【作者】 井玲；

【导师】 李广生；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2006， 博士

【摘要】 骨周软组织钙化和骨化是慢性氟中毒重要的骨病变之一,其发生机制不清。骨周软组织中的主要细胞成分是成纤维细胞,本文将重点研究氟化物对成纤维细胞增殖分化、骨生长因子表达、成骨表型转换以及细胞内Ca2+浓度等方面的影响,探讨氟骨症骨周软组织钙化和骨化的可能机制。以小鼠皮肤成纤维细胞株(L929)为研究对象,应用MTT法、电镜观察及流式细胞术等方法,研究氟化物对成纤维细胞增殖活性、细胞周期和形态结构的变化。结果表明,成纤维细胞染氟后细胞增殖活力增强,合成和分泌功能旺盛,DNA合成明显增加。应用Elisa、RT-PCR和Western blot等方法,证明氟化物刺激成纤维细胞cbfa1、BMP-2、OCN及I型胶原从mRNA到蛋白的表达明显加强,证明成纤维细胞已有成骨表型的表达。染氟成纤维细胞bFGF、PDGF和IGF等生长因子的表达增加,可能在促进成纤维细胞成骨表型表达和成骨作用增强中起重要作用。应用激光扫描共聚焦显微镜检测,证明氟化物可导致成纤维细胞Ca2+一过性升高,其意义可能在于刺激细胞增殖、分化及某些生长因子的表达,进而促进成纤维细胞成骨功能的发挥。对比研究同样染氟条件对小鼠颅骨成骨样细胞的影响,在细胞增殖、cbfa1和OCN表达、BMP-2、bFGF、PDGF和IGF-1含量等方面两种细胞多具有相同的变化趋势,进一步证明了在氟化物的作用下,成纤维细胞向成骨细胞方向的发展。综合分析研究结果,认为氟化物刺激成纤维细胞向成骨细胞方向分化,成骨功能增强,在氟骨症骨周化骨发生机制中起重要作用。更多还原

