| Ossification around bone in fluoride bone injury(FBI) is a special kind of heterotopic ossification(HO), and its main clinical manifestation include extensive hyperostosis osteosclerosis, osteomalacia, osteoporosis, and calcification of interosseous membrane, muscle Tendon and ligament, and so on, which may be the important factor leading to paralysis and mutilation. At home and abroad, there are many reports on FBI, most of which focus on the bone cells injuried by fluoride(F), but neglect that the pathomechanism of arthrosclerosis caused by F, which is the main risk factor seriously endangering the life of people's health.Fibroblast (FB), is derived from embryo mesenchyme cell with multi-differentiation potential. Under the stimulation of F or other pathological conditions, they can be transformated into osteoblasts(OB), which could promote calcification in the non-bone tissue, a kind of HO. Therefore, we choose FB, with osteogenic potential, as experimental subject, establish model of F expousure in vitro, and preliminarily identify the vitality and proliferative activity of FB exposed to fluoride(FBEF) at different times and doses of F-expousure. And on this basis, we detect the transcription and expression of Osterix(Osx) and Osteocalcin (Ocn) at the given time-and dose-F exposure group, to find and analyse the following: 1) optimal time and dose of F exposure in F giving rise to the conversion from FB to OB phenotype; 2) in this process of conversion, to analyse the changes of transcription or expression of Osx and Ocn, and find the best time and dose of their change point caused by F exposure, and the relationship between Osx and Ocn in the level of transcription or expression; 3) inspect and measure the expression level of Osx and Cbfα1 protein in the occupational fluoride exposure crowd; 4) investigate the molecular mechanism of Osx and Cbfα1 in causing ossification around bone of FBI, and interpret the relationship of F-FB-FBI.This study is composed by the following three parts:Part I The proliferative activity of different doses and times of fluoride exposureObjective To investigate the effect of F on the viability and proliferation activity of FB, and determine the appropriate dose and time of F exposure for the second part of the test, we establish the model of subculturing mouse FB L929 exposed to F in vitro. Methods Subculturing mouse FB L929 in vitro, which were respectively exposed to seven pre-seted F-dose group, that is 0.0001, 0.001, 0.01, 0.1, 1, 10, 20 mg/L, and nine F exposure time, that is 1, 2, 4, 8, 12, 24, 48, 72, 96h. We use the test of trypanblau exclusion and MTT to observe survival condition and proliferative activity of FBEF at the abrove seted group. Results 1) viability: compared with the control group, the survival condition of FB in the dose of 0.1, 1, 10 and 20mg/L significantly decrease (p <0.05), except 20mg/L group, in which the viability of FB is under 50%; 2) Activity of proliferation: compared with the control group, in the group of 0.0001 and 0.001mg/L, the proliferation activity of FB takes on increase almost at all time (p <0.05), especial in the time group of 12, 24 and 48h, increase is obvious (p <0.05); while in the group of 0.01 and 0.1mg/L, the obvious increase trend is found only in 12, 24, 48 and 72h (p <0.05), in the rest group, activity of FB proliferating is under a clear downward trend with the increase of F-dose and prolong F exposure time. Conclusion 1) F plays a role of inhibition on the vitality of FB, and there is a clear dose-response relationship between the dose of F and the survival conditional of FB; and 2) it is the same with the activity of FB proliferation, which also shows on a certain dose-time-effect relationship, that is, F significantly enhanced the vitality of FB proliferation in short period of F exposure time and a low dose of F, otherwise decrease with the increase F exposure time and F dose. Except the dose of 20 mg/L and the time for F exposure 1h, the rest groups are all chosen in the follow-up experiment. Part II The expression of Zinc finger transcription factor Osterix and Osteocalcin in fibroblast exposed to fluorideObjective Detection of the transcription and protein expression of Osx and Ocn gene in FBEF, to investigate the molecular mechanism of Osx and Ocn gene in ossification around bone of FBI. Methods The diction of Osx and Ocn protein: Subculturing mouse FB L929 in vitro, and then exposing them to the above seted dose group and F exposure time, using the method of Western blot and Elisa to respectively detect the expression of Osx and Ocn, and the method of fluorescence real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) to relative quantificate the level of transcription of Osx and Ocn mRNA, but we only choose three F exposed time group