胰岛素样生长因子2及受体参与角膜上皮细胞生长和创伤修复的研究

发布时间：2018-01-17 23:23

本文关键词：胰岛素样生长因子2及受体参与角膜上皮细胞生长和创伤修复的研究　出处：《山东大学》2016年博士论文　论文类型：学位论文

【摘要】：背景角膜上皮细胞层是眼球抵御外界有害致病因素侵犯的第一道屏障,在受到创伤后可以快速修复愈合,当愈合延迟时,会造成视力下降,严重时导致失明。目前部分临床及动物实验报道角膜上皮本身存在一定的自我更新及修复能力,但角膜损伤后发挥主要作用的并非角膜上皮而是来自于角膜缘的细胞组织,该组织中含有角膜缘干细胞(LSC),角膜缘干细胞可以通过细胞增殖、分化及迁移来修复损伤的角膜组织。有报道称角膜缘干细胞缺失时角膜的修复功能会受到限制,且角膜修复功能还与角膜结膜化、新生血管生成、慢性炎症刺激及持续性的角膜上皮损害等因素有密切关系,在上述因素等持续存在,角膜最终不能够修复时可产生严重的后果即失明,同时研究表明,我们可通过移植角膜缘组织及角膜缘干细胞进行角膜损伤的治疗,这提示了角膜缘干细胞组织在创伤角膜修复过程中发挥着重要作用。在角膜损伤后,角膜缘干细胞即开始增殖,并分化为过渡性细胞,这种细胞可迁移至损伤部位,诱导细胞表达角膜上皮细胞标记进而促进角膜损伤的修复。然而角膜上皮细胞与角膜缘干细胞组织之间相互作用的分子通路及机制目前尚不清楚。Jia等报道角膜机械或化学损伤后细胞中负责生长及分化的因子基因表达明显增强,包括转化生长因子α (TGF-α)、转化生长因子β (TGF-β)、表皮生长因子(EGF)、肝素样生长因子(HGF)、成纤维细胞生长因子β (FGF-β)等,这些因子均可由角膜上皮细胞生成,它们参与调节细胞的增殖与生长。此外,胰岛素样生长因子2(IGF-2)也被证实能够促使角蛋白细胞增殖,并使黏附连接蛋白N钙黏附素的生成增加,.使细胞外基质增加,从而促使角蛋白胶原的生成。胰岛素样生长因子信号通路已被证实与细胞增殖及分化相关,在细胞及器官水平的生长调节中起重要作用,IGF及以上所述其他细胞调节因子与角膜缘干细胞的增殖、分化及迁移的发生有关。胰岛素样生长因子家族(IGFs))是由两种低分子多肽(IGF-1、IGF-2)、两类特异性受体及六种结合蛋白组成。人体内许多组织都可以合成、分泌胰岛素样生长因子,但循环中的IGF则主要是由肝脏分泌的。胰岛素样生长因子家族的生物学功能是通过与特异性的靶细胞表面的胰岛素样生长因子受体结合而实现的。已发现有两种结构完全不同的IGF受体：IGF-1受体和IGF-2受体(即甘露糖-6磷酸受体)分别又称1型受体和2型受体。正常情况下,IGF与其结合蛋白的亲和力大于或近似等于与其受体的结合,加之高亲和力受体低表达,使少量游离IGF与大量IGF/IGFBP复合物处于平衡状态。IGFs的合成与分泌受血液中生长激素水平的控制,循环中IGFs对生长激素的分泌具有负反馈调节作用,从而形成了一个生长激素—胰岛素样生长因子轴,其对机体组织的分化、增殖及物质代谢具有重要的调节作用。以往研究表明,借助角膜上皮细胞表面的细胞表型标记细胞角蛋白K3及K12或连接蛋白43可对角膜缘干细胞与角膜上皮细胞进行区分,并且此类因子与角膜缘干细胞的增殖及分化亦相关,且还有研究报道说,角膜上皮细胞培养液的上层部分可促使多种干细胞包括毛囊干细胞、间充质干细胞及胚胎干细胞等,分化为携带角膜上皮细胞标记K12的细胞,但相关的分子机制仍不清楚。目的角膜创伤修复的过程通常包括细胞的激活、增殖、分化,细胞因子的释放,细胞外基质的合成与重塑等,都是由多种细胞因子在时间和空间上高度协调而完成的。明确角膜损伤修复过程中关键的调控分子及其作用机制,对于角膜损伤后减少瘢痕形成、促进角膜愈合具有重要意义。本研究中我们利用小鼠角膜机械损伤模型对损伤后角膜上皮细胞与角膜缘干细胞间的相互作用分子机制进行初步探究。方法构建小鼠单侧眼角膜机械损伤模型,对侧角膜为对照组,不同时间点采集小鼠双侧角膜中央及角膜缘组织,进行细胞培养并基因检测EGF、FGF-b、KGF、HGF、TGF-b1、IGF-1及IGF-2等细胞因子不同程度的反应性改变,探究这些细胞因子对角膜缘干细胞分化及成纤维细胞转化的影响,并探讨mTOR/STAT3信号通路在IGF-2调控角膜上皮细胞增殖分化过程中的作用。结果1.角膜上皮损伤后,小鼠眨眼加剧,有畏光的现象,角膜上有不规则的斑块状结构；对损伤角膜组织行苏木精-伊红(HE)染色后显示,针尖破坏角膜上皮层,而基质层未受损,提示我们所采用的角膜机械损伤模型方法可行性良好。2.中央角膜损伤后(0、3、6、12及24h)采集受损侧小鼠角膜组织,实时PCR检测相关细胞因子的基因表达,损伤后3h及6h EGF、FGF-b、KGF、HGF、TGF-b1、IGF-1及IGF-2的基因表达水平升高,IGF-2因子的水平损伤后3h最高,并且显著高于其他各种检测的细胞因子(p0.05)3.角膜损伤后角膜缘干细胞组织较角膜上皮细胞中两种受体的基因表达水平显著增加,3h后其表达差异均具有统计学意义(p0.05)。4.IGF-2对角膜上皮细胞的增殖有促进作用,并且随着培养时间延长,IGF-2干预组的增殖能力与对照组比较有明显的区别。从24h开始,IGF-2干预组的角膜上皮细胞迁移能力明显高于对照组,48h更为明显。通过对细胞骨架进行染色观察,发现IGF-2组较对照组有更强的粘附率。5.胰岛素样生长因子2(IGF-2)在角膜损伤后迅速大量表达,同时促使角膜缘细胞IGF2受体的表达增加并诱导角膜缘干细胞分化为带有K12标记的角膜上皮细胞。6.免疫荧光染色分析显示受损角膜组织细胞中α-平滑肌肌动蛋白水平显著增加,提示可能存在成纤维细胞向肌成纤维细胞的转化。7.检测磷酸化mTOR与磷酸化STAT3蛋白表达的变化,发现p-mTOR与p-STAT3蛋白的表达均随IGF-2浓度的增加而增多。8.使用IGF-2后,检测EGF、FGF-b、KGF、HGF及TGF-bl等细胞因子损伤相关蛋白的表达变化,发现这几个细胞因子在受到IGF-2刺激后,其蛋白表达水平均较对照组有显著的升高,但当同时再给予mTOR/STAT3信号通路抑制剂RAPA50 μmol/L后,发现上述几个细胞因子的蛋白表达被明显抑制。结论1.小鼠单侧眼角膜机械损伤模型具有可重复性,角膜上皮能完全修复,模型制作成功,可用于对角膜上皮损伤修复过程的研究。2.角膜机械损伤后角膜上皮中IGF-2因子表达明显上调,角膜缘组织中IGF-2受体表达增加,其共同作用促使角膜缘干细胞向角膜上皮细胞转化,同时在成纤维细胞向肌成纤维细胞的转化中起一定的作用,对角膜损伤修复有促进作用。3.在体外培养状态下,特异性的阻断mTOR/STAT3信号通路可以阻断IGF-2对角膜上皮细胞的修复作用,IGF-2对角膜上皮细胞修复的这种效应可能是通过激活mTOR/STAT3信号通路而发挥作用。
