的NR可以表達(dá)各類視網(wǎng)膜干細(xì)胞標(biāo)記物,其發(fā)育類似在體視網(wǎng)膜發(fā)育過程。選用改進(jìn)的(BMP4介導(dǎo))三維誘導(dǎo)方法,其誘導(dǎo)過程操作簡單,NR形成更穩(wěn)定,表明改進(jìn)的三維誘導(dǎo)方法更利于后期臨床運(yùn)用。2、氧濃度升高對(duì)于三維誘導(dǎo)hESCs形成NR的主要影響有包括促進(jìn)NR的增殖、促進(jìn)神經(jīng)玫瑰花結(jié)按照正常生理的尖-底端極性發(fā)育、促進(jìn)PAX6陽性RPCs的形成和遷移以及促進(jìn)RGCs的形成、成熟和遷移。表明高氧處理有利于獲得更多、更接近在體發(fā)育的NR。3、低氧培養(yǎng)下,誘導(dǎo)30D分選的hESC-NR-C-Kit+/SSEA4-細(xì)胞的狀態(tài)和增殖活性較常氧培養(yǎng)有明顯改善,表明低氧培養(yǎng)有利于細(xì)胞的穩(wěn)定擴(kuò)增。4、三維誘導(dǎo)hESCs形成的NR中C-Kit陽性細(xì)胞可以同時(shí)表達(dá)Nestin、PAX6、RAX等視網(wǎng)膜干細(xì)胞標(biāo)記,但不表達(dá)胚胎抗原,可分化為感光細(xì)胞,雙極細(xì)胞,Muller 細(xì)胞及 RGCs,表明 hESC-NR-C-Kit+/SSEA4-細(xì)胞為一類 RPCs。5、基因組表達(dá)譜掃描結(jié)果顯示hESC-NR-C-Kit+/SSEA4-細(xì)胞與從人胚眼中分離的RPCs非常類似(90%),但其增殖和遷移相關(guān)基因表達(dá)高于RPCs,提示hESC-NR-C-Kit+/SSEA4-細(xì)胞是接近于在體發(fā)育的一類RPCs。為干細(xì)胞移植治療視網(wǎng)膜變性疾病提供了成瘤風(fēng)險(xiǎn)低、分化潛能好、有標(biāo)準(zhǔn)化產(chǎn)業(yè)化條件新的種子細(xì)胞。
[Abstract]:BACKGROUND: Retinal pigmentosa (RP) is a kind of hereditary blinding ophthalmopathy characterized by photoreceptor cell apoptosis. At present, there is no cure method. Transplantation of stem cell-derived photoreceptor cells or retinal pigment epithelial cells (RPE) is one of the most promising new methods for the treatment of RP. Cell transplantation in vitreoretinal surgery is a mature technique. The function of the retina and the condition of the implanted cells can be detected by non-invasive methods, making the eye an ideal organ for cell transplantation. Human embryonic stem cells (hESCs) have the potential to differentiate inward, outward and outward into three embryonic layers, and are a kind of ideal seed cells. The process of organogenesis in embryonic development has been reproduced, which has made it a good model for developmental research. At present, the induction of hESCs by three-dimensional culture in vitro has been able to form a variety of organs and tissues including optic cups. However, the formation of neural retina (NR) by three-dimensional culture still has the following problems to be solved urgently. Solutions: 1. There are still structural and functional differences between NR induced by hESCs and NR formed during in vivo development; 2. The factors affecting NR induced by hESCs are unknown and the induction efficiency is low. In addition, the NR induced by hESCs is still in the developmental stage, and there is no direct localization and sorting of specific membrane markers. Retinal progenitor cells (RPCs). Based on this, choosing appropriate methods to improve the three-dimensional culture method and selecting appropriate membrane markers to isolate cells is the key point of current research. On the other hand, the development of the body is accompanied by changes in oxygen concentration in the external environment, and the fate of stem cells is closely related to oxygen concentration. The process usually takes place under normal oxygen conditions, but during embryonic development, the central nervous system develops prior to the cardiovascular system, so the development of the retina undergoes a process from low to high oxygen concentration before and after the establishment of blood circulation. Cells should be in the precursor cell stage for in vitro expansion before transplantation and differentiation into target cells after transplantation. 2. There is no embryonic origin, and no infinite proliferation of teratoma after transplantation. Studies have found that organ-derived tyrosine-protein kinase Kit (C-Kit) positive cells are Stage-specific embryonic antigen 4 (SSEA4) is a surface marker expressed in human organs during the embryonic stage. SSEA4 will be helpful to identify embryonic cells. S three-dimensional culture was used to explore the cell and tissue architecture of NR; the effect of elevated oxygen concentration on NR formation efficiency of three-dimensional induction of hESCs was studied to improve the three-dimensional induction method by adjusting oxygen concentration; the biological characteristics of C-Kit+/SSEA4-cells isolated from three-dimensional induction of NR formation of hESCs and the maintenance of their dryness by hypoxia were studied. Methods and Results: This study was divided into three parts: the first part: three-dimensional culture of hESCs and neural retina. Spontaneous formation 1. Immunofluorescence and flow cytometry were used to study the cell characteristics of hESCs cell line H1. It was found that H1-hESCs grew in clonal aggregates with obvious mitotic phase. Visible embryonic bodies (EBs) in about 8 days to form the structure of the optic vesicles, continue to culture 24 days, the morphology of the optic vesicles more clear, and can be observed double-decked cup-like structure, three-dimensional induction of 38D hESCs source NR can express proliferation markers Ki67; RPCs markers: RAX, PAX6, CHX10; neural precursor cell markers; Notes: SOX2, NESTIN; retinal ganglion cells (RGCs) marker Tuj1 and photoreceptor precursor cell marker Crx, in which RAX was expressed in the whole nervous retina. Part II: Effect of oxygen concentration on the development of three-dimensional inducible hESCs to form neural retina 1. Immunofluorescence staining was used to analyze the effect of hyperoxia on three-dimensional inducible hESCs spontaneously. The effect of hyperoxia on the formation of NR was found to promote the proliferation of neuroretinal cells. In 40% O2 group, the proliferation of NR cells increased between apical-basal ends. Hyperoxia could promote the occurrence of nuclear migration. 