【摘要】：目的： 糖尿病角膜病變廣泛存在于糖尿病患者中,危害較大且至今仍缺乏理想的防治措施,目前認為角膜的神經(jīng)病變使其感覺、營養(yǎng)和代謝功能障礙,是引起角膜病反復(fù)發(fā)作、難治療的根本原因。通過PCR array技術(shù)對2型糖尿病模型BKS.Cg-m+/+Leprdb/J小鼠(簡稱BKS-db/db小鼠)三叉神經(jīng)節(jié)中神經(jīng)營養(yǎng)因子及其受體的差異基因篩選,我們發(fā)現(xiàn)神經(jīng)肽FF (Neuropeptide FF, NPFF)表達顯著下調(diào)。本研究旨在進一步研究NPFF在BKS-db/db小鼠角膜和三叉神經(jīng)節(jié)中的表達及定位情況,并探討NPFF在糖尿病角膜病變中的作用及相關(guān)分子機制。 方法： 1、體內(nèi)動物實驗 本研究體內(nèi)動物實驗采用24w齡BKS-db/db小鼠及其對照db/+小鼠各40只,通過角膜知覺檢測、淚液分泌實驗以及β-tublin角膜神經(jīng)染色客觀評價糖尿病角膜病變的發(fā)生情況。通過神經(jīng)逆行示蹤方法,結(jié)膜下注射熒光金標記三叉神經(jīng)節(jié)眼支來源的神經(jīng)節(jié)細胞,采用免疫組化和熒光技術(shù)顯示NPFF及其受體在BKS-db/db小鼠及其對照鼠三叉神經(jīng)節(jié)的細胞定位及表達,并通過ELISA檢測NPFF蛋白在角膜和三叉神經(jīng)節(jié)的表達情況。在已建立好的BKS-db/db小鼠角膜上皮刮除模型上,結(jié)膜下注射NPFF或者空白對照藥物PBS,通過熒光素鈉染色動態(tài)觀察角膜上皮愈合的情況,通過角膜知覺、以及角膜神經(jīng)染色評價角膜末梢神經(jīng)修復(fù)情況。 2、體外細胞實驗 體外培養(yǎng)小鼠角膜緣干細胞系TKE2以及原代培養(yǎng)小鼠三叉神經(jīng)節(jié)細胞,實驗設(shè)置6個組：表皮生長因子(Epidermal Growth Factor, EGF)陽性對照組、甘露醇滲透壓對照、高糖培養(yǎng)、高糖聯(lián)合10μmol/L濃度NPFF,高糖聯(lián)合100μmol/L濃度NPFF、PBS培養(yǎng)組。通過細胞劃痕實驗檢測NPFF對TKE2細胞遷移和增殖能力的影響,通過實時定量PCR、western blot實驗檢測AKT、ERK1/2等信號通路基因和蛋白的表達。通過Live/Dead試劑盒檢測NPFF對體外培養(yǎng)的三叉神經(jīng)節(jié)細胞生長及存活情況的影響。 結(jié)果： 通過血糖及體重等一般生長指標監(jiān)測,以及角膜知覺檢測和神經(jīng)染色,24w齡BKS-db/db小鼠均出現(xiàn)糖尿病角膜病變的典型特征：淚液分泌異常、角膜知覺減退和末梢神經(jīng)異常等。通過熒光金逆行示蹤,成功標記三叉神經(jīng)節(jié)中眼支來源的神經(jīng)節(jié)細胞,并在胞體中檢測到NPFF及其受體的表達。免疫熒光、免疫組化、RT-PCR及ELISA檢測顯示,與db/+對照小鼠比較,NPFF在BKS-db/db小鼠中表達顯著下降。與PBS對照組相比,結(jié)膜下注射NPFF可以顯著促進BKS-db/db小鼠角膜上皮損傷后的愈合,并可以促進角膜末梢神經(jīng)的修復(fù)及其功能恢復(fù)。細胞劃痕實驗證實NPFF能夠顯著促進高糖環(huán)境下TKE2細胞的遷移和增殖,Live/Dead檢測顯示NPFF還能明顯延長體外培養(yǎng)的三叉神經(jīng)節(jié)細胞的存活時間；western blot初步證實NPFF可以在處理5min后特異性激活ERK1/2信號通路,該信號通路與細胞增殖和生長有關(guān),而對AKT信號通路沒有明顯影響。 結(jié)論： 24w齡BKS-db/db小鼠出現(xiàn)典型糖尿病角膜病變的特征,可以作為研究2型糖尿病角膜病變的動物模型。NPFF在2型糖尿病模型BKS-db/db小鼠角膜和三叉神經(jīng)節(jié)中表達顯著下降,體內(nèi)和體外實驗表明NPFF具有促進角膜末梢神經(jīng)損傷修復(fù)和功能恢復(fù)以及延長三叉神經(jīng)節(jié)細胞體外存活時間的作用,NPFF可能是一種潛在的神經(jīng)保護因子,為糖尿病角膜病變的防治提供了新的研究思路,但具體作用機制仍需進一步研究。
[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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R587.2;R772.2

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相關(guān)期刊論文 前1條

1 劉曉燕;朱學(xué)軍;;糖尿病性角膜神經(jīng)病變的研究進展[J];國際眼科雜志;2008年07期

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本文編號：2172322