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ObjectiveTo study the relationship between expression of a3 chain of collagen Ⅸ (COLIXA3)mRNA in the population exposed to fluorine and fluorosis,in order to reveal the role of COLIXA3 gene in the pathogenesis of endemic fluorosis.MethodsTwelve cases of mild drinking water-born skeletal fluorosis were selected as case groups in Regiment 123 and 128 of Xinjiang Production and Construction Corps Seven Division,6cases of healthy people living in fluorosis areas for more than 10 years as a internal control group and 6 heathly cases living in non-fluorosis areas for more than 10 years as a external control group.The expression of COLIXA3mRNA of peripheral blood lymphocyte of skeletal fluorosis patients and control groups were determined by using SYBR Green Ⅰ chimeric fluorescent method for real-time quantitative PCR.ResultsThe results of the relative expression of COLIXA3 mRNA of case group,internal control group and external control group were 2.16 ± 0.62,1.06 ± 0.09 and 1.05 ± 0.12,respectively.The COLIXA3 expression in case group was significantly higher than that of the internal control group and the external control group (all P ＜ 0.05),while the difference of COLIXA3expression between the internal control group and the external control group was not significantly different (P ＞0.05).ConclusionsFluorine contributes to the expression of COLIXA3 mRNA in peripheral blood lymphocyte,and the expression is up to 2 times higher than that of the control groups,meaning potential biomarkers.
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ObjectiveTo detect the concentration and distribution of fluoride ions in osteoblasts exposed to fluoride in vitro culture,and to provide basic information for studying the effect of fluoride on osteoblast injury.MethodsIn vitro cultured osteoblasts were exposed to 0,5,10,20,40 mg/L fluoride for 3,10,30 d (n =6),respectively.Concentration and distribution of fluoride ions in the cytoplasm and the nucleus of these osteoblasts were determined by nuclear magnetic resonance spectroscopy.Results(①) After cultured for 3 d,fluoride ion content of the bone cytoplasm exposed to different concentrations of fluoride 0,5,10,20,40 mg/L were (0.83 ±0.65),(0.54 ± 0.23),(0.65 ± 0.77),(0.59 ± 0.87),(3.64 ± 1.21 )mg/L,respectively,and the values of exposed to 40 mg/L fluoride group was significantly higher than that of exposed to 0,5 mg/L groups (all P ＜ 0.05).(②)after cultured for 10 d,the composition of the fluoride ion in cytoplasm of exposed to fluoride 10,20,40 mg/L groups were (4.03 ± 1.23),(3.66 ± 0.98),(6.26 ± 2.10)mg/L,respectively,which were higher than that of exposed to 0,5 mg/L groups [(0.78 ± 0.75),(2.69 ± 0.89)mg/L,respectively,all P ＜ 0.05].Of fluoride 20,40 mg/L groups,the composition of the fluoride ion in nucleus were (1.63 ± 1.19),(2.17 ± 1.21 )mg/L,respectively,which were higher than that of 0,5 mg/L groups[(0.65 ± 0.46),(1.57 ± 0.33) mg/L,all P ＜ 0.05].(③)After cultured for 30 d,of the exposed to fluoride 10,20,40 mg/L groups,the composition of the fluoride ion in cytoplasm were (3.99 ± 0.84),(4.33 ± 1.67),(5.80 ± 1.38)mg/L,respectively,which were higher than that of 0,5 mg/L groups[(0.88 ± 0.44),(2.84 ± 0.43)mg/L,all P ＜ 0.05].The composition of the fluoride ion in nucleus of the fluoride 20,40 mg/Lgroups were (3.33 ± 1.46),(3.53 ± 1.22)mg/L,respectively,which were significantly higher than that of 0,5mg/L groups [(0.70 ± 0.66),(1.99 ± 0.76)mg/L,all P ＜ 0.05].ConclusionsWhen osteoblasts are exposed to fluoride environment,fluoride ions enter into the osteoblasts quickly,and quickly accumulate in the nucleus,showing a special affinity between fluoride and bone tissue.Intracellular fluoride ions increase with the increase of contact time and exposure dose.