[Abstract]:The background of the corneal epithelium is the first barrier against external harmful pathogenic factors of eye trauma in the invasion, can quickly repair and healing, when healing delay, cause vision loss, leading to severe blindness. At present some animal experiments and clinical reports on the cornea skin itself has a self renewal and repair ability, but the cornea after the injury does not play a major role in corneal epithelium but from the limbal tissue, the tissue contained limbal stem cells (LSC), corneal limbal stem cells through cell proliferation, differentiation and migration to repair the damage of corneal tissue. Reports of corneal limbal stem cell deficiency when the corneal repair function will be limit, and also with the function of cornea and conjunctiva cornea repair, angiogenesis, inflammation and chronic persistent corneal epithelial damage and other factors are closely related, in the Such factors continue to exist, and ultimately can not repair the cornea can have serious consequences that blindness, and research shows that we can treat by the transplantation of limbal and corneal limbal corneal injury, suggesting that the limbal stem cells play an important role in corneal trauma repair process after corneal injury. That limbal stem cells begin to proliferate, and differentiate into transitional cells, these cells can migrate to sites of damage and induce expression of corneal epithelial cell markers and promote corneal wound healing. However, corneal epithelial cells and corneal limbal stem cells and the molecular pathway of the interaction mechanism between tissues is unclear factor gene.Jia reports of corneal mechanical or chemical injury in cells responsible for growth and differentiation was significantly enhanced, including transforming growth factor alpha (TGF- alpha), transforming growth factor (Zi T GF- beta), epidermal growth factor (EGF), heparin like growth factor (HGF), fibroblast growth factor beta (FGF- beta), these factors can be generated by corneal epithelial cells, they are involved in the regulation of cell proliferation and growth. In addition, insulin-like growth factor 2 (IGF-2) has also been shown to promote keratin cell proliferation, adhesion and connection to generate N cadherin protein increased. The increase of extracellular matrix, which prompted the formation of keratin collagen. Insulin-like growth factor signaling pathway has been demonstrated to be associated with cell proliferation and differentiation in cell and organ level growth regulation plays an important role in IGF, and other cells above regulatory factor and limbal stem cell proliferation, differentiation and migration associated insulin-like growth factor family (IGFs)) is composed of two kinds of low molecular peptides (IGF-1, IGF-2), two types and six kinds of specific receptor binding protein White. Many organizations in the human body can synthesis, secretion of insulin-like growth factor, but the cycle of IGF is mainly secreted by the liver. The biological function of insulin-like growth factor family is through the surface and the specific target cells of insulin-like growth factor receptor binding and has been found to have two. The structure