| 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.
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