本文選題：胚胎干細(xì)胞　切入點(diǎn)：誘導(dǎo)分化　出處：《青島大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：目的:誘導(dǎo)人胚胎干細(xì)胞(human Embryonic Stem Cells,h ESCs)向角膜上皮樣細(xì)胞分化,觀察分化細(xì)胞的生物學(xué)特性及其治療角膜緣干細(xì)胞缺乏的效果,并探討分化細(xì)胞的免疫原性及其移植后的免疫排斥反應(yīng)。方法:通過小分子化合物誘導(dǎo)h ESCs向角膜上皮細(xì)胞分化,免疫熒光染色、PCR檢測其上皮細(xì)胞標(biāo)志物的表達(dá),同時培養(yǎng)成體角膜緣上皮細(xì)胞(Limbal Epithelial Cells,LECs),以羊膜為載體制備重組角膜上皮細(xì)胞膜片。建立新西蘭兔和食蟹猴的角膜緣干細(xì)胞缺乏模型,行重組角膜上皮細(xì)胞膜片移植,術(shù)后通過裂隙燈和印跡細(xì)胞學(xué)觀察其治療效果。通過全基因組芯片篩選h ESCs分化細(xì)胞和LECs差異表達(dá)的基因,流式細(xì)胞檢測二者免疫相關(guān)分子的表達(dá),體外T細(xì)胞增殖實(shí)驗(yàn)和NK細(xì)胞殺傷實(shí)驗(yàn)驗(yàn)證其免疫原性,同時在體外模擬炎癥微環(huán)境觀察對細(xì)胞免疫原性的影響,重組角膜上皮細(xì)胞膜片移植后觀察免疫排斥反應(yīng),并用免疫熒光染色觀察免疫細(xì)胞。結(jié)果:h ESCs分化細(xì)胞的細(xì)胞形態(tài)與角膜緣干細(xì)胞相似,并表達(dá)角膜緣干細(xì)胞和角膜上皮細(xì)胞的標(biāo)記物,其多能性標(biāo)志物為陰性,并能在去上皮羊膜上形成復(fù)層的細(xì)胞結(jié)構(gòu),重組角膜上皮細(xì)胞膜片移植到角膜緣干細(xì)胞缺乏的新西蘭兔和食蟹猴體內(nèi)后,能夠顯著改善眼表情況并長時間保持穩(wěn)定。相比于LECs,h ESCs分化細(xì)胞中免疫相關(guān)基因的表達(dá)顯著下調(diào),其MHC分子低表達(dá),在體外不易引起T細(xì)胞增殖也不易被NK細(xì)胞殺傷,并且這些特性不受炎癥微環(huán)境的影響,h ESCs分化細(xì)胞移植到體內(nèi)后未見明顯的免疫排斥反應(yīng),且移植后免疫細(xì)胞的浸潤也較少。結(jié)論:通過小分子化合物誘導(dǎo)的方法可以成功將胚胎干細(xì)胞分化為具有一定增殖能力的角膜上皮樣細(xì)胞,該細(xì)胞體內(nèi)移植后能夠有效治療角膜緣干細(xì)胞缺乏,并且其免疫原性較小,不易引起免疫排斥反應(yīng)。
[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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R772.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

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本文編號：1576500