本文選題：脂多糖 + 眼內(nèi)炎��； 參考：《鄭州大學》2015年博士論文

【摘要】：眼外傷、內(nèi)眼手術(shù)是感染性眼內(nèi)炎的主要原因,多為細菌進入眼內(nèi)導致感染所致,如凝固酶陰性葡萄球菌、金黃色葡萄球菌、鏈球菌、革蘭氏陰性細菌等。細菌性眼內(nèi)炎是嚴重威脅視力的眼科急癥,細菌性眼內(nèi)炎預后受細菌數(shù)量、毒力、患者年齡及免疫狀態(tài)等多種因素影響。雖然及時采取抗生素藥物治療、玻璃體切除術(shù)等治療,但是視力預后仍不理想,常導致嚴重的視功能損害。細菌在增殖、破裂的過程中釋放的毒性物質(zhì)(內(nèi)毒素、外毒素)可誘發(fā)嚴重的炎癥反應及細胞壞死[1,2,3]。其中細菌的裂解產(chǎn)物是誘發(fā)眼內(nèi)炎癥的重要因素之一。脂多糖是革蘭陰性桿菌細胞壁的主要成分,也是其主要毒性致病因子,可用于玻璃體腔注射,誘導感染性眼內(nèi)炎癥反應[4]。炎癥反應導致血眼屏障破壞,炎性細胞向組織間隙遷移浸潤,蛋白滲漏,精密的視網(wǎng)膜結(jié)構(gòu)被破壞,最終導致功能不同程度的喪失。如何消除眼內(nèi)炎中眼內(nèi)組織破壞、改善最終視力預后一直是我們面臨的難題和挑戰(zhàn)。在積極抗菌治療的同時,能否有效并及時地控制眼內(nèi)炎癥反應是治療的關(guān)鍵,也是預后的決定性因素。利魯唑(Riluzole)屬于苯并噻唑類衍生物,1996年開始作為一種新型谷氨酸釋放抑制劑被批準應用于肌萎縮性脊髓側(cè)索硬化癥(amyotrophic lateral sclerosis,ALS)的治療,其穩(wěn)定細胞內(nèi)外離子濃度的作用使其在神經(jīng)保護、抗驚厥、抗抑郁等方面具有廣泛的作用,提示其在更多疾病中的臨床應用價值。根據(jù)其藥理機制,我們推測其可能在眼內(nèi)炎癥控制中起到積極作用。本研究擬觀察利魯唑?qū)χ嗵钦T導大鼠眼內(nèi)炎癥的干預作用,并對其機制進行初步研究。目的通過玻璃體腔注射大腸桿菌脂多糖(LPS,lipopolysaccharide from Escherichia coli)建立大鼠眼內(nèi)炎模型,觀察其炎癥表現(xiàn)、組織病理學特征以及利魯唑的干預作用,然后對其作用機制進行研究,為眼內(nèi)炎的治療提供新線索。材料與方法將Sprague-Dawley(SD)大鼠隨機分為3組,1組:生理鹽水對照組;2組:眼內(nèi)炎組;3組:眼內(nèi)炎利魯唑干預組。生理鹽水組大鼠右眼注入5μl無菌生理鹽水。2、3組大鼠右眼玻璃體腔內(nèi)注入LPS(2μg,5μl),建立LPS誘導的眼內(nèi)炎模型。左眼為正常對照眼。利魯唑干預組在LPS注射前12 h和術(shù)中及術(shù)后連續(xù)3 d腹腔內(nèi)注射利魯唑注射液(0.1%,10mg/kg)。第一部分:造模后6h、12h、24h、48h、3d、5d、7d進行眼部炎癥評分,病理組織學檢查觀察炎癥細胞浸潤,行免疫組化測定谷氨酰胺合成酶(glutamine synthetase,GS)在視網(wǎng)膜組織的表達變化及分布,采用熒光定量聚合酶鏈反應(RT-PCR)技術(shù)檢測視網(wǎng)膜組織中GS的表達情況,觀察利魯唑的干預作用。第二部分:對利魯唑的干預機制進行初步探討,分光光度計法測定玻璃體中谷氨酸(glutamate,Glu)含量,采用TUNEL法檢測視網(wǎng)膜組織細胞凋亡。所有數(shù)據(jù)均用(x±s)表示,應用SPSS21.0統(tǒng)計軟件包進行統(tǒng)計學處理,采用單因素方差分析進行組間比較,采用LSD檢驗進行兩兩比較,P0.05為差異有統(tǒng)計學意義。結(jié)果1.臨床炎癥評分玻璃體腔內(nèi)注射LPS誘導出典型眼內(nèi)炎臨床表現(xiàn),造模后干預組炎癥反應明顯較2組輕,造模后24 h、48h、3d時間點兩組臨床炎癥評分間差異均有統(tǒng)計學意義(P0.05)。1組未見明顯炎癥反應。2.病理組織學檢查常規(guī)HE染色后顯微鏡下觀察白細胞浸潤,對切片中以鋸齒緣為前界的玻璃體腔中的中性粒細胞、淋巴細胞、單核-巨噬細胞等炎癥細胞進行計數(shù)分析。2組、3組在造模后均可觀察到大量外周血白細胞眼內(nèi)的浸潤。HE染色顯示各時間點3組玻璃體內(nèi)炎癥細胞計數(shù)低于2組,差異有統(tǒng)計學意義(P0.05)。而Ⅰ組造模后玻璃體腔、視網(wǎng)膜大部分未見、或散在少量的炎癥細胞浸潤,視網(wǎng)膜形態(tài)基本正常,結(jié)構(gòu)清晰。與1組相比,GS在眼內(nèi)炎組的表達升高,主要表達于內(nèi)顆粒層、神經(jīng)節(jié)細胞層Müller細胞胞漿中。2組、3組在6h、12h、24h、48h、3d GS的表達具有顯著性差異(P0.05),3組GS的表達明顯低于2組,但仍高于對照組。3.實時熒光定量PCR方法檢測視網(wǎng)膜組織GS的表達48h時,對照組視網(wǎng)膜中檢測到低水平表達,眼內(nèi)炎組GSm RNA表達較對照組明顯增強(P0.05),利魯唑干預組GSm RNA表達較眼內(nèi)炎組降低(P0.05),但仍高于對照組。4.谷氨酸濃度2組和3組大鼠玻璃體中谷氨酸含量在炎癥初期就開始升高,48h時達到高峰,分別為324.01±2.47μmol/l、253.95±10.76μmol/l。2組在5d時谷氨酸濃度較之前出現(xiàn)小幅回升(298.04±4.83μmol/l),然后逐漸下降。6h后各時間點,3組造模眼玻璃體谷氨酸濃度明顯低于2組,差異均有統(tǒng)計學意義(P0.05),但仍明顯高于1組。5.LPS誘導眼內(nèi)炎后,TUNEL染色陽性細胞散在分布于視網(wǎng)膜神經(jīng)節(jié)細胞層、內(nèi)顆粒層,3d時出現(xiàn)凋亡細胞數(shù)量明顯增多。2組細胞凋亡數(shù)目明顯多于3組。生理鹽水對照組視網(wǎng)膜未見明顯細胞凋亡,6h,12h,24h時眼內(nèi)炎組與干預組相比細胞凋亡程度無明顯差異,其后各時間點3組凋亡細胞指數(shù)均低于同期眼內(nèi)炎組,差異有統(tǒng)計學意義(P0.05)。結(jié)論利魯唑可以抑制內(nèi)毒素誘導的眼內(nèi)炎中炎癥細胞的眼內(nèi)浸潤,減少組織損害,其機制可能是通過減少視網(wǎng)膜組織細胞凋亡,使玻璃體谷氨酸含量降低,抑制Müller細胞活化。該研究結(jié)果提示利魯唑在細菌性眼內(nèi)炎中的保護作用,為眼內(nèi)炎的輔助治療提供依據(jù)。
[Abstract]:Ocular trauma and internal eye surgery are the main causes of infective endophthalmitis, most of which are caused by bacterial entry into the eye, such as coagulase negative staphylococcus, Staphylococcus aureus, Streptococcus, Gram-negative bacteria, etc. bacterial endophthalmitis is a serious threat to visual acuity, and the prognosis of bacterial endophthalmitis is affected by the number of bacteria, virulence, and patients. A variety of factors such as age and immune status. Although timely use of antibiotics, vitrectomy, and other treatments, the visual prognosis is still not ideal, which often leads to severe visual impairment. The toxic substances released in the process of proliferation and rupture (endotoxin, exotoxin) can