1、博士学位论文角膜内皮细胞新的分子标记及人胚胎干细胞向角膜内皮细胞分化的研究姓名：学号：所在院系：生命科学与技术学院学科门类：理学学科专业：生物化学与分子生物学指导教师： 教授年 月二一二年十二月二一二年九月同济大学博士学位论文摘要I摘要目的(1) 建立人类胎儿和成人角膜内皮细胞（corneal endothelial cells, CEC）mRNA 转录组表达谱。比较角膜内皮细胞在胎儿及成人阶段不同的表达模式，找出角膜内皮细胞组织特异性基因表达谱和若干角膜内皮细胞新的分子标记。 (2) 建立一套有效的的从人胚胎干细胞和可诱导的多能干细胞分化为角膜内皮细胞的方法，为组织工程化角膜内皮细胞治疗角膜
2、内皮损伤提供实验依据。方法(1) 提取胎儿及成人角膜内皮细胞 RNA，运用 RNA-Seq 技术建立人类胎儿和成人角膜内皮细胞转录组表达谱。从 GEO 数据库中收集 97 个不同样品的RNA-Seq 数据，代表其他 12 种不同组织细胞类型。通过比较基因组学，绘制层次聚类图和三维散点图找出胎儿及成人 CEC 与其他组织细胞类型的关系。运用生物信息学，找出 CEC 组织特异性基因表达谱和阶段特异性基因表达谱，用 David 在线生物信息分析系统对 CEC 特异性基因列表进行基因本体分析（Gene Ontology, GO）和 KEGG 通路分析。应用免疫组织荧光化学染色对筛选出的若干新的 CEC
3、 标记分子进行蛋白水平验证。(2) 运用人胚胎干细胞（human embryonic stem cells, hESCs）或可诱导的多能干细胞（induced pluripotent stem cells, iPSCs）作为种子细胞，遵循角膜内皮细胞生长发育规律，将诱导过程分为两个阶段：第一阶段为 hESCs 或 iPSCs 向神经嵴细胞（Neural Crest cells, NCC）分化，该阶段我们尝试了短期诱导法（ 4天）和长期诱导法（2 周），所用的化学因子包括 Noggin 和小分子化合物SB431542。第二阶段为神经嵴细胞向 CEC 转化，我们对比应用了添加特定化学因子组合的方法
4、和利用牛角膜内皮细胞条件培养基进行诱导的方法，所添加的化学因子组合包括:转化生长因子-（Transforming growth factor-beta, TGF-）, 血小板衍生生长因子（ Platelet-derived growth factor，PDGF）和 Dickkopf 2 (DKK2)。诱导期间，通过对各阶段细胞形态学观察，RT-PCR，免疫组织化学染色检测标记分子，建立一套从 hESCs 或 iPSCs 向角膜内皮细胞诱导的最佳方法。将此方法得到的 CEC 样细胞做基因转录表达谱分析，找出和其他组织细胞类型的关系。同济大学博士学位论文摘要II(3) 建立牛角膜内皮细胞培养体系，
5、准备去细胞的牛角膜后弹力层作为细胞载体。建立兔角膜后弹力膜机械损伤模型，移植入以牛角膜后弹力膜为载体的牛角膜内皮细胞。通过术后不同时期兔角膜透明度和厚度的恢复程度来判断移植物对于兔角膜机械损伤的修复效果。实验结束，将兔处死后固定角膜组织，通过 HE 染色判断细胞在受体中存活能力及对角膜结构修复能力的大小。结果(1) 通过和 12 种其他类型的细胞和组织比较，在层次聚类图中我们发现胎儿的角膜内皮细胞表达谱更加接近于一些未成熟的细胞而成人的角膜内皮细胞聚类于一些终末分化的体细胞或组织。通过比较基因组学分析，找出 284 个成人 CEC 和 245 个胎儿 CEC 组织相关性表达基因。GO 分析表明
6、 284 个成人CEC 高表达基因主要参与了其功能相关的无机阴离子跨膜转运活动，转录因子和细胞周期依赖性蛋白激酶抑制剂的活性调控。而 245 个胎儿 CEC 特异性基因则主要参与构建细胞外基质结构成分，与血小板衍生因子及其他生长因子结合的活性。进一步比较胎儿及成人 CEC 阶段特异性基因表达时，分别得到了 518个成人和 668 个胎儿相对高表达基因。GO 和 KEGG 通路分析显示，一些代谢相关的基因在成人 CEC 中呈相对较高的表达水平，而金属肽酶的活性，细胞外结构调节及 TGF- 信号通路则在胎儿 CEC 中相对活跃。免疫组织荧光化学检测若干标记性分子蛋白表达水平显示胎儿（17-18 周
7、）及成人 CEC（37 岁）均表达紧密链接蛋白 ZO-1，但与 CEC 泵功能相关的蛋白 Na+-K+-ATPase，仅在成人 CEC 中有表达。参与 Wnt 信号通路的 Axin2 和 Beta-catenin 在胎儿及成人CEC 中均被检测到，但 Wnt5a 只在胎儿 CEC 中表达。S100 蛋白家族的S100A4 和 S100A6 只表达于成人 CEC。此外，我们还发现即早反应蛋白 IER3在胎儿 CEC 和成人 CEC 转录水平上都呈高表达水平，但在细胞中的定位有明显的区别，IER3 表达在胎儿 CEC 细胞核，却表达于成人 CEC 的细胞质。(2) 通过分组诱导，我们发现在 hES
8、Cs 或 iPSCs 向神经嵴细胞诱导阶段，短期诱导法和长期诱导法都可以获得同时表达神经嵴细胞标记物 P75 和 HNK-1 的细胞，但长期诱导法可以观察到典型的神经玫瑰花状结构，表明其可能产生更为成熟的神经嵴细胞。而在神经嵴细胞向 CEC 样细胞诱导阶段，添加特定细胞因子诱导法可以在一周看到类似内皮细胞样形态的细胞。RT-PCR 显示，该细胞可以表达 AQP-1, Colleagen VIII 等角膜内皮细胞特异性标记分子。免疫组织化学检测，该细胞表达 ZO-1, Vimentin, Wnt5a。但 10-14 天以后，细胞逐渐失同济大学博士学位论文摘要III去内皮样形态，部分细胞甚至死亡。
9、而牛角膜内皮细胞条件培养基的方法显示，当条件培养基添加后 1 至 2 周即可见到内皮细胞样细胞并且随着诱导时间的延长，该细胞可以缓慢扩增，形态上也逐渐成熟。三个月后仍可保持角膜内皮细胞样形态。免疫荧光化学染色表明其可以表达 ZO-1 标记分子。提取该细胞RNA，通过 RNA-Seq 测序技术显示，经诱导的 hESCs 来源的内皮细胞样细胞基因表达谱聚类于胎儿角膜内皮细胞。(3) 运用酶解法得到原代牛角膜内皮细胞，一周后细胞形成紧密连接，维持典型的多角形内皮样结构，增殖能力强，适合连续传代，所得的牛角膜后弹力膜经去细胞处理后可以作为很好的移植载体。而收集每次培养所得的培养基又可为 CEC 样细胞
10、的诱导提供必须的生长因子。(4) 通过部分后弹力膜剥除术，我们成功建立了兔角膜内皮机械损伤模型。结论(1) 通过建立胎儿及成人 CEC 转录组表达谱，我们分别找出了 245 个胎儿CEC 和 284 个成人 CEC 组织特异性基因，以及 518 个成人 CEC 和 668 个胎儿CEC 阶段特异性基因。并通过对胎儿及成人 CEC 角膜组织的免疫荧光化学染色，在蛋白水平上验证了 Wnt5a, S100A4, S100A6 和 IER3 四个 CEC 新的分子标记。(2) 运用分阶段诱导法，我们成功得到了 hESCs 及 iPSCs 来源的角膜内皮细胞样细胞。关键词: 角膜内皮细胞, RNA-Se
