【作者】 孙兆义；

【导师】 洪晶；

【作者基本信息】 中国医科大学 ， 眼科学， 2009， 博士

【摘要】 目的干眼症是一种由各种不同的功能和眼表保护机制紊乱所致的眼表疾病,它是眼科中发病率最高的眼表疾病之一。干眼症的发病是多方面因素导致的,眼表组织结构发生变化和泪膜成分的异常等都会引起干眼症,炎症因子在调节泪液分泌的过程中起重要作用。泪膜的持续异常可损伤眼表正常的修复或防御机制,导致眼表和泪腺处于一种慢性炎症状态。一旦进入进展阶段,炎症成为干眼症发病机制中最关键的因素,而细胞凋亡、神经调节及激素等也共同参与了干眼症的发病过程。肿瘤坏死因子(TNF)-α主要由活化的巨噬细胞、单核细胞和T细胞分泌产生,它以自分泌或旁分泌的方式作用于细胞,可激活相应细胞内的核转录因子NF-κB。TNF-α具有广泛的生物学活性,具有强大的抗肿瘤作用,是迄今发现的抗瘤作用最强的细胞因子,除对肿瘤细胞具有直接的抑制增殖和引起细胞坏死作用外,对其他细胞的生长分化也有影响,同时还有抗病毒及细菌,激活T细胞,促进IL-1、IL-2、IL-6的产生及分泌,诱发炎症反应核转录因子(nuclear transcription factor-κB, NF-κB)通常以非活化形式存在于细胞质中,在NF-KB受到各种活化因素的作用后被激活并促使其合成表达增加,其激活后可转位于细胞核,与相应细胞的靶基因结合而调控其相关基因转录,来拮抗各种有害因素对细胞的损伤作用所产生的促细胞凋亡的影响。NF-κB通路除广泛调控着机体炎症和免疫反应中许多基因的表达之外,还影响细胞的无限增殖和抗凋亡,但NF-κB通路的活化为一网络系统,包括上游和下游基因共同参与,涉及凋亡与抗凋亡基因的相互作用及其平衡。NF-κB的激活能抑制TNF-α柔红霉素或电离辐射引起的细胞凋亡。参与眼部疾病发生和发展的信号传导途径及细胞因子有很多,它们之间的作用机制还不十分明确。煤矿井下工人普遍有眼部干涩、异物感、烧灼感、畏光红痛及有粘丝状分泌物等临床表现,故本实验通过观察特殊人群与煤尘接触后,其干眼症的发生是否与煤尘接触存在相关性,煤矿井下工人结膜囊内TNF-α的表达是否存在异常,以及结膜囊内TNF-α的变化与干眼症病变程度之间的关系；并进一步通过兔动物实验来观察与煤尘接触后,兔结膜囊内TNF-α的表达情况及与兔结膜上皮细胞内NF-κB表达的相关性,来探讨煤尘接触对眼表组织的影响。材料与方法1、通过前瞻性研究来观察：人群与煤尘接触是否可诱发干眼症的发生。2、与煤尘接触的人群与实验兔均分别进行眼表的Schirmer实验、测量泪膜破裂时间(BUT)及泪液溶菌酶含量测量,观察其是否与对照组存在统计学差异,以确定与煤尘接触的职业暴露人群是否具有较高的干眼症发病率。3、煤矿工人结膜囊内的TNF-α的提纯。4、煤矿工人实验分组：与煤尘接触的工人结膜囊提取的200μl液体+5%小牛血清DMEM培养液100μl混合后,取其100μ1混合液作为实验组GT1；来自与煤尘非接触的工人,为实验组GT2；生理盐水200μl和5%小牛血清DMEM培养液100μl混合后,取其100μl混合液作为空白对照组GTO,4℃保存。5、煤矿工人结膜囊内的TNF-α生物学活性测定：取处于对数生长期的L929细胞加入3H-TdR进行孵育48h后,实验各组与TNF-a标准品稀释液分别加入存有L929细胞培养板各孔内,再加入放线菌素D溶液作用24h。所测TNF-a生物学活性=待测样品CPM值(实验各组培养液×标准品活性单位(50U／m1)×待测样品稀释倍数／(标准品稀释液-本底)的CPM值。6、实验兔实验分组：正常饲养的实验兔为空白对照组G0；与煤尘接触四周的实验兔为实验组G1；与煤尘接触二周的实验兔为实验组G2。7、实验兔结膜囊内TNF-a生物学活性检测及兔球结膜组织上皮细胞中NF-κBp65的免疫组化检测：将笼装实验兔放入与煤尘密切接触的环境中,在时间为二周或四周时,分别进行结膜囊液体内TNF-α的提纯及生物学活性测定后,麻醉处死取其眼球穹隆部位的结膜组织,进行石蜡包埋固定,采用SABC法实施免疫组化染色观察NF-κBp65在结膜组织中的表达。每个标本在光学显微镜下观察NF-κB的表达,镜下阳性细胞染色为深浅不一的棕褐色。细胞吸光度×面积(μm)积分大于500者,记为NF-κB表达为阳性的细胞。在200倍视野下随机选取5个视野,计数阳性细胞和总细胞,阳性率=阳性细胞数／总细胞数×100%。8、统计学处理：本文数据用(?)±s表示,采用X2检验、方差分析和t检验进行统计学分析,P<0.05有统计学意义,统计计算过程均用SPSS 10.5软件进行。结果1、Schirmer实验显示：与煤尘接触的人群滤纸被泪液渗湿的长度均短于正常值；而与煤尘非接触的人群,其滤纸被泪液渗湿的长度均大于或等于正常值。与煤尘接触四周的实验兔进行眼表的Schirmer实验显示其滤纸被泪液渗湿的长度短于与煤尘接触两周的实验兔滤纸渗湿长度,与煤尘接触两周的实验兔滤纸渗湿长度又小于正常饲养的实验兔滤纸渗湿长度。2、泪膜破裂时间(BUT)测量结果：与煤尘接触的井下工人泪膜破裂时间均值少于10秒；而与煤尘非接触的井上工人泪膜破裂时间均值大于或等于10秒。与煤尘接触四周的实验兔泪膜破裂时间少于与煤尘接触两周的实验兔泪膜破裂时间,与煤尘接触两周的实验兔泪膜破裂时间又小于正常饲养的实验兔泪膜破裂时间。3、泪液溶菌酶含量测定结果：与煤尘接触的井下工人泪液溶菌酶含量均值少于正常值；而与煤尘非接触的井上工人泪液溶菌酶含量均值大于或等于正常值。与煤尘接触四周的实验兔泪液溶菌酶含量少于与煤尘接触两周的实验兔泪液溶菌酶含量,与煤尘接触两周的实验兔泪液溶菌酶含量又小于正常饲养的实验兔泪液溶菌酶含量。4、煤矿工人结膜囊内的TNF-α生物学活性检测结果：与煤尘接触的井下工人结膜囊内的TNF-α生物学活性为平均值为17.350±1.081U／ml；与煤尘非接触人群结膜囊液体内TNF-α生物学活性平均值为0.023±0.004U／ml,实验数据经t检验显示p<0.05。5、实验兔结膜囊液体内TNF-α生物学活性：与煤尘接触前实验兔结膜囊液体内TNF-α生物学活性平均值为0.014±0.001U／ml；密切接触二周后,实验兔结膜囊液体内TNF-α生物学活性平均值二周为3.051±0.087U／ml；四周为13.096±0.525U／ml,与煤尘非接触的实验兔为0.015±0.001U／ml,实验数据经方差分析显示p<0.05。6、免疫组化显示实验兔结膜上皮细胞表达NF-κBp65的情况：与煤尘非接触的实验兔,结膜上皮细胞表达NF-κBp65的平均细胞阳性率为：1.09±0.22%；与煤尘密切接触时间二周,其结膜组织中结膜上皮细胞表达NF-κBp65的平均细胞阳性率为：8.66±1.07%；粉尘接触时间四周为：21.29±3.74%,实验数据经方差分析显示p<0.05。结论1、与煤尘密切接触可降低泪液分泌水平。2、与煤尘密切接触可破坏泪膜稳定性。3、与煤尘密切接触可降低泪液溶菌酶含量。4、与煤尘密切接触可诱发煤矿工人和实验兔干眼症发生。5、与煤尘密切接触的人群结膜囊液体内TNF-α生物学活性高于非接触人群,TNF-α表达水平与干眼症的发展密切相关。6、实验兔结膜囊液体内的TNF-α生物学活性与其密切接触煤尘的时间呈正相关。7、与煤尘密切接触可促使实验兔结膜上皮细胞出现NF-κB的高表达,并与其结膜囊内TNF-α的表达呈正相关。8、实验兔结膜上皮细胞NF-κB阳性表达率随着其与煤尘接触时间的延长而增加。更多还原