induce severe inflammatory reactions and cell necrosis [1 2,3]. is one of the important factors to induce intraocular inflammation. Lipopolysaccharide is the main component of the cell wall of gram-negative bacilli, and it is also the main toxic factor of the gram negative bacilli. It can be used in the intravitreal injection to induce the infection of the intraocular inflammation and the [4]. inflammatory reaction leads to the destruction of the blood barrier and the inflammatory cells migrate to the interstitial space. It is a difficult problem and challenge to eliminate the destruction of the intraocular tissue in the ophthalmitis and improve the prognosis of the final eyesight. In the meantime, the effect and timely control of the intraocular inflammation is the key to the treatment. Key, also a decisive factor in prognosis. Riluzole is a benzothiazole derivative. It began as a new type of glutamate release inhibitor in 1996 and was approved to be applied to the treatment of amyotrophic lateral sclerosis (amyotrophic lateral sclerosis, ALS), which stabilizes the role of intracellular and extracellular ion concentration to protect it from neuroprotection. The anticonvulsant, antidepressant, and other aspects have a wide range of functions, suggesting its clinical application in more diseases. According to its pharmacological mechanism, we speculate that it may play an active role in the control of intraocular inflammation. This study intends to observe the intervention effect of Lulu on lipopolysaccharide induced endophthalmitis in rats and to make a preliminary study of its mechanism. Objective to establish an endophthalmitis model by injecting LPS (lipopolysaccharide from Escherichia coli) into the cavity of the vitreous body to observe the inflammation, histopathological features and the intervention of lelozole, and then study its mechanism of action to provide new clues for the treatment of endophthalmitis. Materials and methods will apply Sprague- to the treatment of endophthalmitis. Dawley (SD) rats were randomly divided into 3 groups, 1 groups: normal saline control group, 2 group: Endophthalmitis group, 3 group of endophthalmitis leuzole intervention group. The right eye of the normal saline group was injected into the right eye of the right eye of the normal saline group, the right eye of group.2,3 rats were injected LPS (2 mu, 5 mu L), and the LPS induced endophthalmitis model was established. The left eye was the normal eye to the eye. Li Lu azole intervention group. 12 h before LPS injection and intraoperative and postoperative 3 D intraperitoneal injection of ralu injection (0.1%, 10mg/kg). First part: 6h, 12h, 24h, 48h, 3D, 5D, 7d, followed by inflammation scores, histopathological examination of inflammatory cells, and immunohistochemical determination of glutamine synthetase in retina tissue The expression of GS in retinal tissue was detected by fluorescence quantitative polymerase chain reaction (RT-PCR), and the intervention of lurazole was observed. The second part: preliminary discussion on the intervention mechanism of lurazole, spectrophotometric determination of the content of glutamate (Glu) in vitreous body, and the detection of optic network by TUNEL All data were expressed by (x + s), using SPSS21.0 statistical software package for statistical treatment, using single factor analysis of variance to compare groups and using LSD test for 22 comparison, P0.05 was statistically significant. Results 1. clinical inflammation score in vitreous cavity injected LPS to induce typical endophthalmitis The inflammatory