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ObjectiveTo observe the morphological changes of bone in the progress of chronic fluorosis.MethodsWistar rats were randomly divided into three groups,30 rats in each group:normal control group,experimental group Ⅰ and experimental group Ⅱ according to body weight.Rats in normal control group drank distilled water freely.Experimental group Ⅰ and group Ⅱ drunk distilled water with sodium fluoride preparation of fluorine containing ion 100,150 mg/L solution for six months,respectively.Bone mineral density was detected by X-ray,bone morphological changes were observed under light microscope and bone histomorphometric parameters were calculated using image analysis software.ResultsThe bone mineral density values were different statistically between the three groups after feeding for 2 and 4 months(F =19.79,3.28,all P ＜ 0.05).However no significant difference was found after feeding for 6 months(F =1.80,P ＞ 0.05).The bone mineral density of experimental group Ⅰ (0.20 ± 0.03,0.21 ± 0.03) was significantly higher than that of the normal control group(0.17 ± 0.03,0.20 ± 0.04) after feeding for 2 and 4 months.The bone mineral density of experimental group Ⅱ (0.21 ± 0.02) was lower than that of normal control group(0.22 ± 0.03) after feeding for 6 months.The bone lamella in experimental group Ⅰ was arranged disorderly,the number of osteocytes increased with their nucleus atrophy and the osteoblasts were more than that of control grouo which arranged in layers observed under light microscooy.In exoerimental group Ⅱ,the bone lamella was bent deformation,the number of osteocytes had decreased with their nucleus shrinking or even disappeared and the number of osteoclasts had increased significantly observed under light microscopy.In experimental group Ⅰ,the mean trabecular density [(0.33 ± 0.03)％] increased and the mean trabecular separation,thickness [( 163.57 ± 1.99),(59.26 ± 7.18 ) μm] decreased compared with that of normal control group [(0.31 ± 0.02)％,(186.60 ± 2.90)μm,(86.42 ± 1.48)μm,all P ＜ 0.05].In experimental group Ⅱ,the mean trabecular density[(0.26 ± 0.02)％] decreased,the mean trabecular thickness[(71.42 ± 10.77)μm] reduced compared with that of normal control group[(0.31 ± 0.02)％,(86.42 ± 1.48)μm].ConclusionsExcess fluoride can damage bone tissue.Low doses of fluoride can stimulate osteoblast activity and enhance osteogenesis.The activity of osteoblasts is great than that of osteoclasts.High doses of fluoride can stimulate both osteoblasts and osteoclasts activity,but mainly the activity of osteoclasts,and bone resorption increases.
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Objective To study the COLIXA3 gene polymorphism of patients with fluorosis and to explore the pathogenesis of COLIXA3 gene in endemic fluorosis.Methods Fifty one cases of patients with drinking-water borne fluorosis were selected as the case group in Xinzhou city,Shanxi province and 28 cases of healthy people were as the control group.Dental fluorosis was detected by Dean method and skeletal fluorosis was examined by X-ray.COLIXA3 of exon 5 gene product of 103 points was amplified by PCR and the gene locus genotype was sequenced.Results Ten cases of mild dental fluorosis,14 cases of moderate dental fluorosis,15 cases of severe dental fluorosis were detected among the 51 patients.The control group was free of dental fluorosis.All the 51 cases of patients with fluorosis had varying degrees of skeletal fluorosis,mainly osteosclerosis lesions,accounting for 86.27％(44/51 ),and mild skeletal fluorosis patients were all osteosclerosis lesions,and osteosclerosis lesions and multiple skeletal lesions were found among moderate and severe skeletal fluorosis patients in the case group,while control group had no skeletal fluorosis.The differences between genotypes of frequency distribution of AA,Aa,aa of COLIXA3 of case and control groups were not statistically significant [96.08％(49/51 ),3.92％(2/51 ),0.00％(0/51) and 96.43％(27/28),3.57％(1/28),0.00％(0/28),x2 =0.94,P ＞ 0.05].ConclusionsCOLIXA3 gene polymorphism is not significantly correlated to fluorosis.