11、q, 人胚胎干细胞, 可诱导多能干细胞，诱导, 分化Tongji University Doctor of Philosophy AbstractIVABSTRACTObjectives (1) Set up human fetal and adult corneal endothelial cell mRNA transcript expression patterns. Compare stage specific gene expression patterns in fetal and adult corneal endothelial cells. Identify corneal
12、endothelial cell tissue-specific gene expression profiles as well as discover new molecular markers of corneal endothelial cells.(2) Establish an effective method of differentiating human embryonic stem cells and induced pluripotent stem cells into corneal endothelial cells in vitro; provide evidenc
13、e of applying tissue engineered corneal endothelial cells in the treatment of CEC deficiency model.Methods and Materials (1) Extract RNA from the fetal and adult corneal endothelial tissue, using RNA-Seq technology to build up human fetal and adult corneal endothelial cell transcriptome expression p
14、rofiling. We procured 97 samples RNA-Seq data from the GEO database representing 12 different types of cells and tissues. Through comparative genomics, we drew hierarchical clustering and three-dimensional scatter plot to identify the relationship between CEC and other cell types. We applied bioinfo
15、rmatics to identify the tissue-specific gene expression profiles and stage-specific gene expression profiles of adult and fetal CEC, used David online bioinformatics analysis system to analysis the GO and KEGG pathway of those gene lists. Finally, we identified a number of new CEC labeled molecules
16、by immunohistochemical fluorescence staining on the basis of the RNA-Seq data.(2) In order to get hESCs or iPSCs derived CEC, we followed the developmental characteristics of corneal endothelial cells, separated the induced process into two stages. The first stage was getting neural crest cells from
17、 hESCs or iPSCs. We tried short-term induction method (4 days) and long-term (2 weeks) induction method Tongji University Doctor of Philosophy AbstractVbaesd on the treatment of two important factors: Noggin and SB431542. The second stage was neural crest cells differentiate into CEC-like cells, we
18、compared two different ways which including add define combine factors (TGF-, PDGF, and DKK2) or bovine corneal endothelial cell conditioned medium treatment. We kept on observing the morphological changes during the process as well as detecting CEC markers expression and gene expression pattern dur
19、ing the induction process. By comparing the different ways of induction, set up novel method of inducing corneal endothelial cells from hESCs or iPSCs. Finally, construct RNA-Seq library of these CEC-like cells, find out the similarity to the other cell types including fetal and adult CEC.(3) Establ
20、ish bovine corneal endothelial cell culture system. Acellular bovine corneal descemets membrane was prepared as carrier for cell transplantation. Set up rabbit corneal endothelium injury model by peeling cornea descemets membrane. Transplanted with bovine corneal endothelial cells seeded on bovine c
