| Fluorine is one of the necessary microelements of human. It is closely related with vital activity and metabolism of the tissue of tooth and bone of human. Excessive intake can result in fluorosis rather than its necessary effect on human body because of its low relief valve. Fluorosis, being epidemic in over 50 countries of 5 continents, is a worldwide endemic disease closely related with the abundance of fluorine in geographical environment. It is prevalent severely in China. Xinjiang is one of the areas with high prevalence of fluorosis in China. There are endemic areas of 61 counties with about 1,100,000 patients. Since the project of recreational water carried out in 1980s, the health of people in endemic areas has been improved greatly. But there are superstandard indices of water fluoride from some new established waterhead, and patients with fluorosis of bone occurred. In addition, the swift increase of fluoride application in the industry presently leads to the fluoride content enhancing in air, water and soil in the surroundings, and even brings on environmental pollution. Especially, fluoride has been widely used in drinking water and buccal supplies for decayed tooth prevention. Many fluorides are blindly abused so that population expose to high level of fluoride more and more. In this way, people will intake excessive fluorine and their health will be affected. Therefore, endemic fluorosis is undoubtedly one of the endemic diseases endangering people's health at present according to its widely epidemic scope and large amount of sufferingpeople. Developing the study on relationship between fluorine and health to further illuminate the mechanism has been one of the warmly discussed problems in the present medical studies.In the light of the current study on endemic fluorosis, some developments have been obtained in the study of precipitating factor of fluorosis, the effect of excessive fluoride on osteogenesis, osteoclastic action and bone metabolism as well as the mechanism of non-ossifying injury, etc. But there are many researches awaiting repeated verify and deep approach, or some items have not been involved in or involved in too little. The thorough studies are needed. Particularly, it is necessary to study the relative gene of chronic fluorosis, proteome, bone metabolism involved in regulation web, and so on, for the purpose of further approach to the pathogeny of fluorosis.On the basis of general, systematic and exact detection analysis of general skeleton of patients in endemic area with the more advanced digital radiography(DR), the technique of gene chip is taken to screen the relative gene of fluorosis in the study, first reported in this paper, in order to find the differential expression gene of peripheral blood lymphocyte between patients with fluorosis and normal people. And its function in the process of fluorosis is discussed in the study with important theory value and practical significance, besides, flow cytometry (FCM) is used in the materials of patients with fluorosis for the first time, to study the effect of fluorine on distribution of cell cycle of peripheral blood lymphocyte and DNA contents. This study aims at providing basic theory for molecular biology mechanism of bone injury of fluorosis, and scientific basis for the early specific prevention, diagnosis and treatment of fluorosis of bone by epidemiological investigation of molecule on people in endemic areas, and study on skeletal lesion as well as the relative gene and cell cycle.OBJECTIVE:1. To observe pathological process of skeletal lesion in the occurrence and development of fluorosis of bone by general, systematic and exact detection analysis of general skeleton of patients; To discuss relationship between skeletal lesion and skeletal metabolic disorder by detecting and analyzing biochemical markers of some skeletal metabolisms so as to lay a foundation for the further study on relative genes of endemic fluorosis.2. To study DNA contents and distribution of cell cycle of peripheral blood lymphocyte of patients with fluorosis by FCM, and to discuss the effect of fluorine on proliferation ability of non-ossifying histiocytes.3. With the technique of gene chip to screen the relative gene of fluorosis, to find the differential expression gene of peripheral blood lymphocyte between patients with fluorosis and normal people for further approach to its effects on the occurrence and development in the process of fluorosis.METHODS:1. In the endemic area with high incidence, according to the patient's residing years, subjective symptoms and clinical signs, as well as X-ray, epidemiological investigation was carried out to define the patients with fluorosis as case group after eliminating other diseases in clinic. Meanwhile, the inhabitants in neighborhood with normal fluorine content in water, without any symptoms and signs, whose the age, profession, economic situation of the family and so on accorded with the case group, were defined the health as control group by X-ray confirmation.2. Hological digital X-ray apparatus made in America was used to examine and take films of investigated objects' head, chest, lumbar vertebrae, pelvis, forearm, hands and lower limbs. Skeletal density of lumbar vertebrae was surveyed by measurement system QT 3000 of skeletal density. The results shown on X-ray film were analyzed togetherby two radiologists with rich experiences and the findings were taken down in details and conscientiously. Meanwhile, the biochemical markers of some skeletal metabolisms were detected at laboratory.3. FCM was taken to analyze the effect of fluorine on proliferation ability of non-ossifying histiocytes. Lymphocytes isolated from peripheral blood of patients with fluorosis at different degrees and the normal control, were regulated cell concentration to be 1.0×10~6 after it washed by buffer solution and hemolysin and removed impurities. Because of fluorescence emission of nucleus by PI dyeing, cell cycle distribution of lymphocytes, relative contents of DNA and proliferation ability of cells were