| Objective:The study detected the cell cycle regulation molecular(p21)and the key enzyme of DNA methylation(DNMT1)mRNA and protein expression by the epidemiological investigation and laboratory testing?We establish the fluorosis model on the basis of osteoblasts primary cultured in vitro in human to detect the effect of p21 gene promoter methylation level and subsequently mRNA and protein expression in human osteoblasts exposed to fluoride,while detecting the DNMT1 mRNA and protein expression.To explore the role of p21 gene methylation in skeletal fluorosis development.Methods:1 Population based research on fluorosis caused by coal-burning1.1 Objects According to the standard of endemic fluorosis area(GB 17018-2011),we choose the HH village of zhijin county as the burning coal pollution-type fluorosis survey place,and ZG village of Anshun city as control place.We screened 380 subjects(fluorosis patients 295 cases,control 85 cases)according to their time of fluoride exposure,dental health,clinical symptoms,X-rayexamination.1.2 The questionnaire survey,environmental and biological samples Under the principle of informed consent,they will be asked to fill in a questionnaire,collecting environmental samples(Rice,pepper,corn,drinking water,soil,indoor and outdoor air)and biological samples(blood and urine).1.3 The fluorine content of environmental and urine samples The fluorine content of environmental and urine samples were examined with F-ion selective electrode method.The respondengts was divided into 4 groups according the content of urine fluorine.? UF<1.96 mg/gCr group 140 cases(male 61 cases,female 79 cases,age(44 ± 17),? 1.96 mg/gCr ? UF<3.92mg/gCr group 93 cases(male 45 cases,female 48 cases,age(44 ± 18),? 3.92mg/gCr ? UF<7.84mg/gCr group 82 cases(male 44 cases,female 38 cases,age(45 ± 16),? UF ? 7.84mg/gCr group 65 cases(male 29 cases,female 36 cases,age(49 ± 12).1.4 Detecting p21 and DNMT1 mRNA transcription and protein expression Peripheral blood p21 and DNMT1mRNA transcription expression was examined with real-time quantitative PCR(FQ-PCR).Peripheral blood P21 and DNMT1 protein expression was examined with enzyme linked immunosorbent assay(ELISA).2 Fluorosis experiment in vitro2.1 Human primary osteoblasts isolation?culture and identification Human primary osteoblasts were isolated by enzymatic digestion method,They were determined by alkaline phosphatase staining and Mineralization nodes staining.2.2 The cell growth curve and cell cycle of human primary osteoblasts exposed to fluoride The cell growth curve of human primary osteoblasts exposed to 0?mol/L100?mol/L?200?mol/L?400?mol/L?800?mol/L and 1600?mol/L dose of NaF was detected by MTT.The cell cycle distribution in human primary osteoblasts exposed to 0?mol/L?100?mol/L?200?mol/L?400?mol/L?800?mol/L?1000?mol/L?1200?mol/L and 1600?mol/L dose of NaF 72h was detected by flow cytometry.2.3 Making the human primary osteoblasts model of fluorosis The fluoride dose 0?mol/L?125?mol/L?250?mol/L?500?mol/L and 1000?mol/L treatment human primary osteoblasts.The success of fluorosis model of human primary osteoblasts turns on morphologic changes,alkaline phosphatase activity,BGP quantity.2.4 Detecting p21 gene promoter methylation level The fluoride dose 0?mol/L?125?mol/L?250?mol/L?500?mol/L and 1000?mol/L treatment human primary osteoblasts 72h.The p21 gene promoter methylation level in human primary osteoblasts was detected by bisulfate-sequencing polymerase chain reaction(BSP).2.5 Detecting p21 and DNMT1 mRNA transcription and protein expression p21 and DNMT1 mRNA transcription expression was in human primary osteoblasts exposed to fluoride examined with real-time quantitative PCR(FQ-PCR).P21 and DNMT1 protein expression in human primary osteoblasts exposed to fluoride examined with Western blotting(WB).Results:1 Population based research on fluorosis caused by coal-burning 1.1 The fluorine content of environmental and urine samples The fluorine content in environmental samples of the burning coal pollution-type fluorosis survey place than national standards or recommended value,they were obviously higher than the control place(P<0.05).The UF of case group is higher than the control group(=40470.40,P<0.05).UF gradually increase with different ages in case group(x2=18.21,P<0.05).1.2 p21 and DNMT1 mRNA transcription and protein expression