發(fā)布時間：2018-01-10 06:02

  本文關(guān)鍵詞：人尿液來源的誘導多能干細胞定向分化為晶狀體小體的新方法建立及在晶狀體發(fā)育機制研究中的運用　出處：《浙江大學》2017年博士論文　論文類型：學位論文

  更多相關(guān)文章： 尿液細胞 人誘導多能干細胞 荷包蛋法 晶狀體小體 自噬 LncRNA p10540 老年性白內(nèi)障

【摘要】：目的白內(nèi)障是全球最主要的致盲眼病之一,手術(shù)是目前唯一有效的治療途徑。隨著我國老齡化的加劇,白內(nèi)障手術(shù)的需求量每年大幅增加,由此給社會和家庭帶來沉重負擔。近期兩個研究團隊分別發(fā)現(xiàn)兩個對白內(nèi)障可能有治療作用的藥物,并在細胞以及動物模型上獲得良好效果,由此為白內(nèi)障的藥物治療打開新的思路,但由于人源的體外晶狀體模型的缺乏,這類藥物在人源上的驗證未能實現(xiàn)。因此,本學位論文旨在利用人源誘導多能干細胞(h-iPSCs)定向分化獲得人晶狀體小體(LB),構(gòu)建合適的具有晶狀體光學特性的體外晶狀體模型并在此基礎(chǔ)上進行晶狀體發(fā)育機制研究。方法.首先收集分離人尿液中的上皮細胞,用慢病毒做載體將"Yamanaka四因子"轉(zhuǎn)入尿液細胞將其重編程獲得iPSCs,通過堿性磷酸酶染色、多能干細胞標志物(SOX2、SSEA-4、Nanog、TRA1-81、OCT4)的免疫熒光染色及 RT-qPCR、擬胚體形成實驗以及畸胎瘤實驗驗證iPSCs的多能性。其次,我們創(chuàng)立了"荷包蛋法"將iPSCs誘導分化成成熟、具有光學特性的LB,通過免疫熒光染色及RT-qPCR檢驗LB分化過程的晶狀體發(fā)育標志物(DLX3、SIX1、EYA1、PAX6、CRYAA、CRYAB、PROX1、FOXE3、SOX1、CRYB、CRYGC、MIP)的動態(tài)表達,驗證LB分化過程和晶狀體胚胎發(fā)育的對應(yīng)性,用透射電鏡和亞甲藍染色等檢測成熟LB的細微結(jié)構(gòu)驗證其與晶狀體的相似性,通過"X"實驗檢測成熟LB的光學特性(透明度以及放大倍率)。最后通過基因芯片檢測iPSCs和LB中mRNA和lncRNA的差異表達,用RT-qPCR對部分自噬相關(guān)基因及其對應(yīng)lncRNA、晶狀體發(fā)育相關(guān)基因及其對應(yīng)lncRNA進行驗證并從中篩選出lncRNAp10540做進一步功能研究。利用siRNA沉默lncRNAp10540表達,同時檢測LC3B在mRNA和蛋白水平的表達變化。通過對老年性患者以年齡分組并提取其囊膜RNA檢測lncRNAp10540和老年性白內(nèi)障的相關(guān)性。結(jié)果我們利用人尿液來源的上皮細胞成功重編程獲得iPSCs,該iPSCs磷酸酶染色呈強陽性、表達多能干細胞標志物、能形成完美的擬胚體,將其注射入裸鼠中可獲得有三個胚層組織和細胞的畸胎瘤。通過"荷包蛋法"誘導iPSCs可獲得成熟LB,其分化過程有晶狀體板期、早期晶狀體期及成熟晶狀體期,與晶狀體胚胎發(fā)育一一對應(yīng)。成熟LB表達晶狀體纖維細胞標志物(CRYAA、CRYAB、CRYB、CRYGC、MIP),具有晶狀體上皮細胞、晶狀體纖維細胞以及囊膜等結(jié)構(gòu),且具有良好的透明性和1.7倍的放大倍率。自噬存在于LB分化過程中,該現(xiàn)象與小鼠晶狀體胚胎發(fā)育相似�；�因芯片結(jié)果提示自噬基因相對應(yīng)lncRNA在LB分化過程中的差異表達,lncRNA p10540在LB分化過程中通過在細胞胞漿中增強LC3B向活化狀態(tài)LC3BII的轉(zhuǎn)化而影響自噬作用,此外在年齡75歲組中l(wèi)ncRNAp10540的表達明顯下降。結(jié)論人尿液來源的上皮細胞可以作為iPSCs構(gòu)建的理想體細胞來源。利用"荷包蛋法"可將iPSCs分化成立體、直徑可達3mm且具有光學特性的LB,是目前已知的最接近人晶狀體的理想體外晶狀體模型。自噬以及l(fā)ncRNA在LB分化過程中具有重要作用,lncRNAp10540通過影響LC3B的活化影響自噬進而可能影響LB分化發(fā)育且其與老年性白內(nèi)障發(fā)病相關(guān)。
[Abstract]:Objectivecataract is one of the world's leading cause of blindness, surgery is the only effective treatment way at present. With China's aging, the annual demand for cataract surgery increased significantly, thus bring heavy burden to society and family. The recent two research teams are found two drugs on cataract may have therapeutic effects. And get a good effect in cell and animal models, which opens up a new way for cataract drug therapy, but due to the lack of in vitro human lens model, this kind of drug in verification failed to achieve source. Therefore, this thesis aims to use human induced pluripotent stem cells (h-iPSCs) differentiation obtained the lens body (LB), construction of research and development mechanism of the right lens lens lens with optical properties of in vitro model and on the basis of this method. The first charge. Set the separation in human urine epithelial cells using lentiviral vector to do the "Yamanaka four factor" into the urine cell reprogramming iPSCs by alkaline phosphatase staining, pluripotent stem cell markers (SOX2, SSEA-4, Nanog, TRA1-81, OCT4) by immunofluorescence staining and RT-qPCR, embryoid body formation of experimental and experimental verification of teratoma iPSCs. Secondly, we created the "Poached Egg