Wnt信号通路调控角膜缘干细胞治疗角膜缘干细胞缺乏症

文题释义： Wnt 信号通路：Wnt 信号通路是一条高度保守的信号通路，在干细胞调控中发挥重要作用。当细胞外 Wnts 与细胞膜上特异性受体 Frizzled 蛋白结合导致 GSK3β 失活，β-catenin 在胞质中积累继而向胞核转位，标志 着 Wnt 通路被激活。β-catenin 在细胞核中与转录因子 Tcf /LEF 结合启动下游基因而发挥功能。 角膜缘干细胞：角膜缘为角膜和结膜、巩膜交界部分，角膜缘干细胞存在于角膜缘的 Vogt 栅栏结构中，属于 单能干细胞，在角膜损伤修复中发挥重要作用。角膜缘干细胞缺乏是由于眼表各类损伤(化学伤、严重的感染 等)或者基质微环境异常(先天性无虹膜、放射线所致角膜病)导致角膜上皮被结膜上皮侵犯和替代。
Corresponding author: Han Bo, Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China
Abstract BACKGROUND: Wnt signaling pathway plays an important role in the regulation of stem cells, but its regulatory effect on limbal stem cells is not well defined. OBJECTIVE: To investigate the regulation of limbal stem cells via Wnt signaling pathway and its function in the treatment of limbal stem cell deficiency. METHODS: Rat limbal segments were digested with Dispase and trypsin/EDTA and then single limbal stem cells were seeded in 3D-Matrigel. In experimental group LiCl (500 μmol/L) was added to the culture system. Control group received no LiCl. The expression levels of p63α, CK12, CEBPδ and Ki67 in limbal stem cells were detected by qRT-PCR on the 7th day of culture. The expression level of β-catenin in limbal stem cells was detected by immunofluorescence staining. The rat model of limbal stem cell deficiency was made by alkali burn method. Rats in treatment group were treated with Wnt-activated limbal stem cells by subconjunctival injection. Rats in control group were treated with PBS. Rats were checked by slit lamp every day. On the 4th day after treatment, the immunofluorescence staining and hematoxylin-eosin staining were applied to evaluate the repair of limbus. RESULTS AND CONCLUSION: (1) The limbal stem cells aggregated in 3D-Matrigel. β-Catenin was negative in the control group. β-Catenin was found in the cytoplasm and nucleus of the limbal stem cells in the experimental group. (2) qRT-PCR results showed there was no significant difference in the levels of p63α, CK12 or CEBPδ between control and experimental groups (P > 0.05). The Ki67 level in the experimental group was significantly higher than that in the control group (P < 0.05). (3) The rat models of limbal stem cell deficiency were established. The degree of corneal opacity in the treatment group was significantly lower than that in the control group (P < 0.05). Hematoxylin-eosin staining results observed that the corneal epithelial cells in the treatment group were neatly arranged, the cell size was uniform, and repair was good. Immunofluorescence staining showed that β-catenin was found in the cytoplasm and nucleus of most corneal epithelial cells in the treatment group. While β-catenin was only weakly positive in the control group, and was invisible in the cytoplasm or nucleus. (4) These results indicate that activation of Wnt signaling pathway in limbal stem cells enhances their proliferation and keeps them in an undifferentiated self-renewal