21、orneal descemets membrane. The effect of transplantation will be determined by corneal transparency and thickness after surgery. One month after surgery, the rabbits were sacrificed and the corneal tissue were fixed for HE staining to determine cell vitality and repair capacity of corneal structural
22、 remodeling. Results(1) By comparing with 12 other tissue and cell types, hierarchal clustering revealed that fetal CECs cluster close to relatively immature cell types, In contrast, adult CECs cluster grouped together with other terminally differentiated somatic cell types. Through the comparative
23、genomics analysis, we identied a total of 284 and 245 genes that are specically enriched in adult and fetal CECs, respectively. Gene ontology (GO) analysis revealed that the 284 up-regulated genes in adult CECs mainly participate in inorganic anion transmembrane transporter activity, transcription f
24、actor activity and cyclin-dependent protein kinase inhibitor activity. Genes unique to fetal CECs are involved in extracellular matrix structural constituent, platelet-derived growth factor binding and growth factor activity. We next directly compared adult and fetal CECs stage specific gene express
25、ion profile, identied 518 and 668 genes that are highly expressed in adult and fetal CECs respectively. GO and kyoto encyclopedia Tongji University Doctor of Philosophy AbstractVIof genes and genomes (KEGG) pathway analysis indicated that many genes involved in metabolic processes are up-regulated i
26、n adult CECs, whereas fetal CECs showed GO terms enriched in metallopeptidase activity, extracellular structure and TGF-signaling pathway. We also collected fetal and adult corneal tissues for protein expression level analysis. As expected, immunostaining results shown expression of tight junction p
27、rotein ZO-1 in both fetal (17-18 weeks in gestation) and adult CECs (37 years old). Na+-K+-ATPase, which is one of the most important markers for the pump function in CECs, is highly expressed in adult CECs but not in fetal CECs. Wnt signaling molecules such as Axin2 and Beta-catenin were detected i
28、n both fetal and adult CECs. However, Wnt5a only expressed in fetal CECs. We also found sub-members of the S100A protein family (S100A4 and S100A6) expressed in adult CECs. In addition, Immediate Early Response 3 (IER3) can be readily detected in both fetal and adult CECs, but with a stark contrast
29、in subcellular localization. IER3 localizes in the cytoplasm of adult CECs, whereas it is found in the nucleus of fetal CECs.(2) By comparing different ways of differentiation, we found that in the first stage, both of the cells after short-term or long-term induction treatment could express neural
30、crest markers P75 and HNK-1. However, we only observed typical neural rosettes structure after long-term induction method, which shown that more mature neural crest cells were obtained after long-term treatment with noggin and SB431542. During the second stage, CEC-like cells were shown after 7 days