of different IGF receptors: IGF-1 receptors and IGF-2 receptors (i.e. -6 phosphate mannose receptor) were also called type 1 receptor and type 2 receptor. Under normal circumstances, the IGF binding protein affinity is greater than or equal to the approximate binding to its receptor, coupled with the high affinity receptor of low expression, so that a small amount of free IGF and a large number of IGF/IGFBP composite the thing is in equilibrium state of synthesis and secretion of.IGFs by controlling the level of growth hormone in blood circulation in IGFs has a negative feedback effect on the secretion of growth hormone, thus forming a student Growth hormone - insulin-like growth factor axis, the body tissue differentiation, plays an important role in the regulation of proliferation and metabolism. Previous studies showed that the cell phenotypic markers of cell surface corneal epithelial cells and keratin K3 K12 or connexin 43 of limbal stem cells and corneal epithelial cells were distinguished, and such factors and limbal stem cell proliferation and differentiation are related, and there are reports that the upper part of corneal epithelial cell culture solution can promote a variety of stem cells including hair follicle stem cells, mesenchymal stem cells and embryonic stem cells, differentiate into corneal epithelial cells with labeled K12 cells, but the molecular mechanism the purpose of the process is still not clear. Corneal wound repair usually include cell activation, proliferation, differentiation, cytokine release, extracellular matrix synthesis and remodeling, are made of a variety of cells Factor in time and space are highly coordinated and completed. Clear corneal injury and its mechanism of key regulatory molecules in the repair process, to reduce the scar formation after corneal injury, which is of great significance to the corneal healing. In this study, we use the model of corneal mechanical damage on mice interaction molecular mechanism between cells of corneal epithelial cells and corneal limbal stem injury study. Methods corneal mechanical injury model of mice unilateral construction, contralateral cornea as control group were collected at different time points of bilateral central cornea and limbus tissue, cell culture and gene detection of EGF, FGF-b, KGF, HGF, TGF-b1, IGF-1 and IGF-2 cytokine responses in different degrees change into these cytokines of limbal stem cell differentiation and the effects of fibroblast transformation, and to explore the mTOR/STAT3 signaling pathway in the regulation of IGF-2. Membrane epithelial cell proliferation and differentiation process. Results of the 1. corneal epithelium after injury, mice have the phenomenon of increased blink, photophobia, a plaque like structure of irregular cornea; damage to corneal tissue by hematoxylin eosin (HE) staining showed that the needle destruction of corneal epithelium. The stromal layer is not damaged, the feasibility that corneal mechanical injury model we adopt the method of good.2. central cornea after injury (0,3,6,12 