本文選題：糖尿病 + 丙酮醛　； 參考：《廣州醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景晚期糖基化終末產(chǎn)物(Advanced glycation end products,AGEs)堆積是糖尿病(Diabetes mellitus,DM)皮膚并發(fā)癥的重要原因。丙酮醛(Methyglyoxal,MGO)是一種活性二羰基化合物,是AGEs產(chǎn)生的重要中間體,在糖尿病的病理過程中能生成AGEs。N-乙酰-L-半胱氨酸(N-acetyl-cysteine,NAC)是某些臨床常用藥的主要活性成分,參與內(nèi)源性還原型谷胱甘肽(reduced glutathione,GSH)和硫化氫(hydrogen sulfide,H2S)的生成。然而,NAC可否通過抑制AGEs的產(chǎn)生或其毒性,減輕DM誘導(dǎo)的皮膚損傷仍鮮有報道。因而,本研究旨在觀察NAC對MGO誘導(dǎo)的糖尿病性皮膚細(xì)胞損傷的影響及分子機(jī)制。方法應(yīng)用酶聯(lián)免疫吸附試驗(enzyme-linked immunosorbent assay,ELISA)法檢測糖尿病患者和健康志愿者血漿中AGEs的含量。用牛血清白蛋白(bovine serum albumin,BSA)與MGO于37℃孵育3 d制備AGEs。根據(jù)AGEs具有自發(fā)熒光和形成棕色溶液的特點(diǎn),分別用熒光酶標(biāo)儀檢測340/465 nm反應(yīng)體系中熒光強(qiáng)度以及觀察BSA與MGO混合溶液的顏色;進(jìn)一步觀察NAC對MGO誘導(dǎo)的AGEs生成的影響。人永生化角質(zhì)形成細(xì)胞(Human adult low calcuim kertinocytes,Ha Ca T)給予MGO處理,模擬糖尿病血液或組織中高M(jìn)GO狀態(tài),建立糖尿病皮膚傷口愈合障礙的體外模型。從細(xì)胞活力、線粒體膜電位、細(xì)胞的粘附和遷移功能、炎癥因子的分泌、核因子κB(nuclear factor kappa B,NF-κB)亞基p65的核轉(zhuǎn)位、基質(zhì)金屬蛋白酶9(matrix metalloproteinase 9,MMP-9)的表達(dá)以及細(xì)胞外基質(zhì)的含量進(jìn)行評價。在MGO處理Ha Ca T細(xì)胞前,用NAC預(yù)處理1 h,觀察NAC對MGO誘導(dǎo)的上述指標(biāo)改變的影響,以明確其皮膚細(xì)胞保護(hù)效應(yīng)。最后,通過檢測AGEs的受體(receptor for advanced end products,RAGE)的表達(dá),以及其中和抗體(Neutralizing antibody against RAGE,N-ABR)預(yù)處理對MGO誘導(dǎo)的Ha Ca T細(xì)胞損傷的影響,最終揭示MGO誘導(dǎo)的細(xì)胞損傷以及NAC的細(xì)胞保護(hù)效應(yīng)是否依賴于RAGE的激活。結(jié)果1.糖尿病患者血漿中AGEs的含量高于正常人,差異具有統(tǒng)計學(xué)意義(P0.05)。2.MGO和BSA反應(yīng)后,溶液顏色加深,熒光增強(qiáng),提示MGO可誘導(dǎo)AGEs的生成。NAC處理可以降低溶液的顏色和熒光強(qiáng)度(P0.01),提示其可以抑制MGO誘導(dǎo)的AGEs生成。另外,用MGO處理Ha Ca T細(xì)胞,可使細(xì)胞培養(yǎng)基中AGEs含量增加,NAC預(yù)處理能明顯抑制MGO誘導(dǎo)的AGEs生成,差異具有統(tǒng)計學(xué)意義(P0.01)。3.用MGO處理Ha Ca T細(xì)胞,可以劑量依賴性地降低細(xì)胞活力、損害線粒體膜電位和細(xì)胞的粘附和遷移等行為學(xué)功能,這種損害效應(yīng)可以被NAC預(yù)處理所減弱。提示NAC可減弱MGO誘導(dǎo)細(xì)胞損傷。4.Ha Ca T細(xì)胞經(jīng)MGO處理后,炎癥因子IL-6和IL-8的分泌增加、NF-κB亞基p65核轉(zhuǎn)位增強(qiáng),NAC預(yù)處理能抑制MGO誘導(dǎo)的炎癥因子分泌和NF-κB亞基p65核轉(zhuǎn)位。提示NAC能抑制MGO誘導(dǎo)的炎癥反應(yīng)。5.MGO處理Ha Ca T細(xì)胞可導(dǎo)致MMP-9的表達(dá)上調(diào)以及I型膠原的生成減少,這種效應(yīng)能部分被NAC預(yù)處理逆轉(zhuǎn)。提示NAC改善MGO誘導(dǎo)的細(xì)胞遷移障礙可能與改善細(xì)胞外基質(zhì)的水平有關(guān)。6.MGO處理可上調(diào)Ha Ca T細(xì)胞內(nèi)RAGE受體的表達(dá),NAC預(yù)處理可以抑制MGO誘導(dǎo)的RAGE受體過表達(dá)。重要的是,用RAGE中和抗體阻礙其作用后,MGO引起的細(xì)胞受損,炎癥因子釋放,MMP-9過度表達(dá)以及NF-κB亞基p65核轉(zhuǎn)位均被部分改善。提示MGO誘導(dǎo)的皮膚細(xì)胞炎癥損傷與RAGE受體的激活有關(guān),而NAC拮抗MGO的損傷作用是通過抑制RAGE受體實現(xiàn)的。結(jié)論本文研究證實,NAC可減弱MGO誘導(dǎo)的皮膚細(xì)胞損傷,其機(jī)制與抑制AGEs的生成和AGEs/RAGE/NF-κB信號通路的活化有關(guān)。本文的研究為臨床應(yīng)用NAC相關(guān)的藥物防治糖尿病皮膚并發(fā)癥提供了基礎(chǔ)資料與理論支持。
[Abstract]:The accumulation of advanced glycosylation end products (Advanced glycation end products, AGEs) is an important reason for the skin complications of diabetes (Diabetes mellitus, DM). The acetaldehyde (Methyglyoxal, MGO) is a kind of active two carbonyl compound, an important intermediate in AGEs production, and can produce cysteine acetyl acetate in the pathological process of diabetes. N-acetyl-cysteine (NAC) is the main active component of some common clinical drugs, and participates in the production of endogenous reduced glutathione (GSH) and hydrogen sulfide (hydrogen sulfide, H2S). However, it is still rarely reported that NAC can reduce DM induced skin damage by inhibiting the production of AGEs or its toxicity. The effects of NAC on MGO induced diabetic skin cell damage and its molecular mechanism were observed. Methods the content of AGEs in plasma of diabetic patients and healthy volunteers was detected by enzyme linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) and incubated with bovine serum albumin (bovine serum albumin, BSA) and MGO at 37. Es. based on the characteristics of AGEs with spontaneous fluorescence and the formation of brown solution, the