response in the intervention group was significantly lighter than that of the 2 groups after the model, 24 h, 48h, and 3D time points in the two groups. There was a significant difference between the two groups of clinical inflammation scores (P0.05) there was no obvious inflammatory reaction in the.1 group and.2. histopathological examination was observed under the routine HE staining, and the leukocyte infiltration was observed under the microscope and the serrations in the anterior boundary of the glass body were in the section. Inflammatory cells such as neutrophils, lymphocytes, mononuclear macrophages and other inflammatory cells were counted and analyzed in.2 group. The 3 groups could observe the infiltration of.HE in the eyes of a large number of peripheral blood leucocytes in the 3 groups. The count of inflammatory cells in the vitreous body of 3 groups was lower than that of the 2 groups, and the difference was statistically significant (P0.05). As compared with the 1 groups, the expression of GS in the endophthalmitis group increased, mainly in the inner granular layer, the.2 group in the ganglion cell layer M u ller cell cytoplasm, and the 3 groups in 6h, 12h, 24h, 48h, 3D GS (P0.05), 3 groups of GS tables. It was significantly lower than the 2 groups, but still higher than the control group.3. real-time fluorescence quantitative PCR method to detect the expression of GS in the retinal tissue 48h, the control group detected a low level of expression in the retina, the expression of GSm RNA in the group of endophthalmitis was significantly enhanced (P0.05), and the RNA table of the rizole intervention group was lower than that of the endophthalmitis group (P0.05), but still higher than the control group.4.. The glutamic acid content in the vitreous body of the 2 group and the 3 group of rats began to rise in the early stage of the inflammation, and reached the peak at 48h, 324.01 + 2.47 mu mol/l respectively. The glutamic acid concentration in the 253.95 + 10.76 mol/l.2 group appeared slightly higher than before (298.04 + 4.83 Mu mol/l), and then gradually decreased at each time point after.6h, and the vitreous valleys of the 3 groups were made. The concentration of ammonia acid was significantly lower than that of the 2 group (P0.05), but it was still significantly higher than the 1 groups of.5.LPS induced endophthalmitis. The TUNEL staining positive cells were scattered in the retinal ganglion cell layer, the inner granular layer, and the number of apoptotic cells in the 3D group increased obviously in.2 group more than in the 3 groups. No obvious apoptosis was found in the membrane. There was no significant difference in the degree of apoptosis between the endophthalmitis group and the intervention group at 6h, 12h and 24h. The 3 groups of apoptotic cells in each time point were lower than those in the same period of endophthalmitis. The difference was statistically significant (P0.05). Conclusion raluzole could inhibit the intraocular infiltration of inflammatory cells in endotoxin induced endophthalmitis and the reduction group. The mechanism may be to reduce the content of vitreous glutamic acid and inhibit the activation of M u ller cells by reducing the apoptosis of retinal tissue cells. The results suggest that the protective effect of raluzole in bacterial endophthalmitis provides a basis for the adjuvant treatment of endophthalmitis.
【學位授予單位】：鄭州大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R77

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相關(guān)期刊論文 前4條

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