神经肽FF在糖尿病角膜病变中的作用及分子机制研究
发布时间：2018-08-08 16:11
【摘要】：目的： 糖尿病角膜病变广泛存在于糖尿病患者中,危害较大且至今仍缺乏理想的防治措施,目前认为角膜的神经病变使其感觉、营养和代谢功能障碍,是引起角膜病反复发作、难治疗的根本原因。通过PCR array技术对2型糖尿病模型BKS.Cg-m+/+Leprdb/J小鼠(简称BKS-db/db小鼠)三叉神经节中神经营养因子及其受体的差异基因筛选,我们发现神经肽FF (Neuropeptide FF, NPFF)表达显著下调。本研究旨在进一步研究NPFF在BKS-db/db小鼠角膜和三叉神经节中的表达及定位情况,并探讨NPFF在糖尿病角膜病变中的作用及相关分子机制。 方法： 1、体内动物实验 本研究体内动物实验采用24w龄BKS-db/db小鼠及其对照db/+小鼠各40只,通过角膜知觉检测、泪液分泌实验以及β-tublin角膜神经染色客观评价糖尿病角膜病变的发生情况。通过神经逆行示踪方法,结膜下注射荧光金标记三叉神经节眼支来源的神经节细胞,采用免疫组化和荧光技术显示NPFF及其受体在BKS-db/db小鼠及其对照鼠三叉神经节的细胞定位及表达,并通过ELISA检测NPFF蛋白在角膜和三叉神经节的表达情况。在已建立好的BKS-db/db小鼠角膜上皮刮除模型上,结膜下注射NPFF或者空白对照药物PBS,通过荧光素钠染色动态观察角膜上皮愈合的情况,通过角膜知觉、以及角膜神经染色评价角膜末梢神经修复情况。 2、体外细胞实验 体外培养小鼠角膜缘干细胞系TKE2以及原代培养小鼠三叉神经节细胞,实验设置6个组：表皮生长因子(Epidermal Growth Factor, EGF)阳性对照组、甘露醇渗透压对照、高糖培养、高糖联合10μmol/L浓度NPFF,高糖联合100μmol/L浓度NPFF、PBS培养组。通过细胞划痕实验检测NPFF对TKE2细胞迁移和增殖能力的影响,通过实时定量PCR、western blot实验检测AKT、ERK1/2等信号通路基因和蛋白的表达。通过Live/Dead试剂盒检测NPFF对体外培养的三叉神经节细胞生长及存活情况的影响。 结果： 通过血糖及体重等一般生长指标监测,以及角膜知觉检测和神经染色,24w龄BKS-db/db小鼠均出现糖尿病角膜病变的典型特征：泪液分泌异常、角膜知觉减退和末梢神经异常等。通过荧光金逆行示踪,成功标记三叉神经节中眼支来源的神经节细胞,并在胞体中检测到NPFF及其受体的表达。免疫荧光、免疫组化、RT-PCR及ELISA检测显示,与db/+对照小鼠比较,NPFF在BKS-db/db小鼠中表达显著下降。与PBS对照组相比,结膜下注射NPFF可以显著促进BKS-db/db小鼠角膜上皮损伤后的愈合,并可以促进角膜末梢神经的修复及其功能恢复。细胞划痕实验证实NPFF能够显著促进高糖环境下TKE2细胞的迁移和增殖,Live/Dead检测显示NPFF还能明显延长体外培养的三叉神经节细胞的存活时间；western blot初步证实NPFF可以在处理5min后特异性激活ERK1/2信号通路,该信号通路与细胞增殖和生长有关,而对AKT信号通路没有明显影响。 结论： 24w龄BKS-db/db小鼠出现典型糖尿病角膜病变的特征,可以作为研究2型糖尿病角膜病变的动物模型。NPFF在2型糖尿病模型BKS-db/db小鼠角膜和三叉神经节中表达显著下降,体内和体外实验表明NPFF具有促进角膜末梢神经损伤修复和功能恢复以及延长三叉神经节细胞体外存活时间的作用,NPFF可能是一种潜在的神经保护因子,为糖尿病角膜病变的防治提供了新的研究思路,但具体作用机制仍需进一步研究。
[Abstract]:Objective:
Diabetic keratopathy is widely found in diabetic patients, and it is very harmful and still lacks the ideal control measures. At present, corneal neuropathy is considered as the root cause of recurrent attacks of keratopathy and difficult to treat. PCR array technique is used for the model of type 2 diabetes mellitus (BKS.Cg-m+/+Leprdb/J). We found the differential expression of neuropeptide FF (Neuropeptide FF, NPFF) in the trigeminal ganglia of mice (BKS-db/db mice). The purpose of this study was to further study the expression and localization of NPFF in the cornea and trigeminal ganglia of BKS-db/db mice, and to explore NPFF in the diabetic angle. The role of the membrane lesions and the related molecular mechanisms.
