本文選題：角膜 + 損傷修復(fù)��； 參考：《南華大學(xué)》2015年碩士論文

【摘要】：目的:通過(guò)機(jī)械刮除角膜上皮及Na OH處理角膜建立兔角膜損傷模型,檢測(cè)中性粒細(xì)胞在損傷修復(fù)過(guò)程中的浸潤(rùn)及凋亡數(shù)量情況,研究中性粒細(xì)胞凋亡對(duì)角膜損傷修復(fù)的影響。方法:1、標(biāo)準(zhǔn)環(huán)境下飼養(yǎng)新西蘭大白兔20只,隨機(jī)分成5組,每組4只。第1組為空白對(duì)照組,第2組為機(jī)械損傷組,即在局麻下刮除兔角膜中央8mm直徑上皮,建立機(jī)械損傷模型;第3.4.5組分別用1%、2%、4%氫氧化鈉處理兔角膜中央8mm直徑上皮,再用生理鹽水沖洗干凈,建立堿燒傷模型。正常喂養(yǎng)一個(gè)星期,誘發(fā)兔角膜炎癥發(fā)應(yīng),啟動(dòng)角膜損傷修復(fù)程序。分別在損傷后12小時(shí)、24小時(shí)、48小時(shí)、96小時(shí)空氣栓塞處死兔子;2、取新鮮兔角膜以4%多聚甲醛固定,OCT包埋后行冰凍切片。HE染色觀察兔角膜組織形態(tài)學(xué)的變化,利用免疫熒光化學(xué)染色觀察中性粒細(xì)胞在兔角膜組織中的浸潤(rùn)數(shù)量變化。3、TUNEL法檢測(cè)兔角膜組織中性粒細(xì)胞凋亡情況,了解中性粒細(xì)胞凋亡與角膜損傷修復(fù)機(jī)制的關(guān)系。結(jié)果:1.新西蘭大白兔角膜組織HE染色可見(jiàn):空白對(duì)照組角膜上皮完整,膠原纖維粗細(xì)均勻,排列整齊;機(jī)械損傷組可見(jiàn)角膜上皮細(xì)胞脫落,基質(zhì)水腫,膠原纖維排列不規(guī)則,炎性細(xì)胞浸潤(rùn);堿燒傷組可見(jiàn)角膜上皮細(xì)胞脫落,基質(zhì)水腫,膠原纖維排列不規(guī)則,炎性細(xì)胞浸潤(rùn)增多。2.中性粒細(xì)胞DNA電泳結(jié)果:各組中性粒細(xì)胞DNA經(jīng)1%瓊脂糖凝膠電泳和紫外分光光度檢測(cè),完整性很好。反轉(zhuǎn)錄產(chǎn)物經(jīng)PCR檢測(cè),擴(kuò)增產(chǎn)物均為單一條帶,分子量與目的條帶大小一致,無(wú)非特異性條帶。3.免疫熒光染色見(jiàn)空白對(duì)照組角膜緣有少量中性粒細(xì)胞出現(xiàn),中央角膜區(qū)沒(méi)有發(fā)現(xiàn)。機(jī)械損傷組12小時(shí)后可發(fā)現(xiàn)中性粒細(xì)胞浸潤(rùn),72小時(shí)可見(jiàn)中性粒細(xì)胞出現(xiàn)脫顆粒和核降解,96小時(shí)后角膜上皮完全修復(fù)。堿燒傷組角膜12小時(shí)后可以發(fā)現(xiàn)有大量中性粒細(xì)胞浸潤(rùn),隨時(shí)間推移逐漸減少,至96小時(shí)達(dá)到最低。不同的堿濃度下中性粒細(xì)胞數(shù)量也不相同。隨著Na OH濃度升高,在12小時(shí)、24小時(shí)、48小時(shí)、96小時(shí)觀察發(fā)現(xiàn)中性粒細(xì)胞數(shù)量也相應(yīng)有所增加。4.TUNEL法檢測(cè)中性粒細(xì)胞凋亡情況可見(jiàn),機(jī)械損傷組與堿燒傷組相比較,中性粒細(xì)胞凋亡數(shù)量前者多于后者。各種濃度堿燒傷角膜組間比較,Na OH濃度越高,中性粒細(xì)胞凋亡的數(shù)量越少。5.計(jì)數(shù)各組不同時(shí)間點(diǎn)角膜中性粒細(xì)胞浸潤(rùn)及凋亡數(shù)量。中性粒細(xì)胞浸潤(rùn)數(shù)量:4%Na OH處理組2%Na OH處理組1%Na OH處理組機(jī)械損傷組空白對(duì)照組(P0.05)。中性粒細(xì)胞凋亡數(shù)量:各時(shí)間點(diǎn)4%Na OH處理組中性粒細(xì)胞凋亡數(shù)量較其他濃度Na OH處理組低(P0.05),堿燒傷組中性粒細(xì)胞凋亡數(shù)量較機(jī)械損傷組中性粒細(xì)胞凋亡數(shù)量低(P0.05),各組與空白對(duì)照組相比有統(tǒng)計(jì)學(xué)差異(P0.05)。結(jié)論:1、實(shí)驗(yàn)表明角膜損傷修復(fù)包含復(fù)雜的炎癥機(jī)制。成功建立角膜機(jī)械損傷及堿燒傷模型,觀察到角膜損傷后有大量的中性粒細(xì)胞的浸潤(rùn),且損傷越嚴(yán)重,中性粒細(xì)胞浸潤(rùn)的數(shù)量也越多。在角膜損傷修復(fù)過(guò)程中發(fā)現(xiàn)有中性粒細(xì)胞凋亡,角膜損傷越嚴(yán)重,凋亡中性粒細(xì)胞反而越少,炎癥持續(xù)發(fā)展,損傷修復(fù)的速度變慢。2、角膜損傷修復(fù)過(guò)程與角膜中性粒細(xì)胞凋亡有很大的關(guān)聯(lián),且與角膜損傷嚴(yán)重程度相關(guān),嚴(yán)重堿燒傷時(shí)中性粒細(xì)胞凋亡減少更明顯。中性粒細(xì)胞適度適量的存在及在基因調(diào)控下的正常凋亡,對(duì)于角膜創(chuàng)傷后損傷組織的增殖修復(fù)有很重要的作用。
[Abstract]:Objective: to establish a rabbit corneal injury model by mechanical scraping of corneal epithelium and Na OH treatment, and to detect the number of infiltration and apoptosis of neutrophils during the repair process, and to study the effect of neutrophils apoptosis on the repair of corneal injury. Methods: 1, 20 New Zealand white rabbits were fed under standard conditions and were randomly divided into 5 groups, each group was 4. Only first groups were blank control group and second groups were mechanical injury groups, that is to scrape the central 8mm diameter epithelium of rabbit cornea under local anesthesia and establish a mechanical damage model. Group 3.4.5 used 1%, 2%, 4% sodium hydroxide to treat the central 8mm diameter epithelium of rabbit cornea, and then rinse the rabbit's cornea with normal saline to establish the alkali burn model. Keratitis should be made to start the corneal injury repair procedure. Rabbits were killed 12 hours, 24 hours, 48 hours and 96 hours after injury. 