胚胎干细胞来源的角膜上皮样细胞治疗角膜缘干细胞缺乏的实验研究

发布时间：2018-03-06 21:09

本文选题：胚胎干细胞　切入点：诱导分化　出处：《青岛大学》2016年博士论文　论文类型：学位论文

【摘要】：目的:诱导人胚胎干细胞(human Embryonic Stem Cells,h ESCs)向角膜上皮样细胞分化,观察分化细胞的生物学特性及其治疗角膜缘干细胞缺乏的效果,并探讨分化细胞的免疫原性及其移植后的免疫排斥反应。方法:通过小分子化合物诱导h ESCs向角膜上皮细胞分化,免疫荧光染色、PCR检测其上皮细胞标志物的表达,同时培养成体角膜缘上皮细胞(Limbal Epithelial Cells,LECs),以羊膜为载体制备重组角膜上皮细胞膜片。建立新西兰兔和食蟹猴的角膜缘干细胞缺乏模型,行重组角膜上皮细胞膜片移植,术后通过裂隙灯和印迹细胞学观察其治疗效果。通过全基因组芯片筛选h ESCs分化细胞和LECs差异表达的基因,流式细胞检测二者免疫相关分子的表达,体外T细胞增殖实验和NK细胞杀伤实验验证其免疫原性,同时在体外模拟炎症微环境观察对细胞免疫原性的影响,重组角膜上皮细胞膜片移植后观察免疫排斥反应,并用免疫荧光染色观察免疫细胞。结果:h ESCs分化细胞的细胞形态与角膜缘干细胞相似,并表达角膜缘干细胞和角膜上皮细胞的标记物,其多能性标志物为阴性,并能在去上皮羊膜上形成复层的细胞结构,重组角膜上皮细胞膜片移植到角膜缘干细胞缺乏的新西兰兔和食蟹猴体内后,能够显著改善眼表情况并长时间保持稳定。相比于LECs,h ESCs分化细胞中免疫相关基因的表达显著下调,其MHC分子低表达,在体外不易引起T细胞增殖也不易被NK细胞杀伤,并且这些特性不受炎症微环境的影响,h ESCs分化细胞移植到体内后未见明显的免疫排斥反应,且移植后免疫细胞的浸润也较少。结论:通过小分子化合物诱导的方法可以成功将胚胎干细胞分化为具有一定增殖能力的角膜上皮样细胞,该细胞体内移植后能够有效治疗角膜缘干细胞缺乏,并且其免疫原性较小,不易引起免疫排斥反应。
[Abstract]:Objective: to induce human Embryonic Stem cells to differentiate into corneal epithelioid cells, and to observe the biological characteristics of differentiation cells and the effect of treating limbal stem cell deficiency. Methods: h ESCs was induced to differentiate into corneal epithelial cells by small molecular compound, and the expression of markers in epithelial cells was detected by immunofluorescence staining. At the same time, limbal Epithelial cells were cultured to prepare recombinant corneal epithelial cell membranes using amniotic membrane as the carrier. The limbal stem cell model of New Zealand rabbit and crab monkey was established, and the recombinant corneal epithelial cell membrane was transplanted. After operation, the therapeutic effect was observed by slit lamp and blotting cytology. The differentially expressed genes of h ESCs differentiation cells and LECs were screened by whole genome microarray, and the expression of immune-related molecules was detected by flow cytometry. The immunogenicity of T cell proliferation test and NK cell killing test in vitro was verified, and the immunogenicity was observed by simulating inflammatory microenvironment in vitro, and immunological rejection was observed after transplantation of recombinant corneal epithelial cell membrane. Results the cell morphology of ESCs differentiated cells was similar to that of limbal stem cells, and the markers of corneal limbal stem cells and corneal epithelial cells were expressed. The pluripotent markers were negative. The laminated cell structure can be formed on the epithelially removed amniotic membrane, and the recombined corneal epithelial cell membrane can be transplanted into the limbal stem cells of New Zealand rabbits and crab-eating monkeys lacking in limbal stem cells. Compared with the down-regulated expression of immune-related genes in ESCs differentiation cells, the expression of MHC molecules was low, and T cell proliferation was not easy to be killed by NK cells in vitro. Moreover, these characteristics were not affected by the inflammatory microenvironment. There was no obvious immune rejection after transplantation of ESCs differentiation cells in vivo. Conclusion: embryonic stem cells can be successfully differentiated into corneal epithelioid cells with certain proliferative ability by small molecular compound induction. This cell can effectively treat limbal stem cell deficiency after in vivo transplantation, and its immunogenicity is relatively small, so it is not easy to cause immune rejection.
【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R772.2

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