【Abstract】 Extraperiosteal calcification and ossification plays an important role in thedevelopment of severe disabled skeletal fluorosis. We will focus our attention onfibroblast which is the main type of cell exists in the extraperiosteal soft tissue includingtendon and the point linking up the ligament. Fibroblast is in the category of inducibleosteogenic precursor cell（IOPC）because of its osteogenesis function under thenon-physiological conditions. It is possible that fluoride can induce expression ofosteogenic phenotype and enhance the potent osteogenesis function of FB. Althoughmany details remain to be studied,the interaction between fluoride and FB would bestrongly analogous to the mechanism of extraperiosteal ossification.The effects and importance of fluoride on FB remain be unclear. Based on ourinferences above, we investigated the effects of fluoride on FB lin（eL929）, including cellproliferation and differentiation, expression of osteogenic phenotype, and the level ofintracellular ionic calcium ([Ca²⁺]_i).1. The effects of fluoride on the proliferation, differentiation,synthesis and secretionfunction of FB and OB（1）The MTT method was used to detect the proliferation of FB and OB in vitro. Resultsshowed that the proliferous activity increased significantly in FBs exposed to certainrange of fluoride concentration （0.001mgF-/L） at certain time （1h and 2hs）. Afterwards,the proliferous activity was decreased with the time of culture and the doses increase offluoride. The effect of fluoride on the proliferous activity of FB was dose-andtime-dependent. The same tendency was found in OB and stronger enhancement wasobserved during the longer period.（2）The ultrastructural changes were observed in FB exposed to fluoride for 48h. Somefindings including lots of enlarged rough endoplasmic reticula, Golgi complexes, fatdrops and glycogen were observed in the fluoride-treated groups. No obvious cytotoxicitywas found even in the group of 10mg F-/L. The results suggest that fluoride can stimulatethe functions of FB in cell proliferation, differentiation,synthesis and secretion.（3）DNA synthesis and cell apoptosis were studied by using flow Cytometry. Thepercentage of cell in G0-G1 phase was 54.0% in the group of 0 mg F-/L and 48.8% in thegroup of 0.1mg F-/L;the percentage of S phase was 31.7% in the group of 0 mg F-/L and32.4%, 46.1%, 32.4%, 35.1%, and 33.7% in the groups of 0.0001, 0.001, 0.1,1,10 and20mg F-/L respectively. The percentage of apoptotic cell was 3.1% in the group of 0 mgF-/L and 2.6%, 2.5%, 1.3%, 0.8%, 0.5%, 1.9% in the groups of 0.0001, 0.001, 0.1, 1, 10and 20 mg F-/L respectively. These data indicated that the DNA synthesis of FB waspromoted,but the cell apoptosis was significantly inhibited by certain concentration offluoride.（4）A major product of OB is type I collagen. By using the methods of RT-PCR andElisa, we investigated the expression of Col I in FB and OB following the treatment withfluoride. Compared with the group of 0 mg F-/L, the content of Col I mRNA and proteinand the formation of mineralized nodule were increased obviously. The results means thatFB is in a higher activity state in preparing for the calcium deposition, under thestimulation of fluoride.2. The expression of osteogenic phenotype in fluoride-treated FB（1）Cbfa1 is the earliest and most specific marker of osteogenesis and an essentialtranscriptional factor for osteoblast differentiation, synthesis and deposition of boneextracellular matrix. Osteocalcin, a major noncollagenous matrix protein of bone, dentin,and cementum, is found tight association with the calcium phosphate mineral phase ofthese tissues. The expression of OCN suggests the activity of OB and the level of boneturnover. The gene expression of OCN is controlled by cbfa1. The expression of bothmRNA and protein of cbfa1 and OCN were increased significantly in FB stimulated byfluoride. It can be concluded that FB have been transformed into OB under the influenceof fluoride.（2）It is of interest to note that the relationship between expression of proto oncogenesc-fos, c-jun and osteogenetic function of fluoride-treated FB. Proto oncogenes areinducible transcriptional factors and regulate growth factors such as bFGF,TGF-β,ColI,c-myc and endothelin in cooperation with other transcriptional factors and play the keyroles in cell proliferation, differentiation and signal transduction. The higher expressionof c-fos and c-jun in fluoride-treated FBs were found in this study. It is possible that protooncogenes have cooperated with fluoride in the inducible expression of cbfa1, OCN andbone derived growth factors in FB.（3）The development, differentiation, synthesis and functions of OB includingangiogenesis, DNA synthesis and bone extracelluar matrix deposition, etc. , arecontrolled by bone derived growth factors（BDGFs）. By using the methods of Elisa,RT-PCR and Western blot,the higher expression of BMP-2、bFGF、PDGF, and IGF-1protein were found in fluoride-treated FB. Although many details remain to be studied,itis supposed that BDGFs should play an important role in promoting the osteogeneticaction of FB. Based on the references and our work,It is inferred that the higherexpression of BMP-2 may mediate higher level of cbfa1 and OCN induced by fluoride. Itis our further work to reveal the interrelations between higher expression of BDGF andosteogenetic function of FB exposed to fluoride.3.The effect of fluoride on the level of intracellular ionic calcium （[Ca2+]i） in FBSkeletal fluorosis was regarded as a “calcium paradox disease”. The elevation of[Ca2+]i may be one of earlier mechanism in activating OB in the exist of fluoride. It issupposed that change of [Ca2+]i in FB induced by fluoride may promote thetransformation from FB into OB by enhancing the expression of cbfa1 and other bonegrowth factors. The level of [Ca2+]i in FB was examined by using laser scanning confocalmicroscope. We found that a significant increase in [Ca2+]i in FB treated with 1mg F-/L.The elevation of FB [Ca2+]i plays an very important role in regulating the coorperationand restriction among the cbfa1 and other growth factors including BMPs, Smads, TGF-β, PDGF, bFGF, IGF-1 and promoting the proliferation, differentiation, expression ofosteogenesis phenotype in FB.4. Effects of fluoride on OBUnder the same experimental condition as showed in FB, we investigated the effectsof fluoride on OB. The similar tendency were showed on the expression of cbfa1, OCN,BMP-2, bFGF, PDGF and IGF-1 in OB. These data not only provide an experimentalsupport that osteoblast can be activated by fluoride in vitro,but also indicate that FBexposed to fluoride has the similar phenotype and function as OB.Fluoride could stimulate the expression of osteogenesis phenotype in FB was firstreported in this paper. These results coincided in our hypothesis that the osteogenic actionof FB, i.e. the non-physiological osteogenesis leading to extraperiosteal ossificationwould be induced by fluoride. The studies above will demonstrate the critical function ofcbfa1 in the pathogenesis of extraperiosteal ossification. To elucidate the mechanisms bywhich fluoride promotes osteogenesis of FB may help to find the causal relations offluoride—FB—extraperiosteal ossification. The project will not only explain thepathogenesis of calcification and osteogenesis in extraperiosteal tissues and contribute toestablish the scientific policy for protecting disabled skeletal fluorosis, but also give cluesto the studies of other similar diseases.Several points need to be put forward for the better understanding of the effects ofosteogenesis stimulated by fluoride in FB in our further study. For example, theinterrelations between [Ca2+]i and bone derived growth factors, the mediated function ofBMP-2 in elevating the expression of cbfa1 in FB exposed to fluoride, and so on.更多还原