中性粒细胞凋亡对角膜损伤修复的影响研究

发布时间：2018-07-07 12:39

本文选题：角膜 + 损伤修复　；参考：《南华大学》2015年硕士论文

【摘要】：目的:通过机械刮除角膜上皮及Na OH处理角膜建立兔角膜损伤模型,检测中性粒细胞在损伤修复过程中的浸润及凋亡数量情况,研究中性粒细胞凋亡对角膜损伤修复的影响。方法:1、标准环境下饲养新西兰大白兔20只,随机分成5组,每组4只。第1组为空白对照组,第2组为机械损伤组,即在局麻下刮除兔角膜中央8mm直径上皮,建立机械损伤模型;第3.4.5组分别用1%、2%、4%氢氧化钠处理兔角膜中央8mm直径上皮,再用生理盐水冲洗干净,建立碱烧伤模型。正常喂养一个星期,诱发兔角膜炎症发应,启动角膜损伤修复程序。分别在损伤后12小时、24小时、48小时、96小时空气栓塞处死兔子;2、取新鲜兔角膜以4%多聚甲醛固定,OCT包埋后行冰冻切片。HE染色观察兔角膜组织形态学的变化,利用免疫荧光化学染色观察中性粒细胞在兔角膜组织中的浸润数量变化。3、TUNEL法检测兔角膜组织中性粒细胞凋亡情况,了解中性粒细胞凋亡与角膜损伤修复机制的关系。结果:1.新西兰大白兔角膜组织HE染色可见:空白对照组角膜上皮完整,胶原纤维粗细均匀,排列整齐;机械损伤组可见角膜上皮细胞脱落,基质水肿,胶原纤维排列不规则,炎性细胞浸润;碱烧伤组可见角膜上皮细胞脱落,基质水肿,胶原纤维排列不规则,炎性细胞浸润增多。2.中性粒细胞DNA电泳结果:各组中性粒细胞DNA经1%琼脂糖凝胶电泳和紫外分光光度检测,完整性很好。反转录产物经PCR检测,扩增产物均为单一条带,分子量与目的条带大小一致,无非特异性条带。3.免疫荧光染色见空白对照组角膜缘有少量中性粒细胞出现,中央角膜区没有发现。机械损伤组12小时后可发现中性粒细胞浸润,72小时可见中性粒细胞出现脱颗粒和核降解,96小时后角膜上皮完全修复。碱烧伤组角膜12小时后可以发现有大量中性粒细胞浸润,随时间推移逐渐减少,至96小时达到最低。不同的碱浓度下中性粒细胞数量也不相同。随着Na OH浓度升高,在12小时、24小时、48小时、96小时观察发现中性粒细胞数量也相应有所增加。4.TUNEL法检测中性粒细胞凋亡情况可见,机械损伤组与碱烧伤组相比较,中性粒细胞凋亡数量前者多于后者。各种浓度碱烧伤角膜组间比较,Na OH浓度越高,中性粒细胞凋亡的数量越少。5.计数各组不同时间点角膜中性粒细胞浸润及凋亡数量。中性粒细胞浸润数量:4%Na OH处理组2%Na OH处理组1%Na OH处理组机械损伤组空白对照组(P0.05)。中性粒细胞凋亡数量:各时间点4%Na OH处理组中性粒细胞凋亡数量较其他浓度Na OH处理组低(P0.05),碱烧伤组中性粒细胞凋亡数量较机械损伤组中性粒细胞凋亡数量低(P0.05),各组与空白对照组相比有统计学差异(P0.05)。结论:1、实验表明角膜损伤修复包含复杂的炎症机制。成功建立角膜机械损伤及碱烧伤模型,观察到角膜损伤后有大量的中性粒细胞的浸润,且损伤越严重,中性粒细胞浸润的数量也越多。在角膜损伤修复过程中发现有中性粒细胞凋亡,角膜损伤越严重,凋亡中性粒细胞反而越少,炎症持续发展,损伤修复的速度变慢。2、角膜损伤修复过程与角膜中性粒细胞凋亡有很大的关联,且与角膜损伤严重程度相关,严重碱烧伤时中性粒细胞凋亡减少更明显。中性粒细胞适度适量的存在及在基因调控下的正常凋亡,对于角膜创伤后损伤组织的增殖修复有很重要的作用。
[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.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R779.6

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