人尿液来源的诱导多能干细胞定向分化为晶状体小体的新方法建立及在晶状体发育机制研究中的运用

发布时间：2018-01-10 06:02

本文关键词：人尿液来源的诱导多能干细胞定向分化为晶状体小体的新方法建立及在晶状体发育机制研究中的运用　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：目的白内障是全球最主要的致盲眼病之一,手术是目前唯一有效的治疗途径。随着我国老龄化的加剧,白内障手术的需求量每年大幅增加,由此给社会和家庭带来沉重负担。近期两个研究团队分别发现两个对白内障可能有治疗作用的药物,并在细胞以及动物模型上获得良好效果,由此为白内障的药物治疗打开新的思路,但由于人源的体外晶状体模型的缺乏,这类药物在人源上的验证未能实现。因此,本学位论文旨在利用人源诱导多能干细胞(h-iPSCs)定向分化获得人晶状体小体(LB),构建合适的具有晶状体光学特性的体外晶状体模型并在此基础上进行晶状体发育机制研究。方法.首先收集分离人尿液中的上皮细胞,用慢病毒做载体将"Yamanaka四因子"转入尿液细胞将其重编程获得iPSCs,通过碱性磷酸酶染色、多能干细胞标志物(SOX2、SSEA-4、Nanog、TRA1-81、OCT4)的免疫荧光染色及 RT-qPCR、拟胚体形成实验以及畸胎瘤实验验证iPSCs的多能性。其次,我们创立了"荷包蛋法"将iPSCs诱导分化成成熟、具有光学特性的LB,通过免疫荧光染色及RT-qPCR检验LB分化过程的晶状体发育标志物(DLX3、SIX1、EYA1、PAX6、CRYAA、CRYAB、PROX1、FOXE3、SOX1、CRYB、CRYGC、MIP)的动态表达,验证LB分化过程和晶状体胚胎发育的对应性,用透射电镜和亚甲蓝染色等检测成熟LB的细微结构验证其与晶状体的相似性,通过"X"实验检测成熟LB的光学特性(透明度以及放大倍率)。最后通过基因芯片检测iPSCs和LB中mRNA和lncRNA的差异表达,用RT-qPCR对部分自噬相关基因及其对应lncRNA、晶状体发育相关基因及其对应lncRNA进行验证并从中筛选出lncRNAp10540做进一步功能研究。利用siRNA沉默lncRNAp10540表达,同时检测LC3B在mRNA和蛋白水平的表达变化。通过对老年性患者以年龄分组并提取其囊膜RNA检测lncRNAp10540和老年性白内障的相关性。结果我们利用人尿液来源的上皮细胞成功重编程获得iPSCs,该iPSCs磷酸酶染色呈强阳性、表达多能干细胞标志物、能形成完美的拟胚体,将其注射入裸鼠中可获得有三个胚层组织和细胞的畸胎瘤。通过"荷包蛋法"诱导iPSCs可获得成熟LB,其分化过程有晶状体板期、早期晶状体期及成熟晶状体期,与晶状体胚胎发育一一对应。成熟LB表达晶状体纤维细胞标志物(CRYAA、CRYAB、CRYB、CRYGC、MIP),具有晶状体上皮细胞、晶状体纤维细胞以及囊膜等结构,且具有良好的透明性和1.7倍的放大倍率。自噬存在于LB分化过程中,该现象与小鼠晶状体胚胎发育相似。基因芯片结果提示自噬基因相对应lncRNA在LB分化过程中的差异表达,lncRNA p10540在LB分化过程中通过在细胞胞浆中增强LC3B向活化状态LC3BII的转化而影响自噬作用,此外在年龄75岁组中lncRNAp10540的表达明显下降。结论人尿液来源的上皮细胞可以作为iPSCs构建的理想体细胞来源。利用"荷包蛋法"可将iPSCs分化成立体、直径可达3mm且具有光学特性的LB,是目前已知的最接近人晶状体的理想体外晶状体模型。自噬以及lncRNA在LB分化过程中具有重要作用,lncRNAp10540通过影响LC3B的活化影响自噬进而可能影响LB分化发育且其与老年性白内障发病相关。
[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.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R776.1

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