method" iPSCs induced differentiation into mature, with the optical properties of LB by immunofluorescence staining and RT-qPCR test LB differentiation of lens development markers (DLX3, SIX1, EYA1, PAX6, CRYAA, CRYAB, PROX1, FOXE3. SOX1, CRYB, CRYGC, MIP) the dynamic expression of corresponding verification of LB differentiation and embryonic development of the lens, and the lens similarity verification with fine structure of transmission electron microscope and methylene blue staining to detect the maturation of LB, through the "X" test of mature LB The optical properties (transparency and magnification). The difference between mRNA and lncRNA gene chip for detection of iPSCs and LB in the expression of RT-qPCR on autophagy related gene and its corresponding lncRNA lens development related genes and their corresponding lncRNA verified and selected lncRNAp10540 for further functional studies. The expression of siRNA silencing of lncRNAp10540 expression detection of LC3B in mRNA and protein levels. The correlation of elderly patients in the age group and extract the envelope RNA detection of lncRNAp10540 and senile cataract. Results we use human urine derived epithelial cells obtained successful reprogramming of iPSCs, the iPSCs phosphatase staining was strongly positive expression of pluripotent stem cell markers, can the formation of embryoid body perfect, it can be injected into the nude mice can obtain teratoma three germ layers of tissues and cells. Through the "Lotus egg pack method "IPSCs can be induced mature LB, differentiation of lens in early stage and mature stage, lens lens, lens and corresponding embryos. Mature LB expression of lens fiber cell markers (CRYAA, CRYAB, CRYB, CRYGC, MIP), with lens epithelial cells, lens fiber cells and capsule etc. the structure, magnification and has good transparency and 1.7 times. Autophagy exists in LB differentiation process, the phenomenon and embryonic development of mouse lens are similar. The microarray results suggest that autophagy gene corresponding to lncRNA in LB differentiation process differences expression of lncRNA p10540 during the differentiation of LB cells by enhanced LC3B in the pulp into the activation state of LC3BII and the effect of autophagy, in addition to the expression of lncRNAp10540 at the age of 75 in the group decreased significantly. As iPSCs can construct conclusion urine derived epithelial cells of Want to use somatic cell sources. "Poached Egg method" iPSCs can be differentiated into three-dimensional, diameter up to 3mm and has the optical properties of LB, is now known to be the most close to the ideal in vitro human lens lens model. Autophagy and lncRNA play an important role during the differentiation of LB, lncRNAp10540 by LC3B and then affect the activation effect of autophagy may effect of differentiation and development of LB and the incidence of senile cataract.

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R776.1

【參考文獻】

相關(guān)期刊論文 前1條

1 Yi-ye Zhou;Fanyi Zeng;;Integration-free Methods for Generating Induced Pluripotent Stem Cells[J];Genomics,Proteomics & Bioinformatics;2013年05期

，

本文編號：1404105