【Abstract】 IntroductionDry eye is a common disorder of the ocular surface and tear film, which there is a higher disease incidence and is caused by many factors. The severe dry eye can compromise corneal integrity by causing epithelial defects, stromal infiltration, and ulceration, and can also result in visually significant scarring. Inflammatory factors play an important role on regulation of lacrimal secretion secretion. The continuous abnormality on lacrimal secretion can damage the mechanism of recovery and defense for ocular surface function, finally, it make ocular surface and glandula lacrimalis to be on situation of chronic imflammation. In the progresive process of chronic imflammation, it becomes a key factor on the development of xerophthalmia in which apoptosis of conjunctival epithelium cells, neural regulation and hormone regulation participate.Tumor necrosis factor (TNF)-a is secreted by many cells, and it influences other cells through its autocrine and paracrine secretion. TNF-αcan activates NF-κB, however, its transcription is regulated by NF-κB. TNF-αpossesses many function, including defending to virus and bacterium, to activate T cells, to promote synthesis and secretion of interleukin-1, interleukin-2 and interleukin-6, finally, induces inflammatory reaction.As known, there are a lot of signal pathways to mediate the development of many diseases for eye, however, their interactive mechanisms are not clear. In our study, their interaction, between some signal pathways and cell factors, and TNF-αand NF-κB on development of xerophthalmia induced by coal dust contiguity. There are syndromes for coal miners such as foreign body sensation, burning sensation, phengophobia and filamentous secretion. This study is to investigate whether there is a dependability between occurrence of xerophthalmia and coal dust contiguity or not, and to investigate expression of TNF-αin conjunctival sac of coal miners, and to investigate the relationship between the expression of TNF-αin conjunctival sac of coal miners and the degree of xerophthalmia. Further more, experimental rabbits are taken into coal well to get in touch with coal dust, and then to observed whether there is a dependability between expression of TNF-αin conjunctival sac of rabbits between expression of NF-κB in conjunctival epithelial cells of rabbits or not, and to investigate effects of coal dust contiguity on ocular tissue.Methods and Materials 1. The perspective study was use to observe whether people xerophthalmia of occurrence could induced by coal dust contiguity or not.2. Detection of Shirmer test, BUT and lysozyme were performed on rabbits and coal miners, and