and 24h) acquisition damaged corneal tissue side of mice, expression was detected by real-time PCR cytokines related genes, 6h and 3h after injury EGF, FGF-b, KGF, HGF, TGF-b1, IGF-1 and IGF-2 gene expression level increased, the level of the damage factor IGF-2 after 3H is the highest, and was significantly higher than that of other kinds of cytokine detection (P0.05) 3. corneal limbal stem cells with two receptors of corneal epithelial cells in a tissue gene expression level increased 3 After h the expression differences were statistically significant (P0.05.4.IGF-2) on the proliferation of corneal epithelial cells has a role in promoting, and with the extension of the culture time, the proliferation of IGF-2 in the intervention group compared with the control group have significant difference. Starting from 24h, corneal epithelial cell migration ability of IGF-2 in the intervention group was significantly higher than the control group, 48h the staining is obvious. Through the cytoskeleton, found in IGF-2 group than in the control group has a stronger adhesion rate of.5. insulin-like growth factor 2 (IGF-2) quickly expressed in corneal limbal cells and induce the expression of IGF2 receptor increased and induce limbal stem cell differentiation into corneal epithelial cells.6. staining immunofluorescence with the mark of K12 analysis show that the damaged corneal tissue cells of alpha smooth muscle actin levels increased significantly, suggesting the transformation of fibroblasts fibroblasts to muscle. 7. expression detection of phosphorylated mTOR and phosphorylated STAT3 protein, expression of p-mTOR and p-STAT3 protein was increased with the concentration of IGF-2 and.8. increased after using IGF-2, FGF-b, KGF, EGF detection, the expression of HGF protein and cytokines such as TGF-bl damage, found that several cytokines in response to IGF-2 later, the expression level of the protein were higher than those in control group were significantly increased, but also when given the mTOR/STAT3 signaling pathway inhibitor RAPA50 mol/L, found that the expression of several cytokines protein was inhibited. Conclusion 1. mice with unilateral corneal mechanical injury model with repeatability, corneal epithelium can be fully restored, the success of the model that can be used for the upregulated expression of IGF-2 factor of.2. on the process of corneal wound healing of the cornea after mechanical injury of corneal epithelium, the expression of IGF-2 receptor increased the total limbal tissues The same effect to limbal stem cells into corneal epithelial cells, and in fibroblasts to muscle plays a certain role in transforming fiber cells, promote the role of.3. in vitro on corneal wound healing, the specificity of blocking the mTOR/STAT3 signaling pathway can inhibit the effect of IGF-2 on the repair of corneal epithelial cells, the effect of IGF-2 on the repair of corneal epithelial cells may play a role through the activation of the mTOR/STAT3 pathway.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R77

【相似文献】