and all F dose groups to detect. Results 1) The results of Osx protein:①Compared with the control group, Osx protein significantly increase in follow-up group(p <0.05), the group 0.0001 and 1mg/L with F exposure 12 and 48h, the group 0.0001, 0.01 and 0.1 mg/L with F exposure 24h, the group 1 and 10mg/L with F exposure 72h, and the group 0.001mg/L with F exposure 96h;②Compared with the group with F exposed 8h, Osx protein significantly increases in follow-up group(p <0.05), the group 0.0001 and 1mg/L with F exposed 12h, the group 1mg/L with F exposed 48h, and all the groups with F exposed 24h; but in most of groups with F exposed 96h, Osx protein significantly decreases (p <0.05); Overall, the expression of Osx protein in all dose groups reached the highest at F exposed 24h, and then gradually decreases with F dose increasing and F exposed time prolonging; 2) The results of Ocn protein:①Compared with the 0 mg/L group, Ocn protein significantly increases in follow-up group(p <0.05), the group 0.001, 0.1 and 10mg/L with F exposed 24h, the group 10mg/L with F exposed 48 ~ 96h ;②Compared with the group with F exposed 8h, Ocn protein significantly increases in the majority of groups with F exposed 24h, and the 10mg/L with most F exposed 72h, but the concentration of Ocn significantly downward in all groups with F exposed 48 ~ 96h; Overall, the concentration of Ocn shows decrease trend in all the dose groups with the extension of F exposure, only few groups significantly increase when FB is exposed to F 24h; 3) The transcription level of Osx mRNA: Compared with the control group, Osx mRNA increases in all groups and time groups, especially in the group 1 and 10mg/L with F exposed 24 and 48h, increasing obviously (p <0.05); 4) The transcription level of Ocn mRNA: Compared with the control group, the change trend is almost the same with that of Osx mRNA, only when FB is exposed to F 72h, the former increases at first and then shows a downward trend, while the latter Osx mRNA decreases all the time, but the level of transcription Ocn mRNA and Osx mRNA are higher than the control group.Conclusion 1) F can promote the transcription and expression of Osx and Ocn gene in FBEF, and the effect shows a time-effect relationship; 2) the group 0.0001mg/L at 24h and the group 1mg/L at 48h are the best conditions to promote the expression Osx, and the group 10mg/L at 24 and 48h to the transcription level of Osx mRNA; 3) F can stimulate FB convert into OB, promote the transcription and expression of Osx gene, and then further promote the transcription and expression of Ocn, which belongs to the OB special marker genes; 4) Osx gene is likely to be an important regulatory factor in the course of FBI.Part III The detection of Zinc finger transcription factor Osterix and core transcription factor Cbfα1 in different fluoride burden groupsObjective In the level of gene expression, to detect the concentration of serum Osx and Cbfα1 in different occupational crowd of fluoride exposure, to investigate the molecular mechanism of Osx and Cbfα1 in ossification around bone of fluoride bone injury. Methods Choose male people who have worked more than five years in the Aluminum plant in Hubei Province. According to the concentrations of their serum F and urine F, the subjects are divided into three groups: control group (serum-F<0.16mg/L and urineF<2 mg/L), low-load group fluoride (0.16mg/L≤serum-F<0.25mg/L and 2mg/L≤urine-F <4mg/L) and high-F load group (0.25mg/L≤serum-F and 4mg/L≤urine-F). The concentration of serum Osterix and cbfα1 are detected by the method of Elisa. Results 1) The result of serum-F and urine-F: Compared to the control group, the urine-F significantly increases in both low and high fluorine burden group (p <0.05), but and the serum-F significantly increases only in high group (p <0.05); Compared to the low group, both serum-F and urine-F significantly increase in high group (p <0.05). 2) The result of serum Osx and Cbfα1: Compared to the control group, both serum Osx and Cbfα1 in low group increase, but there is significant statistical significance to the serum Osx (p <0.05); Compared to the low group, both serum Osx and Cbfα1 significantly decrease in high group (p <0.05). Conclusion In the process of ossification around bone of FBI, Osx and Cbfα1 may be two more important controlling genes than other transcription factors. At the same time, we also found that the change of serum Osx is more sensitive and specific than that of Cbfα1 in the process of regulation in ossification. Therefore, it can be initially assumed that serum Osx and Cbfα1 may be a reference indicator for early diagnosis of FBI, and especially Osx may be one of important control points in the causing of ossification around bone of FBI. |
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