Method:
1, in vivo animal experiment
In this study, 24W aged BKS-db/db mice and 40 control db/+ mice were used in this study. The occurrence of diabetic keratopathy was objectively evaluated by corneal perception detection, tear secretion experiment and beta -tublin corneal nerve staining. The source of trigeminal ganglion eye branches was marked by subconjunctival injection of fluorescent gold. In the ganglion cells, the localization and expression of NPFF and its receptor in the trigeminal ganglia of BKS-db/db mice and their control rats were demonstrated by immunohistochemistry and fluorescence technique, and the expression of NPFF protein in the cornea and trigeminal ganglia was detected by ELISA. In the established corneal epithelial curettage model of the BKS-db/db rat, NPF was injected under the conjunctiva. F or a blank control drug, PBS, was used to dynamically observe the healing of corneal epithelium by fluorescein sodium staining, and to evaluate the repair of the peripheral nerve by corneal sensation and corneal nerve staining.
2, in vitro cell test
The mouse corneal limbal stem cell line TKE2 and the primary culture mouse trigeminal ganglion cells were cultured in vitro. The experiment set 6 groups: epidermal growth factor (Epidermal Growth Factor, EGF) positive control group, mannitol osmotic pressure control, high sugar culture, high glucose combined with 10 micron mol/L concentration NPFF, high glucose combined with 100 u mol/L concentration NPFF, PBS culture group. The effect of NPFF on the migration and proliferation of TKE2 cells was detected by cell scratch test. The expression of AKT, ERK1/2 and other signaling genes and proteins were detected by real-time quantitative PCR and Western blot. The effect of NPFF on the growth and survival of trigeminal ganglion cells cultured in vitro was detected by Live/Dead kit.
Result:
Through the monitoring of general growth indicators such as blood sugar and weight, as well as corneal perception detection and nerve staining, the typical characteristics of diabetic keratopathy in 24W BKS-db/db mice were characterized by abnormal secretion of tear, degeneration of corneal perception, and abnormality of peripheral nerve. The nerve of the trigeminal ganglia was successfully traced by retrograde tracing of fluorescent gold and the nerve of the branches of the branches of the trigeminal ganglia were successfully labelled The expression of NPFF and its receptor in the cell body was detected. The immunofluorescence, immunohistochemistry, RT-PCR and ELISA showed that the expression of NPFF in the BKS-db/db mice decreased significantly. Compared with the PBS control group, subconjunctival injection of NPFF could significantly promote the healing of corneal epithelial injury in BKS-db/db mice. Promoting the repair and functional recovery of the peripheral nerve of the cornea. The cell scratching proved that NPFF could significantly promote the migration and proliferation of TKE2 cells in high glucose environment. Live/Dead detection showed that NPFF could significantly prolong the survival time of the trigeminal ganglion cells in vitro; Western blot preliminarily confirmed that NPFF could be specific after 5min. Activation of ERK1/2 signaling pathway is related to cell proliferation and growth, but has no significant effect on AKT signaling pathway.
Conclusion:
24W age BKS-db/db mice have the characteristics of typical diabetic keratopathy, which can be used as an animal model for the study of type 2 diabetic keratopathy. The expression of.NPFF in the corneal and trigeminal ganglia of type 2 diabetes model mice is significantly decreased. In vivo and in vitro experiments show that NPFF can promote the repair of corneal end nerve injury and function recovery. As well as the effect of prolonging the survival time of trigeminal ganglion cells in vitro, NPFF may be a potential neuroprotective factor, which provides new research ideas for the prevention and treatment of diabetic keratopathy, but the specific mechanism still needs to be further studied.