then investigated whether there was a statistics difference compared with control group in order to observe whether there was a high xerophthalmia incidence on coal miners.3. Extraction of TNF-αfrom coal miners’s conjunctival sac4. Grouping of experimentGroup GT1was 100μl mixed liquor which came from coal miners and 5% calf serum DMEM culture solution, and group GT2 was 100μl mixed liquor which came from 5% calf serum DMEM culture solution and general miners without coal dust contiguity, and GT0 was 100μl mixed liquor which came from 5% calf serum DMEM culture solution and normal saline. They were preserved at 4℃.5. Detection of TNF-αactivityThe L929 cell cytotoxic assay was used to determine the blood serum TNF-αactivity. After L929 cells, which were in exponential phase of growth, had been incubated with 3H-TdR for 48 h, 100μl mixed liquor of every group and actinomycin D solution were added into 24-well flat-bottomed plates for 24 h. TNF-αactivity was expressed in units per milliliter by CPM of every group.6. Grouping of rabbits Rabbits were bred in normal surroundings as blank control Go, and rabbits were bred in surrounding with coal dust contiguity for 4 weeks as G1, and rabbits were bred in surrounding with coal dust contiguity for 2 weeks as G2.7. Expression of TNF-αof conjunctival sacs and NF-κBp65 in ocular conjunctival epithelial cells on rabbitsAfter all rabbits with or without coal dust contiguity were extracted TNF-αfrom conjunctival sacs to detect the activity of TNF-α, rabbits were anesthetized and then their eyes were removed, finally, eyes were made into paraffin sections and processed for the demonstration of NF-κB. After endogenous peroxidase blocking, sections were treated with blocking buffer for 30 minutes and incubated at 4℃in the following primary antibodies diluted in blocking buffer:rabbit anti-rabbit NF-κB p65 antibody. Sections processed for the demonstration of NF-κB were quantified as previously described. Briefly, sections were digitized by a video camera mounted on a Leitz microscope and interfaced to a Macintosh computer. Image software was used to quantify the immunocytochemical staining. The quantification measure, referred to as the reactivity grade, was defined as density value of a cell. The density exceed a standard value was defined as positive expression of a cell. The ratio of NF-κBp65 positive expression= positive expression cells/total cells 8. Statistical analysesAll analyses were performed with the SPSS 10.5 statistical package and all results were presented as mean±SD. Interclass analyses of difference were performed with analysis of variance, X2 test and two sets were performed with independent-samples T tests.Results1. As showed in Schirmer tear test, the average length of paper strips which were excreting dampnessed by lacrimal fluid was shorter in rabbits with coal dust contiguity for 2 weeks or 4 weeks than that of