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Objective To study the effect of fluoride on expression of osteoblast Runx2, Osterix and their downstream COL I A2 in vitro. Methods Human osteoblast Saos-2 was cultured in vitro. The cells were grouped according to fluoride(NaF) dose used: 0(control ), 0.625,1.250,2.500,5.000,10.000,20.000,40.000,80.000,160.000 mg/L. Cells were collected after 24 h culture, RNA extracted, and the mRNA expression of Runx2 and Osterix and downstream genes COL I A2 was detected using fluorescent quantitative reverse transcription polymerase chain reaction [Real-time (RT)-PCR]). Results After 24 h in vitro cell cultivation with NaF, the expression of Runx2 in 0.625,1.250,2.500,5.000,10.000,20.000 mg/L groups(388.00 ± 41.80,209.00 ± 25.80,42.80 ±4.52,63.00 ± 16.10,24.30 ± 4.23,16.20 ± 4.32) was higher than that of the control group( 1.00 ± 0.12, all P ＜0.05). The expression of Runx2 in 40.000,80.000,160.000 mg/L groups(0.40 ± 0.05,1.91 ± 0.28,4.87±1.36)compared with that of control group, the difference was statistically insignificant(all P ＞ 0.05).The expression of Osterix mRNA in 1.250,2.500,5.000 mg/L groups(4.04 ± 1.67,229.00 ± 51.00,46.40 ± 10.60) was higher than that of the control group( 1.00 ± 0.42,all P ＜ 0.05). The expression of Osterix mRNA in 10.000,20.000,40.000,80.000,160.000 mg/L groups(0. 16 ± 0.07,0.13 ± 0.01,1.73 ± 0.54,0.01 ± 0.01, 0.09 ± 0.01) compared with that of control group, the difference was statistically insignificant (all P ＞ 0.05). The expression of COL I A2 mRNA in 0.625,1.250,2.500,5.000,10.000,20.000 mg/L groups (2.27 ± 0.89,8.03 ± 2.31,14.20 ± 2.75,7.66 ± 1.34,8.96 ±2.30) was higher than that of the control group (1.00 ± 0.04, all P ＜ 0.05). The expression of COL I A2 mRNA in 160.000 mg/L(0.54 ± 0.01 ) was lower than that of the control group(P ＜ 0.05). Conclusions Fluoride may affect mRNA expression of Osterix and Runx2 in osteoblast and their expression level is related to fluoride concentration.Runx2 and Osterix can also regulate the expression of COL I A2 mRNA.
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Objective To explore whether different degrees of fluorosis influence the expression of cartilage COLIXA3 protein in fluorosis model rats. Methods Forty male Wistar rats 3 to 4 weeks old were randomly divided into 5 groups according to body mass, and these rats were fed with distilled water containing sodium fluoride(NaF) of 0(control), 25, 50, 100 and 150 mg/L for 6 months, respectively, in order to establish the animal model of drinking water type fluorosis. Pathomorphologieal changes of the osseous tissues of rats were analyzed under light microscope and transmission electron microscope, and the expression of COLIXA3 protein of femur metaphysis was examined by immunohistochemistry. Results HE staining showed different degrees of femoral metaphyseal ossification of cartilage in each experimental group, bone density increased, with sclerotic lesions of skeletal fluorosis. The control group showed no abnormal cartilage. Electron microscopy showed that the experimental groups with varying degrees of cartilage cell swelling, cell matrix fades, 50 mg/L group .showed hyperplasia, and 100,150 mg/L groups were observed with organelles decreased, part of the disintegration of the cartilage cell lacunae, lmmunohistochemical staining of rat chondrocytes COLIXA3 was positive, cytoplasm with brown granules, cartilage COLIXA3 protein expression(23.3 ± 4.5, 41.2 ± 5.6, 26.4 ~ 7.5) in the 25, 50 and 100 mg/L groups enhanced. Compared to the control group (6.1 ± 3.5), the expression of 50 and 100 mg/L groups was significantly increased, and the differences were statistically significant(all P 0.05). Conclusions There has pathological changes of sclerosing skeletal fluorosis in animal model. Low-dose fluoride promotes while high-dose inhibits cartilage cell proliferation. When fluorine concentration in external environment is too high and with extended exposure to fluoride, direct toxic effects of fluoride on cartilage cells is observed. Fluorine affects and promotes the expression of COLIXA3 protein in cartilage. Low-dose fluoride can promote COLIXA3 protein expression, as the dose increases (over 100 mg/L), the effect decreases.
