【作者】 马俊香；

【导师】 刘克俭；

【作者基本信息】 华中科技大学 ， 劳动卫生与环境卫生学， 2009， 硕士

【摘要】 氟性骨损伤(fluoride bone injury, FBI)骨周化骨是一种特殊的异位骨化形式(Heterotopic ossification,HO),其主要临床表现为广泛性骨质增生/硬化、骨软化、骨质疏松、骨间膜、肌键和韧带钙化等。国内外关于FBI的研究大多集中在氟(fluoride, F)对骨细胞的损伤方面,对F导致骨关节硬化方面的研究较少,而后者才是导致慢性FBI患者致瘫致残的重要原因。成纤维细胞(fibroblast,FB)起源于胚胎间充质干细胞(mesenchymal stem cells, MSCs),具有多分化潜能,在在F的刺激作用或某些病理条件下,这些FB还可进一步向成骨细胞(osteoblast, OB)转化,进一步在非骨组织内发生钙化,引起HO。因此,本实验选择具有成骨潜能的FB作为研究对象,通过建立体外染F模型,并对不同染F剂量组和时间段的染氟成纤维细胞(fibroblast exposed to fluoride, FBEF)的活力和增殖活性进行了初步鉴定,并在此基础上,进一步研究了F对锌指结构转录因子Osterix(Osx)和骨钙素(Osteocalcin, Ocn)转录和表达的影响。初步分析了:1)引起FB向成骨表型转换的最佳染F时间剂量;2)Osx和Ocn在此转换过程中转录或表达的变化情况,分析引起二者变化的最佳染F时间和剂量,并进一步分析二者转录或表达之间的相关性;3)检测Osx和Cbfα1在职业性F暴露人群中的表达水平;4)探讨Osx和Cbfα1在FBI骨周化骨过程中的作用机制,进一步阐明F-FB-FBI骨周化骨三者之间的关系。本研究主要由以下三部分组成:第一部分不同染氟剂量组和染氟时间段染氟成纤维细胞的增殖活性目的:建立体外传代培养小鼠FB L929染F模型,探讨F对小鼠FB L929活力和增殖活性的影响,为第二部分的试验确定合适的染F剂量和染F时间。方法:传代培养的小鼠FB L929分别预设七个染F剂量组:0.0001、0.001、0.01、0.1、1、10、20mg/L,9个染F时间段:1、2、4、8、12、24、48、72、96h,采用台盼蓝排斥试验法,观察不同染F剂量组和不同染F时间段FB的存活情况;用MTT法测定FBEF的增殖活性。结果:1)细胞活力:与对照组相比,0.1、1、10和20mg/L剂量组活细胞率明显下降(p<0.05),仅20mg/L剂量组的活细胞率小于50%;2)细胞增殖活性:0.0001mg/L和0.001mg/L剂量组,几乎在各染F时间段FB的增殖活力均增加(p<0.05),其中在12、24和48细胞活性增加比较明显(p<0.05);0.01mg/L和0.1mg/L剂量组,只有12、24、48和72h细胞活性增加(p<0.05),其余各组均随染F时间的延长和染F剂量的增加,细胞增殖活性呈现明显下降趋势。结论:1)F对FB的活力有抑制作用,染F剂量与FB活细胞率呈现明显的剂量-效应关系;2)F对FB的增殖活性的影响呈现一定的剂量-时间-效应关系,即短时间-低剂量的F明显增强FB的增殖活力,随着染F剂量的增加和染F时间的延长,FB的增殖活力明显减弱。确定后续实验的染F剂量为0.0001、0.001、0.01、0.1、1、10mg/L,染F时间为2、4、8、12、24、48、72、96h。第二部分锌指结构转录因子Osterix和骨钙素在染氟成纤维细胞中的表达目的:检测Osx和Ocn基因在FBEF中的转录和蛋白表达,探讨Osx和Ocn在FBI骨周化骨中作用机制。方法:1)Osx和Ocn蛋白的检测:将传代培养小鼠FB L929分别暴露于以下6个不同的染F剂量组:0.0001、0.001、0.01、0.1、1、10mg/L,分别按8个时间段进行染F:2、4、8、12、24、48、72、96h,分别采用Western blot和Elisa法,分别检测Osx和Ocn蛋白在每个染F时间段和染F剂量组的FBEF中的表达量;2)Osx和Ocn mRNA基因的检测:将传代培养小鼠FB L929分别暴露于以下6个不同的染F剂量组:0.0001、0.001、0.01、0.1、1、10mg/LNaF,分别染F 24、48、72h后,提取各个剂量组和染F时间段的mRNA进行逆转录,然后采用荧光实时定量PCR的方法,对各个剂量组和染F时间段的mRNA进行相对定量。