2, fresh rabbit cornea was fixed with 4% polyformaldehyde. After OCT embedding, the histomorphology of rabbit cornea was observed by frozen section.HE staining, and neutrophils were observed by immunofluorescence staining. The changes in the number of infiltration in rabbit cornea tissue were.3, TUNEL method was used to detect the apoptosis of neutrophils in rabbit cornea tissue, and to understand the relationship between neutrophil apoptosis and corneal injury repair mechanism. Results: 1. the HE staining of corneal tissue in New Zealand white rabbits showed that the corneal epithelium in the blank control group was complete, the thickness of collagen fiber was uniform, and the arrangement of the cornea was uniform; The injury group showed corneal epithelial cells falling off, matrix edema, irregular arrangement of collagen fibers, infiltration of inflammatory cells, corneal epithelial cells shedding, matrix edema, irregular arrangement of collagen fibers, and increased inflammatory cell infiltration by.2. neutrophils DNA electrophoresis results: DNA of each group of neutrophils in each group was 1% agarose gel electrophoresis and purple. The reverse transcriptional products were detected by PCR, and the products were all single band, and the molecular weight was the same as the size of the target band. There was a small number of neutrophils in the corneal limbus of the blank control group, and the central kernels were not found in the blank control group, and the mechanical injury group could be found 12 hours later. Neutrophil infiltration and neutrophils were degranulation and nuclear degradation in 72 hours. Corneal epithelium was completely repaired after 96 hours. A large number of neutrophils were found after 12 hours in the alkali burn group, gradually decreasing with time, reaching the lowest level to 96 hours. The number of neutrophils at different alkali concentrations was also different. The number of neutrophils was observed at 12 hours, 24 hours, 48 hours and 96 hours, and the number of neutrophils was increased by.4.TUNEL method at 12 hours, 24 hours, 48 hours and 96 hours. The number of neutrophil apoptosis in the mechanical injury group was more than that in the alkali burn group, and the number of neutrophil apoptosis was more than that of the latter. The Na OH concentration was compared with the concentration of alkali burn cornea group. Na OH concentration was compared. The higher the number of neutrophils, the less the number of neutrophils apoptosis.5. counts the number of neutrophils infiltration and apoptosis at different time points. The number of neutrophil infiltration: the blank control group (P0.05) in the 1%Na OH treatment group of the 1%Na OH treatment group of the OH treatment group of the 2%Na OH treatment group. The number of neutrophil apoptosis: the neutrophils in the 4%Na OH treatment group at each time point. The number of apoptotic cells was lower than that of the other concentration Na OH treatment group (P0.05). The number of neutrophils apoptosis in the alkali burn group was lower than that of the neutrophils (P0.05) in the mechanical injury group (P0.05), and there was a statistical difference between the groups and the blank control group (P0.05). Conclusion: 1, the experimental results show that the repair of the angular membrane injury contains a complex inflammatory mechanism. A large number of neutrophil infiltration was observed after corneal injury, and the more serious the damage was, the more neutrophils infiltrated, the more the neutrophils infiltrated, the more serious the corneal injury was, the more serious the corneal injury, the less the apoptotic neutrophils, the continuous development of inflammation, and the repair of injury. The speed of.2 is slow. The repair process of corneal injury is closely related to the apoptosis of corneal neutrophils, and it is related to the severity of corneal injury. The decrease of neutrophils apoptosis is more obvious when severe alkali burns. The moderate amount of neutrophils and the normal withering under the regulation of gene can be used for the proliferation of the injured tissue after corneal trauma. It has an important role to play.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R779.6

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