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Objective To study the effects of fluoride on minichromosone maintenance(MCM)3 mRNA and the bone formation-related gene:bone sialoprotein(BSP),osteocalcin(OC),osteopontin(OP)mRNA expression on human osteoblast cells.The expression of MCM3 was tested for diagnosis and surveillance value on osteoblast treated with excess fluoride.Methods Human osteoblast cell(Saos-2)was cultured in McCoy5A medium and treated with fluoride(sodium fluoride,NaF).There were eight groups including:0(control),0.625,1.250,2.500,5.000,10.000,20.000,40.000 mg/L groups.Expression of MCM3,BSP,OC,OP mRNA were detected by real-time PCR.Dual-standard curve method was used for analysis.ALPase was determined by measuring the absorbance using a micro titer plate reader. Results Expression of MCM3 mRNA was lower in the 0.625,1.250,2.500,5.000,20.000, 40.000 mg/L groups(0.059 ± 0.003,0.027 ± 0.001,0.272 ± 0.004,0.115 ± 0.002,0.137 ± 0.004,0.754 ±0.002, all P > 0.05) and was higher in10.000 mg/L group(21.300 ± 1.200, P < 0.01 ) than control group( 1.000 ±0.020), especially 10.000 mg/L group was higher than groups treated with fluoride(all P < 0.01 ), the differences among groups were significant(F = 305.842, P < 0.01 ). Expression of BSP mRNA was significantly higher in 0.625,1.250,2.500,5.000,10.000 mg/L groups(71.80 ± 3.60,133.00 ± 7.20,85.50 ± 0.60,80.90 ± 1.20,304.00 ± 21.00)than the control group( 1.00 ± 0.04), especially 10.000 mg/L group was higher than others groups treated with fluoride(all P < 0.01 ), the differences among groups were signifieant(F = 159.531, P < 0.01 ). Expressions of OC mRNA were higher in 0.625,1.250,2.500,5.000 mg/L groups(110.00 ± 12.00,143.00 ± 2.10,90.60 ± 4.10,23.70±1.20) than control group(1.00 ± 0.01, all P < 0.01), and the differences among groups were significant (F = 158.734, P < 0.01 ). Expression of OP mRNA were higher in 0.625,1.250,2.500,5.000,10.000,20.000 mg/L groups(167.00 ± 11.20, 111.00 ± 12.10,72.50 ± 3.50,134.00 ± 14.00,42.30 ± 2.40,45.20 ± 3.30) than the control group(1.00 ± 0.04, all P < 0.05 or < 0.01 ), the differences among groups were significant(F = 60.226, P < 0.01 ).Compared with control group(4.2 ± 1.2), the ALPase activity was increased in all groups treated with fluoride (6.0 ± 0.4,5.8 ± 0.1,5.7 ± 0.4,7.7 ± 1.1,19.2 ± 2.4,8.5 ± 3.0,18.1 ± 4.2), but only 10.000 mg/L and 40.000 mg/L groups were higher than control group and other groups treated with fluoride(all P < 0.01 ), the differences among groups were signifieant(F = 7.806, P < 0.01 ). Conclusions Irregular expression of MCM3 mRNA is not suitable as a diagnostic and monitoring biomarker of osteoblasts exposed to excessive fluoride. Fluoride may affect the osteoblast-related gene expression and to promote osteogenic differentiation.