The difference of p21 mRNA transcription and protein expression with statistical significance(Fp21mRNA=10.32,FP21 protein=3.62,P<0.05),The p21 mRNA transcription and protein expression decresed with UF increase(rp21mRNA =-0.10,rP21protein =-0.16,P<0.05).The difference of DNMT1 mRNA transcription and protein expression with statistical significance(FDNNT1mRNA=6.66,FDNMTprotein=3.11,P<0.05),The DNMT1 mRNA transcription and protein expression increases with the UF increasing(r=0.17,P<0.05).With UF increasing the DNMT1 protein expression dose-effect relationship was not observed.2 Fluorosis experiment in vitro2.1 Human primary osteoblasts isolation?culture and identification Enzyme assisted isolate cells derived from human cancellous bone after cultured 24h vitro,the shapes of cell bodys are fusiform or spherical.Fusiform cells increased along with the cell proliferation.After cell growth fusion,swirling and radiating patterns and was observed under microscope.Alkaline phosphatase staining and alizarin red staining presented positive results.The results indicated that the cells we cultured was Human primary osteoblasts.2.2 The cell growth curve of human primary osteoblasts exposed to fluoride Human primary osteoblasts exposed to fluoride 72h,100?mol/L?200?mol/L?400?mol/L and 800?mol/L dose group cells proliferation with the prolonged fluoride action period.Human primary osteoblasts exposed to fluoride 96h,each dose group cells were markedly reduced.1600?mol/L dose group cells reduced with the prolonged fluoride action period.The same time points and different dose groups compared,Human primary osteoblasts exposed to fluoride 48h and 72h,100?mol/L200?mol/L?400?mol/L and 800?mol/L dose group proliferation rate obviously higher than control group(P<0.05),Human primary osteoblasts exposed to fluoride 96h,each group proliferation rate lower than control group,800?mol/L and 1600?mol/L dose group proliferation rate obviously lower than control group(P<0.05).2.3 The cell cycle of human primary osteoblasts exposed to fluoride Human primary osteoblasts exposed to fluoride 72h,G0/G1 phase cells reduced,proliferation index and S phase cells increased the 1000?mol/L dose range of fluoride.1000?mol/L dose group S phase cells and proliferation index obviously higher than each other groups,show proliferative state.2.4 The human primary osteoblasts model of fluorosis Human primary osteoblasts increased with increasing dose of exposed to fluoride,and cell morphological changes at the same time.AKP activity and BGP content increased with increasing dose of exposed to fluoride(FAKP=77.40,FBGP= 11.26,P<0.05).The experiment show that human primary osteoblasts proliferation activity was increased under the rxpreriment conditions,successfully been constructed human primary osteoblasts model of fluorosis.2.5 p21 gene promoter methylation level Human primary osteoblasts exposed to fluoride with 0?mol/L?125?mol/L?250?mol/L?500?mol/L and 1000?mol/L 72 later,the p21 gene promoter methylation level were 0%(0/288),0.7%(2/288),3.8%(11/288),11.8%(34/288)and 17.7%(51/288),respectively.There were significant difference among groups(?2=107.368,P<0.05).The p21 gene promoter methylation level increased with exposing to fluoride increase(?2?=96.273,P<0.05).2.6 Human primary osteoblasts p21 and DNMT1 mRNA transcription and protein expression The p21 mRNA transcription and protein expression decreased with increasing dose of exposed to fluoride(Fp21mRNA=3.90,FP21 protein=22.18,P<0.05),DNMT1 mRNA transcription expression increased with increasing dose of exposed to fluoride(F=3.43,P<0.05).The p21 mRNA transcription and protein expression decreased,DNMT1 mRNA transcription and protein expression increased,with human primary osteoblasts expose to fluoride increase.Conclusions:? Vivo and in vitro showed that fluoride can cause p21 mRNA transcription and protein expression decreased,its possible cause osteoblast through G1 quickly into the S phase?? Fluoride induced p21 gene promoter methylation,inhibit subsequently mRNA transcription and protein expression,it maybe the one of molecular mechanisms that skeletal fluorosis developmen.? Fluoride induced DNMT1mRNA transcription and protein expression enhanced,it was maybe participated in p21 gene promoter high methylation process... |
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