fluorescence intensity of 340/465 nm reaction system and the color of BSA and MGO mixed solution were observed by fluorescent enzyme labeling, and the effect of NAC on MGO induced AGEs formation was further observed. T) give MGO treatment, simulate the high MGO state of diabetic blood or tissue, establish an in vitro model of diabetic skin wound healing disorder. From cell viability, mitochondrial membrane potential, cell adhesion and migration, secretion of inflammatory factors, nuclear factor kappa B (nuclear factor kappa B, NF- kappa B) subunit p65, matrix metalloproteinase 9 (matr) The expression of IX metalloproteinase 9, MMP-9) and the content of extracellular matrix were evaluated. Before MGO Ha Ca T cells were treated with NAC, 1 h was pretreated with NAC to observe the effect of NAC on MGO induced changes of the above index, in order to clarify the protective effect of its skin cells. As well as the effects of Neutralizing antibody against RAGE (N-ABR) pretreatment on MGO induced Ha Ca T cell damage, and finally revealed whether MGO induced cell damage and NAC cell protection effect depended on RAGE activation. Results 1. diabetic patients were higher than normal people in plasma, and the difference was statistically significant. After the reaction of P0.05.2.MGO and BSA, the color of the solution was deepened and the fluorescence enhanced, suggesting that MGO could induce the formation of AGEs by.NAC treatment to reduce the color and fluorescence intensity of the solution (P0.01), suggesting that it could inhibit the AGEs generation induced by MGO. Furthermore, the MGO processing of Ha Ca cells could increase the content of the cell culture medium, and the pretreatment could be obvious. Inhibition of MGO induced AGEs generation, the difference has statistical significance (P0.01),.3. using MGO to treat Ha Ca T cells, can reduce cell viability in a dose-dependent manner, damage mitochondrial membrane potential and cell adhesion and migration and other behavioral functions, this damage effect can be reduced by NAC preconditioning. NAC can weaken MGO induced cell damage.4.Ha. The secretion of inflammatory factors IL-6 and IL-8 increased after MGO treatment, and the nuclear transposition of NF- kappa B subunit was enhanced. NAC preprocessing could inhibit the secretion of inflammatory factors induced by MGO and NF- kappa B subunit nuclear transposition. Less, this effect can be partially reversed by NAC pretreatment. Suggesting that NAC improves MGO induced cell migration disorders may be associated with the improvement of the level of extracellular matrix,.6.MGO treatment can up regulate the expression of RAGE receptors in Ha Ca T cells. NAC preconditioning can inhibit RAGE receptor overexpression induced by MGO. It is important that the action of RAGE neutralize antibodies to obstruct its action. Later, MGO induced cell damage, inflammatory factors release, MMP-9 overexpression and NF- kappa B subunit p65 nuclear transposition were partially improved. It suggests that MGO induced skin cell inflammation damage is related to the activation of RAGE receptor, and NAC's antagonistic effect on MGO is achieved by inhibiting RAGE receptor. Conclusion this study confirms that NAC can weaken MGO induced. The mechanism of skin cell damage is related to the inhibition of the formation of AGEs and the activation of the AGEs/RAGE/NF- kappa B signaling pathway. This study provides basic information and theoretical support for the clinical application of NAC related drugs in the prevention and treatment of diabetic skin complications.

【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R96

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