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Objective To detect the influence of fluoride on the expression TM9SF1 mRNA and Ras mRNA of osteoblasts. Methods The third generation of primary cultured osteoblasts were exposed to a series concentrations of 0,2.5,5.0, 10.0,20.0 mg/L fluoride for 10 days. The influence of different doses of fluorine on the expression of TM9SF1 mRNA and Ras mRNA of osteoblasts cultured in vitro was investigated by SYBR Green I methods. Results The osteoblasts of the control group and the 2.5 mg/L group were in the shape of long spindle, triangle or irregular polygon and had processes, and the cytoplasm was translucent, adjacent cells affixed to each other under light microscope. Those of the 20.0 mg/L group shaped as long spindle or irregular polygon, and some vacuolization and granular materials appeared in cytoplasm. The number of the cells decreased and the volume increased significantly. After exposed to fluoride for 10 days, osteoblasts of 2.5 mg/L group morphologically proliferated. There were statistical siguificances between each groups of TM9SF1 mRNA in human osteoblasts(F = 322.82, P < 0.01). The highest in the 2.5 mg/L group(9326.0 ± 115.97), the expression of TM9SF1 mRNA decreased along with the increasing dose of fluorine. There were statistical significances between 5.0, 10.0,20.0 mg/L groups(6495.0 ± 323.9, 4387.5 ± 545.2, 5962.5 ± 536.7) and control group(9221.0 ± 107.5, all P< 0.01). There was a statistical significance between each groups of Ras mRNA in human osteoblasts(F = 703.28, P < 0.01). The highest in the control group, the expression of Ras mRNA decreased along with the increasing of dose of fluorine. There were statistical significance between 2.5, 5.0, 10.0, 20.0 mg/L groups(6144.5 ± 270.82,5603.5 ± 88.39,3181.0 ± 159.81,4067.5 ± 37.4) and control group(6571.0 ± 196.58). Conclusion The influence on TM9SFI mRNA and Ras mRNA expression in osteoblasts correlates with the dose of fluorine.
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Objective To observe the effects of vitamin C (VC) and E (VE) on the uhrastrueture of liver, kidney and brain tissue of fluorosis rats. Methods One hundred and twenty Wistar rats were chosen as the experimental animals and were divided into 9 groups randomly. The control group were given distilled water and the fluoride exposed group were given distilled water containing sodium fluoride 150 mg/L. The throe VC-fluoride exposed groups were given VC orally in a dose of 50,100,150 mg·kg-1.d-1, respectively, and the three VE-fluoride exposed groups were given VE of 25,50,75 mg·kg-1·d-1, respectively. The VC-VE-fluoride exposed group were given VC of 100 mg·kg-1·d-1and VE of 50 mg·kg-1·d-1at the same time of high fluoride water intake. The rats were sacrificed after 9 months and the ultrastructure changes on liver, kidney and brain tissues of each group were observed under transmission electron microscope(TEM). Results The uhrastrueture of liver, kidney and brain showed pathologic changes in the rats that drank water containing high eoneentrations of sodium fluoride. ①Edema of hepatocytes, smeared mitochontria and nuclear matrix, lipid droplet in eytoplasm of hepatocytes, margination of nueleohs as well as obvious swelling of liver sinusoidal endothelial were observed in fluoride exposed group. ② There were marginafion of heterochromatin, expansion of cell space and endoplasmic reticulum in the kidney after the exposure to excess fluoride.③Signifieant changes were found on glial eells on the brain, including cell swelling, increase and marginafion of heterochromatin in the fluoride exposure group. There were no significant uhrastrueture changes in the VC or VE intervention group, while the VC-VE-fluoride exposure group was almost the same as the control group. Conclusions Fluoresis may cause damage on liver, kidney and brain in rats. VC and VE, alone or combined, have protective effects, and the combined supplementation was stronger than single supplementations.