state. Wnt-activated limbal stem cells can promote the regeneration of corneal epithelium and reduce the degree of corneal opacity in the treatment of limbal stem cell deficiency. The regulation of limbal stem cells via Wnt signaling pathway is expected to provide new ideas for the treatment of limbal stem cell deficiency. Key words: Wnt signaling pathway; limbal stem cells; limbal stem cell deficiency; self-renewal Funding: the National Natural Science Foundation of China, No. 81400378 (to HB)
对照组： 结膜下注射等量 PBS
PCR 检测 p63α、CK12、 Ki-67、CEBPδ 的表达
克隆形成实验
免疫荧光检测 β-catenin 的表达
裂隙灯、荧光染色观察 角膜组织
角膜石蜡切片苏木精伊红染色
免疫荧光检测角膜组织 β-catenin 的表达
结论： (1)Wnt 信号通
路激活使得 角膜缘干细 胞进入自我 更新状态； (2)自我更新状 态的角膜缘 干细胞在角 膜缘干细胞 缺乏症的治 疗中能发挥 显著作用。
《中国组织工程研究》 Chinese Journal of Tissue Engineering Research
Wnt信号通路调控角膜缘干细胞治疗角膜缘干细胞缺乏(华中科技大学同济医学院附属协和医院眼科，湖北省武汉市 430022)
DOI:10.3969/j.issn.2095-4344.2028
Han Bo, MD, Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China
得角膜缘干细胞进入自我更新状态； (3)建立大鼠角膜缘干细胞缺乏症模型，以结膜下注射的方式给予 Wnt 信号通路激活的角膜缘干细胞，
发现有明显的治疗效果。
大鼠角膜 缘干细胞 培养
大鼠角膜 缘干细胞 缺乏症模 型
实验组： 培养液中加入氯化 锂
对照组： 常规培养液
治疗组： 结膜下注射上述实 验组角膜缘干细胞
Regulation of limbal stem cells via Wnt signaling in the treatment of limbal stem cell deficiency
Han Bo, Yang Zhe, Li Jing, Zhang Mingchang (Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China)
韩波，女，1977 年生，湖 北省武汉市人，汉族，2007 年华中科技大学毕业，博 士，主要从事角膜病、青 光眼、白内障研究。
通讯作者：韩波，博士， 华中科技大学同济医学院 附属协和医院眼科，湖北 省武汉市 430022
文献标识码:B 来稿日期：2019-07-10 送审日期：2019-07-22 采用日期：2019-08-23 在线日期：2019-11-25
摘要 背景：Wnt 信号通路在干细胞的调控中发挥重要作用，但是其对于角膜缘干细胞的调控作用尚不十分明确。 目的：探讨 Wnt 信号通路对角膜缘干细胞的调控作用及其在角膜缘干细胞缺乏症治疗中的疗效。 方法：大鼠角膜缘组织中依次加入 Dispase 和 Trypsin/EDTA 进行消化，将消化后的角膜缘干细胞悬液接种 于 3D-Matrigel 微环境中培养，实验组培养体系中加入 Wnt 信号通路激活剂氯化锂(500 μmol/L)，对照组不加 氯化锂，培养第 7 天 qRT-PCR 检测角膜缘干细胞中 p63α、CK12、CEBPδ 和 Ki67 的表达，免疫荧光染色 检测角膜缘干细胞中 β-catenin 的表达。采用碱烧伤法建立大鼠角膜缘干细胞缺乏模型，治疗组大鼠结膜下注 射激活了 Wnt 信号通路的角膜缘干细胞，对照组大鼠结膜下注射等量 PBS，随后每天使用裂隙灯观察，治疗 后第 4 天取角膜缘组织进行免疫荧光染色、苏木精-伊红染色。 结果与结论：①角膜缘干细胞在 3D-Matrigel 培养微环境中呈球形聚集生长，对照组角膜缘干细胞中 β-catenin 呈阴性，实验组角膜缘干细胞中 β-catenin 在细胞浆和细胞核内聚集；②qRT-PCR 结果显示：对照组与实验 组之间干性指标 p63α、CK12 和功能指标 CEBPδ 差异均无显著性意义(P > 0.05)；实验组增殖指标 Ki-67 较 对照组水平明显升高，差异有显著性意义(P < 0.05)；③建立大鼠角膜缘干细胞缺乏模型，治疗组大鼠角膜组 织混浊评分较对照组明显减低，差异有显著性意义(P < 0.05)，角膜组织苏木精-伊红染色可见治疗组大鼠角 膜上皮细胞排列整齐，细胞大小较均一，修复情况较良好，免疫荧光结果显示治疗组大部分角膜上皮细胞中 β-catenin 在细胞浆以及细胞核内聚集，而对照组角膜上皮细胞中 β-catenin 仅是弱阳性，未见在胞浆聚集或 者进核；④结果表明，Wnt 信号通路的激活增强了角膜缘干细胞的增殖能力，同时能够维持细胞干性，角膜 缘干细胞进入干细胞自我更新状态；激活 Wnt 信号通路后的角膜缘干细胞可促进角膜缘干细胞缺乏模型大鼠 角膜上皮的修复，减轻角膜混浊程度；通过调控 Wnt 信号通路改变角膜缘干细胞的功能特性可能成为治疗角 膜缘干细胞缺乏症的新途径。 关键词： Wnt 信号通路；角膜缘干细胞；角膜缘干细胞缺乏症；自我更新
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韩波，杨喆，栗静，张明昌. Wnt 信号通路调控角膜缘干细胞治疗角膜缘干细胞缺乏症[J]. 中国组织工程研究，2020，24(7):1057-1062. DOI:10.3969/j.issn.2095-4344.2028
中图分类号：R459.9；R394.2；R772.2 基金资助： 国家自然科学基金(81400378)，项目负责人：韩波
ORCID: 0000-0003-0698-3653(韩波)
文章快速阅读：
文章创新— (1)文献报道 Wnt 信号通路在维持多种干细胞特性中起重要作用，并参与调控干细胞分化，但 Wnt
信号通路是否能对角膜缘干细胞的细胞特性及分化产生重要的调控作用尚无更多信息支持； (2)通过氯化锂激活 Wnt 信号通路，研究其对角膜缘干细胞的调控作用，发现 Wnt 信号通路激活可使