31、 treated with specific cytokines. RT-PCR results showed the expression of corneal endothelial cell-specific markers AQP-1 and Colleagen VIII. Immunohistochemistry staining detected ZO-1, Vimentin, Wnt5a expression. But 10-14 days later, the cells gradually lose their endothelial-like morphology and
32、even death. When we using bovine corneal endothelial cell conditioned medium. The cells showed endothelial-like morphology one to two weeks after neural crest cell inducement. Followed by the extension of the induction, the percentage of the polygonal endothelial-like cell becoming more and more in
33、the whole well. Immunofluorescence staining showed that these cells can express ZO-1. After extracting RNA from the induced CEC-like cells, we established the RNA-Seq liberary for transcriptome analysis, by comparing with 17 other cell type gene expression pattens , it shown that these hESCs derived
34、 CEC-like cells were cluster together with fetal CEC. Tongji University Doctor of Philosophy AbstractVII(3) We successfully set up primary bovine CEC culture system, these cells can easily proliferated, suitable for continuous passage. Meanwhile, Acellularized bovine corneal descemets membrane can b
35、e used as a ideal carrier for cell transplantation. The culture medium could also been collected as conditional medium for CEC-like cells inducement. (4) By peeling rabbit descemets membrane, we successfully established a rabbit corneal endothelial injury model.Conclusion(1) By establishing the feta
36、l and adult CEC transcriptome profiles, we identified 245 fetal CEC and 284 adults CEC tissue-specific genes, as well as 518 adults CEC and 668 fetal CEC stage-specific genes. By immunohistochemistry analysis of fetal and adult ocular tissues, we demonstrated Wnt5a, S100A4, of S100A6 and IER3 as fou
37、r new CEC molecular markers in fetal and adult CECs at protein level. (2) The neural crest cells inducement method together with bovine corneal endothelial cells conditioned medium can get hESCs or iPSCs derived corneal endothelial-like cells.Key words: Corneal endothelial cells, RNA-Seq, human embr
38、yonic stem cells, induced pluripotent stem cells, induction, differentiation同济大学博士学位论文英文缩略词索引VIII英文缩略词缩略词 英文全称 中文翻译ANOVA Analysis Of Variance 方差分析Asc-2P Ascorbic acid 2-phosphate 抗坏血酸-2 -磷酸bFGF basic Fibroblast Growth Factor 成纤维细胞生长因子CECs Corneal endothelial cells 角膜内皮细胞CHED1 Congenital hereditary e
39、ndothelial dystrophy 1 先天性遗传性内皮营养 不良 1CHED2 Congenital hereditary endothelial dystrophy 2 先天性遗传性内皮营养 不良 2Cdkn1a Cyclin-dependent kinase inhibitor 1A 细胞周期依赖性蛋白激 酶抑制剂 1ACOL8A2 Collagen, Type VIII, alpha 2 型胶原 2CS Chondroitin sulfate 硫酸软骨素CDK Cyclin-dependent kinase 细胞周期蛋白依赖性激 酶DALK Deep Anterior Lamellar Keratoplasty 前部深板层角膜移植DMEK Descemets Membrane Endothelial Keratoplasty 后弹力膜内皮移植术DSEK Descemets stripping with endothelial keratoplasty 后弹力层剥除内皮移植 术EB Embryonic Body 拟胚体ESCs Embryonic Stem Cells 胚胎干细胞EDTA Ethylenediaminetetraacetic acid 乙二胺四乙酸FDR False discovery rate 错误发现率