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Objective To study the proliferation of in vitro cultured mouse chondrocytes exposed to different doses of fluoride.Methods The third generation of primary cultured chondrocytes were exposed to the concentrations of 0,5,10,20,40 mg/L fluoride for 10 days to observe the morphological changes under light microscope and electron microscope to counter the numbers of ehondrocytes and proliferating rote with the growth curve and MTT.Results After exposed to fluoride for 10 days,the proliferation was present in the chondrocytes of the 5,10,20 mg/L groups,and shrinked chromatine and apoptosed ehondrocytes were seen in 40 mg/L group.The absorbance was not significantly different between all groups(F=2.313,P＞0.05);after exposed to fluoride for 48 and 72 hours,there was a significant difference of proliferating ability among 0 mg/L(the contr01)group[(23.5±4.6)%,(29.9±1.7)%],5 mg/L group[(34.6±4.7)%,(45.3±5.9)%],10 mg/L group[(39.9±4.8)%.(56.8±5.5)%],20 mg/L group[(31.8±4.1)%,(38.3±6.5)%]and 40 mg/L group[(28.3±4.3)%,(33.4±4.8)%](F=11.401,25.671,P＜0.05).There wss a significant difference compared with the control group(P＜0.05)with that of 5 and 10 mg/L groups higher than that of 40 mg/L groups(P＜0.05).Conclusions Lower doses of fluoride improve the proliferation of in vitro mouse chondrocyte in a short exposing time,higher doses result in the opposite.
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<p><b>OBJECTIVE</b>To understand the differentially expressed genes in human T lymphocytes induced by arsenic trioxide (As(2)O(3)) and to explore mechanism of its immunotoxicity and immune suppression.</p><p><b>METHODS</b>Human Jurkat T cell line was treated by arsenic trioxide (5 micromol/L, 24 h) in vitro, as a sample model. Then, the differentially expressed genes were cloned and the subtractive cDNA library from Jurkat T cell line was constructed by suppression subtractive hybridization (SSH). Polymerase chain reaction (PCR) and sequencing techniques were applied to identify positive clones.</p><p><b>RESULTS</b>The forward subtracted cDNA library contained differentially expressed genes from Jurkat T cell line induced by arsenic trioxide was constructed, including 29 different gene fragments and only replicated one in the subtracted cDNA library identified by PCR and sequencing analysis. These gene sequences were 95%-100% analogous to the genes in public database (GenBank/EMBL). The cDNA library contained oxidative metabolic genes in mitochondria (triose phosphate dehydrogenase, NADH4, pyrophosphate synthase, 16S rRNA ribosome, succinate-CoA ligase and ATP synthase 6); transcriptional and translation genes poly (A) binding protein, t-RNA-guanine transglycoslase, ribosomal protein L23, ribosomal protein S15A, eukaryotic translation initiation factor 3, Rab interaction protein 5, splicing factor-arginine serine rich 5, and ADP-ribosylation factor-like 6 interacting protein), oxide stress related genes (ferritin high chain and high-mobility group protein 2); protein activating and signaling pathway related genes (casein kinase, serine kinase 2 and phosphatidylinositol-four-phosphate adaptor protein-1-associated protein); cell differentiation and apoptosis associated genes (NB4 cell apoptosis related protein and myeloid differentiation primary response protein) and five genes with unknown function (KIAA0092, CGI-147protein, GCI-35, nucleolar phosphoprotein Nopp34 and Mus muscular partial mRNA for hypothetical protein), as well as a novel gene unmatched to the sequence in GenBank.</p><p><b>CONCLUSIONS</b>The forward subtracted cDNA library contained differentially expressed genes from Jurkat T cell line induced by arsenic trioxide was successfully constructed. And, genes not involved in previous research on arsenic were found. Results of analysis for these genetic function suggested that there should be many genes involved in process of T lymphocytes apoptosis or injury induced by arsenic trioxide and that there should still be many genes associated with arsenic that were not reported in the past.</p>