normal condition, so results showed in BUT. In lacrimal fluid’s lysozyme essay, the mean amount of lysozyme was fewer in rabbits with coal dust contiguity for 2 weeks or 4 weeks than that of normal condition.2. As showed in Schirmer tear test, the average length of paper strips which were excreting dampnessed by lacrimal fluid was shorter in coal miners with coal dust contiguity than that of normal value, but the average length of paper strips which were excreting dampnessed by lacrimal fluid was longer in general workers without coal dust contiguity than that of normal value, or near to normal value, so results showed in BUT and in lacrimal fluid’s lysozyme essay.3. Expression of TNF-αactivity in conjunctival sac for workersThe average value of TNF-αactivity in conjunctival sac was 17.350±1.081U/ml for workers who got in touch with coal dust in their working time, and was 0.032±0.004U/ml for workers without coal dust contiguity, p＜0.05.4. Expression of TNF-αactivity in conjunctival sac for rabbitsThe average value of TNF-αactivity in conjunctival sac was 0.014±0.001U/ml before rabbits began to get in touch with coal dust. The average value of TNF-αactivity in conjunctival sac was 3.051±0.087U/ml after 12 rabbits got in touch with coal dust for 2 weeks, and 13.096±0.525U/ml for 4 weeks, and 0.015±0.001U/ml for rabbits without coal dust contiguity, p＜0.05.5. Expression of NF-κBp65 by immunohistochemistry stainingRabbits without coal dust contiguity, the ratio of NF-κB p65 positive expression cells was 1.09±0.22% in conjunctival epithelium cells. The ratio of NF-κB p65 positive expression cells was 8.66±1.07% in conjunctival epithelium cells after rabbits got in touch with coal dust for 2 weeks, and 21.29±3.74% for 4 weeks,p＜0.05.Conclusions1、Lacrimal secretion of coal miners and rabbits will decrease because of them to get in touch with coal dust.2、The stability of lacrimal film of coal miners and rabbits will be broken down because of them to get in touch with coal dust. 3、The tear fluid lysozyme amount of coal miners and rabbits will decrease because of them to get in touch with coal dust.4、Development of dry eye of coal miners and experimental rabbits can be induced through coal dust contiguity.5、The level of TNF-αactivity in conjunctival sac of people is higher than that of people without coal dust contiguity. Expression of TNF-αactivity in conjunctival sac of people show a direct correlation to development of dry eye.6、Expression of TNF-αactivity in conjunctival sac of rabbits show a direct correlation to period of them to get in touch with coal dust.7、The level of TNF-αactivity in conjunctival sac of rabbits with coal dust contiguity showes positive correlation with NF-κB expression in conjunctival epithelium cells.8、The higher expression of NF-κB in conjunctival epithelium cells of rabbit show a direct correlation to period of them to get in touch with coal dust.更多还原