结果:1)Osx蛋白的检测结果:①与对照组相比:染F 12和48h,0.0001和1mg/L剂量组Osx表达量升高比较明显(p<0.05);染F 24h,0.0001、0.01和0.1 mg/L剂量组Osx表达量明显升高(p<0.05);染F 72h,1和10mg/L剂量组Osx表达量明显升高(p<0.05);染F 96h,仅在0.001mg/L剂量组Osx表达量明显升高(p<0.05);②与8h相比:染F 12h的0.0001和1mg/L剂量组和染F 48h的1mg/L剂量组Osx表达量明显升高(p<0.05);染F 24h时几乎所有剂量组的Osx表达量均明显升高(p<0.05);染F 72～96h时大部分剂量组的Osx表达量均明显降低(p<0.05);总的来看,在各染F剂量组Osx表达量在24h时达最高,其余剂量组,随着染F时间的延长Osx蛋白的表达量呈逐渐降低的趋势;2)Ocn蛋白的检测结果:①与0 mg/L组相比,染F 24h,0.001、0.1和10mg/L剂量组的Ocn浓度显著性增高(p<0.05);染F 48～96h,10mg/L剂量组的Ocn浓度明显升高(p<0.05);②与8h相比,染F 24h的大部分剂量组和染F 72h的10mg/L剂量组的Ocn浓度呈明显升高(p<0.05);染F 48～96h,各剂量组中的Ocn浓度呈明显下降趋势;总的来看,各剂量组的Ocn浓度随着染F时间的延长有降低的趋势,个别剂量组在24h时则明显上升;3)Osx mRNA基因的检测结果:染F 24和48h时,各个剂量组Osx mRNA的转录水平均呈增高趋势,尤其在1和10mg/L剂量组中增加比较明显(p<0.05);染F 72h时Osx mRNA的转录水平总体呈现下降趋势,仅在1和10mg/L剂量组转录水平有所增加;4)Ocn mRNA基因的检测结果:染F 24h,Ocn mRNA的转录水平较对照组明显增高(p<0.05);染F 48h,各剂量组的Ocn mRNA转录水平总体呈现先下降后升高的趋势;染F 72h,各个剂量组Ocn mRNA的转录水平则呈现先升高后下降的趋势,但Ocn mRNA的转录水平均较对照组高。结论:1)F可促进Osx和Ocn基因在FBEF的转录和表达,而且Osx和Ocn基因的转录和表达呈现时间-效应关系,24h的0.0001mg/L剂量组和48h的1mg/L剂量组是引起Osx蛋白表达的最佳条件,引起Osx mRNA转录的最佳时间-剂量则为24h和48h的10mg/L剂量组;2)染F可刺激FB向OB的转化,并促进Osx基因的转录和表达,并进一步促进成骨标志性基因Ocn的转录和表达,Osx基因可能是FBI骨周化骨过程中重要的调控基因。第三部分锌指结构转录因子Osterix和核心转录因子Cbfα1在不同职业性氟负荷人群中的检测目的:在蛋白表达水平上,观察Osx和Cbfα1在不同职业性F负荷人群中的表达,探讨Osx和Cbfα1在FBI骨周化骨中的作用机制。方法:选取湖北某铝业集团连续工作5年以上的男性工人为研究对象,然后根据F负荷(血F和尿F浓度)将观察对象分为三组:对照组(血F<0.16mg/L且尿F<2mg/L),低F负荷组(0.16mg/L≤血F<0.25mg/L且2mg/L≤尿F<4mg/L)和高F负荷组(0.25mg/L≤血F且4mg/L≤尿F),采用Elisa法分别检测血清Osx和Cbfα1的含量。结果:1)血氟和尿氟检测结果:中F负荷组和高F负荷组的尿F浓度与对照组相比明显升高(p<0.05),而血清F仅在高F负荷组升高较明显(p<0.05);与中F负荷组相比,高F负荷组的血清F和尿F均明显升高(p<0.05);2)血清Osx和Cbfα1检测结果:①与对照组相比,中F负荷组的血清Osx浓度明显升高(p<0.05),而血清Cbfα1升高却不太明显;②与中F负荷组相比,高F负荷组血清Osx和Cbfα1的浓度均明显降低(p<0.05)。结论:Osx和Cbfα1基因可能是FBI骨周化骨过程中两个比较重要的调控基因,而且血清Osx变化较Cbfα1更明显,在成骨过程中更具特异性。因此,可以初步推测血清Osx和Cbfα1可能是FBI早期损伤诊断的参考指标,尤其是Osx可能是FBI骨周化骨过程中重要的调控点之一。更多还原

【Abstract】 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.更多还原