2. Immunofluorescence staining was used to study the effect of hyperoxia on three-dimensional induction. The effect of nerve rossetes tip-to-bottom polarity on spontaneous formation of NR in hESCs was observed. In 20% O2 group, the medial tip of the nerve rossetes turned to the lateral side, while in 40% O2 group, the nerve rossetes developed according to the normal physiological tip-to-bottom polarity. 3. Immunofluorescence staining was used to study the effect of hyperoxia on three-dimensional induction of hESCs self-polarity. The changes of oxygen concentration did not change the expression pattern of neuroretinal markers, but the number of PAX6-positive retinal progenitor cells increased significantly in 40% O2 compared with 20% O2, and the migration of RPCs to the inner layer of the nerve was more obvious. 40% O2 significantly promoted the formation of RGCs. Compared with 20% O2, the three-dimensional induction of hESCs in hyperoxia induced the formation of hESCs. The process of RGCs was longer and the migration to basal side increased. Part 3: The effect of oxygen concentration on the proliferation of NR-derived C-Kit+/SSEA4-cells induced by hESCs and its biological characteristics were analyzed. Part 1: The NR formation of hESCs was induced by an improved three-dimensional induction method. It was found that the formation of EBs by the improved three-dimensional induction method was relatively slow, and the characteristics of NR cells and tissue architecture were not significantly changed. Hyperoxia still promoted the growth of EBs. 2. Immunofluorescence staining was used to study the temporal and spatial distribution of C-Kit expression in NR induced by improved three-dimensional induction of hESCs. C-Kit positive cells expressed stem cell markers Nestin, PAX6 and RAX but not CHX10 in the inner layer of the retina. With the development of induction time, the expression of C-Kit in NR decreased. 3. Cell survival assay was used to analyze the proliferation characteristics of hESC-NR-C-Kit+/SSEA4-cells. It was found that hESC-NR-C-Kit+/SSEA4-cells were fine under 3% O2 hypoxia. Cell proliferation was significantly increased in the induction of 30D, 45D and 60D sorted cells, and 30D sorted cells had the best proliferative activity. 4. Immunofluorescence staining was used to study the characteristics of hESC-NR-C-Kit+/SSEA4-cells. It was found that hESC-NR-C-Kit+/SSEA4-cells could express markers and proliferative markers Ki67 including Nestin, PAX6, RAX and other RPCs. RGCs, bipolar cells, photoreceptor cells and Muller cells were induced to differentiate into RGCs. Pearson correlation coefficients between hESC-NR-C-Kit+/SSEA4-retinal precursor cells and RPCs isolated from human embryonic eyes were found by genome-wide transcription analysis. HESC-NR-C-Kit+/SSEA4-cells expressed more genes related to proliferation and migration than RPCs, and the activation of p53 signaling pathway increased, but the activation of cell adhesion molecule pathway decreased. Conclusion: 1. NR induced by three-dimensional hESCs can express various kinds of retinal stem cell markers, and its development is similar to that of retinal development in vivo. The improved three-dimensional (BMP4-mediated) induction method is simple and stable, which indicates that the improved three-dimensional induction method is more conducive to the later clinical application. 2. The main effects of elevated oxygen concentration on three-dimensional induction of NR in hESCs include promoting the proliferation of NR and promoting the rosette formation in accordance with the normal physiological tip-to-bottom. The results showed that hyperoxia treatment was conducive to the formation, maturation and migration of PAX6-positive RPCs, which was closer to NR.3 in vivo. Under hypoxia culture, the state and proliferative activity of 30D-sorted hESC-NR-C-Kit+/SSEA4-cells were significantly improved than those in normoxia culture. C-Kit positive cells could express Nestin, PAX6, RAX and other retinal stem cell markers at the same time, but did not express embryonic antigen. They could differentiate into photoreceptor cells, bipolar cells, Muller cells and RGCs, indicating that hESC-NR-C-Kit+/SSEA4-cells were a class of RPCs. The results showed that hESC-NR-C-Kit+/SSEA4-cells were very similar to RPCs isolated from the eyes of human embryos (90%), but the expression of proliferation and migration-related genes was higher than that of RPCs, suggesting that hESC-NR-C-Kit+/SSEA4-cells were a class of RPCs close to in vivo development. There are new seed cells with standardized industrialization conditions.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R774.1

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