【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R587.2;R772.2
本文编号：2172322
[Abstract]:Objective:
Diabetic keratopathy is widely found in diabetic patients, and it is very harmful and still lacks the ideal control measures. At present, corneal neuropathy is considered as the root cause of recurrent attacks of keratopathy and difficult to treat. PCR array technique is used for the model of type 2 diabetes mellitus (BKS.Cg-m+/+Leprdb/J). We found the differential expression of neuropeptide FF (Neuropeptide FF, NPFF) in the trigeminal ganglia of mice (BKS-db/db mice). The purpose of this study was to further study the expression and localization of NPFF in the cornea and trigeminal ganglia of BKS-db/db mice, and to explore NPFF in the diabetic angle. The role of the membrane lesions and the related molecular mechanisms.
Method:
1, in vivo animal experiment
In this study, 24W aged BKS-db/db mice and 40 control db/+ mice were used in this study. The occurrence of diabetic keratopathy was objectively evaluated by corneal perception detection, tear secretion experiment and beta -tublin corneal nerve staining. The source of trigeminal ganglion eye branches was marked by subconjunctival injection of fluorescent gold. In the ganglion cells, the localization and expression of NPFF and its receptor in the trigeminal ganglia of BKS-db/db mice and their control rats were demonstrated by immunohistochemistry and fluorescence technique, and the expression of NPFF protein in the cornea and trigeminal ganglia was detected by ELISA. In the established corneal epithelial curettage model of the BKS-db/db rat, NPF was injected under the conjunctiva. F or a blank control drug, PBS, was used to dynamically observe the healing of corneal epithelium by fluorescein sodium staining, and to evaluate the repair of the peripheral nerve by corneal sensation and corneal nerve staining.
2, in vitro cell test
The mouse corneal limbal stem cell line TKE2 and the primary culture mouse trigeminal ganglion cells were cultured in vitro. The experiment set 6 groups: epidermal growth factor (Epidermal Growth Factor, EGF) positive control group, mannitol osmotic pressure control, high sugar culture, high glucose combined with 10 micron mol/L concentration NPFF, high glucose combined with 100 u mol/L concentration NPFF, PBS culture group. The effect of NPFF on the migration and proliferation of TKE2 cells was detected by cell scratch test. The expression of AKT, ERK1/2 and other signaling genes and proteins were detected by real-time quantitative PCR and Western blot. The effect of NPFF on the growth and survival of trigeminal ganglion cells cultured in vitro was detected by Live/Dead kit.
Result:
Through the monitoring of general growth indicators such as blood sugar and weight, as well as corneal perception detection and nerve staining, the typical characteristics of diabetic keratopathy in 24W BKS-db/db mice were characterized by abnormal secretion of tear, degeneration of corneal perception, and abnormality of peripheral nerve. The nerve of the trigeminal ganglia was successfully traced by retrograde tracing of fluorescent gold and the nerve of the branches of the branches of the trigeminal ganglia were successfully labelled The expression of NPFF and its receptor in the cell body was detected. The immunofluorescence, immunohistochemistry, RT-PCR and ELISA showed that the expression of NPFF in the BKS-db/db mice decreased significantly. Compared with the PBS control group, subconjunctival injection of NPFF could significantly promote the healing of corneal epithelial injury in BKS-db/db mice. Promoting the repair and functional recovery of the peripheral nerve of the cornea. The cell scratching proved that NPFF could significantly promote the migration and proliferation of TKE2 cells in high glucose environment. Live/Dead detection showed that NPFF could significantly prolong the survival time of the trigeminal ganglion cells in vitro; Western blot preliminarily confirmed that NPFF could be specific after 5min. Activation of ERK1/2 signaling pathway is related to cell proliferation and growth, but has no significant effect on AKT signaling pathway.
Conclusion:
24W age BKS-db/db mice have the characteristics of typical diabetic keratopathy, which can be used as an animal model for the study of type 2 diabetic keratopathy. The expression of.NPFF in the corneal and trigeminal ganglia of type 2 diabetes model mice is significantly decreased. In vivo and in vitro experiments show that NPFF can promote the repair of corneal end nerve injury and function recovery. As well as the effect of prolonging the survival time of trigeminal ganglion cells in vitro, NPFF may be a potential neuroprotective factor, which provides new research ideas for the prevention and treatment of diabetic keratopathy, but the specific mechanism still needs to be further studied.
【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R587.2;R772.2
【参考文献】
相关期刊论文 前1条
1 刘晓燕;朱学军;;糖尿病性角膜神经病变的研究进展[J];国际眼科杂志;2008年07期
，本文编号：2172322
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