detected with FACSCalibur? FCM analyzer.4. The technique of cDNA chip was taken to screen relative gene of fluorosis. RNA extracted from peripheral blood lymphocyte of patients with fluorosis at different degrees and the normal control, was carried out reverse transcription, marking ~-33P probe, purification, and hybridization with cDNA chip at last. The result of hybridization was scanned to be image and inputted into computer. The data were analyzed with Pathways ?3.0 software, and the differential expression gene was obtained after screening by T-test and Ratio value. Ratio value over 2 indicates gene expressed upregulation and Ratio value less than 0.5 refers to gene expressed downregulation.RESULTS:1. It was shown by epidemiological investigation that two or more than two subjective symptoms occurred in all patients with fluorosis of bone, and the incidence rate of subjective symptoms and detectable rate of each relative signs increased along with the patient's aggravated condition (P<0.05). And one or two serious patients occurred joint flexion, deformity, limited activity and so on.2. There were skeletal lesions of different degrees in 69 patients withfluorosis of bone diagnosed by X-ray which showed various bony abnormity. According to the injury position, the incidence rate was the highest of 69.57% (48/69) in head injury, the second of 49.28% in pelvis. Out of the four kinds of bony injury (osteosclerosis, rarefaction, osteomalacia and the mixed), whether the injury was serious or not, osteosclerosis was the major lesion (65.22%), which was much more than the other types. Particularly, it was high to be 83.33% in slight patients. At the same time, the detected results of laboratory indicated skeletal metabolism change by urinary fluorine increase, blood Calcium decrease and the variety of hormonal level and cytokine.3. The detection of cell cycle showed that, there was no obvious differences in distribution of cell cycle of peripheral blood lymphocyte, relative contents of DNA and proliferation ability of cells between the patients with fluorosis and the normal control (P>0.05).4. It was shown by screening differential expression gene that, compared with the control, there were 32 genes expressed upregulation (Ratio of 2.0039~ 4.0467) and 31 genes expressed down regulation (Ratio of 0.0918~0.4881) in the slight case group, 53 genes expressed upregulation (Ratio of 2.0040—3.7180) and 19 genes expressed down regulation (Ratio of 0.0886—0.4934) in the severe case group. Compared with the slight group, there were only 4 genes expressed upregulation (Ratio of 2.3630— 2.7040) and 11 genes expressed down regulation (Ratio of 0.3341—0.4950) in the severe group. When compared with the control, Ras protein and transmembrane protein (TM9SF1) expressed upregulation both in the slight and severe groups (Ratio of 2.0462— 2.9936). There were 14 genes expressed down regulation at the same time (Ratio of 0.0918-0.4934): ATP synthase (ATP5I), keratin 4 (KRT4/K4), adenosine acceptor A2b (ADORA2b), human DNA mismatch repair gene (hMLHl), transcription factor (p38IP), single-stranded DNA binding protein (PURA/Pur a), class II MHC (HLA-DMB), corticosteroid-binding globulin (CBG), lactotransferrin (LTF/LF), mitochondrion translation initiation factor 2 (MTIF2), oligoadenylate synthetase 1(OAS1), double cortex protein (DCX), crystalline (CRYM), cytotoxic-granule-associated protein (TIA1).CONCLUSION:1. Results of image and relative physical examination confirmed the existence of skeletal lesion of fluorosis. Fluorosis of bone is one of the characteristic pathological changes. Whether skeletal lesion is serious or not, head lesion is the most injury position and osteosclerosis is the major bony injury, especially in slight patients high to be 83.33%. All these indicate that osteosclerosis in the occurrence and development of fluorosis of bone be the early and characteristic appearance and accompany with multiple skeletal lesions in the middle-late pathological changes. It is suggested that head should be the routine radiography position in the diagnosis of fluorosis of bone.2. High urinary fluorine and low blood calcium in the detected results of relative indices of skeletal metabolism indicate that "calcium contradictory" exists and plays significant role in the pathogenesis of endemic fluorosis, and suggest that combination of recreational water to reduce fluorine and calcium supplement in diet be the key to the control of endemic fluorosis. Meanwhile, abnormities of hydroxyproline in urine, osteocalcin and interleukins-6 illuminate certain effects of fluorosis on bone formation and the process of skeletal absorb and metabolism. But its mechanism of effect needs further study.3. The concentration of fluorine in patients' humor with chronic fluorosis cannot result in the change of cell cycle of peripheral blood lymphocyte, and cannot obviously affect proliferation ability of cells and relative contents of DNA. It may be related with the difference between the effect of fluorine on non-ossifying cell proliferation and on bone cellproliferation. Meanwhile, it indicates that there are cycle dependence and species specificity as well as dose-effect relationship in the influence of fluorine on cell cycle.4. High level of fluorine can influence human gene expression to some degrees, especially the high and continue expression of Ras protein and TM9SF1 in slight and severe patients with fluorosis, as well as low and continue expression of 14 genes (ATP5I, KRT4, AD0RA2b, hMLHl, p38IP, PURA, HLA-DMB, CBG, LTF, MTIF2, OAS1, DCX, CRYM, TIA1). These genes relate with many functions of human body. The continually differential expression in the occurrence and process of fluorosis will affect cell signal conduction, immune defence, protein synthesis, energy metabolism, susceptibility to tumour and cardiovascular function as well as nervous system upgrowth, while it indicates its important function in study on the effect of high level of fluorine on injury mechanism of human body.
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