Role Of CIC-7in The Development Of Dental Fluorosis And Skeletal Fluorosis In Fluorosis Animal Models

Endemic fluorosis is a kind of disease which causes the damage of the hard tissues,suchas teeth and bones, which are named enamel fluorosis and skeletal fluorosis. When peopleare exposed to the fluorosis environment for a long time, they would get high fluoridepollutionfrom air, food and water. We got the preliminary understanding about fluoride ionsin and out of the cell by previous in vitro experiments ways and mechanism of the disease.We surveyed fluorosis patients from the high fluoride areas in Shaanxi province and found aclose relationship between dental fluorosis and genetic background factor. Here weestablished the fluorosis animal model, and the relationship between endemic fluorosis andchloride ion channel were further interpretated.We used fluorosis mice models to observe their teeth to certain period of changes, todetermine the model success. We tested the biological characteristics of voltage-gatedchloride channel7(ClC-7). The main materials and methods were as follows: PartⅠ: We chose the SPF C57BL/6J mice as experimental animals. The treatmentgroup was treated with100mg/L NaF. We observed the general condition of mice includeingsize, weight and tooth appearance according to the enamel fluorosis classification standardof mice. We used HE staining (Hematein&Eosin stainng) method to find the shape andfunction damage by high fluoride to ameloblast.PartⅡ: We used Micro-CT to rebuild the bone trabecular micro morphology and bonedynamic parameters from mice femur, then analyzed the bone microstructure in order todefine the damage to mice femur. HE staining of mice femur was used to observe thechanges of its structure.Part Ⅲ: We extracted the total mRNA from the mice incisors and femur, and usedinverse transcription, real-time polymerase chain reaction (Real-time PCR) technique andimmunohistochemistry (IHC) to test Clcn7mRNA expression level and ClC-7changesprotein expression level, to determine whether ClC-7is involved in the occurrence offluorosis.PartⅣ: In order to further clarify whether the shape and function of offspring tooth andClC-7relevant biological characteristics will be changed by parental generation treated withhigh fluoride stimulates, we used Real-time PCR to test Clcn7mRNA expression level oftooth germ in P1mice which are born by fluorosis mice. HE staining was used to observemorphological changes of tooth germ.The main results were as follows:1.2months later, the treatment group appeared dental fluorosis, fluorosis teeth indexreached a magnitude9;2months and6months mice in treatment group appeared curvedspine, depression, loss of appetite, mobility and other symptoms. The weight of mice wassignificantly reduced than the control group (p<0.05). Through HE staining was used toobserve ameloblast apoptosis and hypoplasia of enamel in treatment group. Above situationshowed that the treatment group mice got enamel fluorosis.2. Micro-CT dynamic observation found that bone fluorosis mice characteristics ofbone mineral density (BMD) was reduced,bone trabecular structure was changed, bone strength was declined; The TRAP and HE staining found high-speed bone status and activityof osteoporosis in treatment group. These results showed that we have successfully set up askeletal fluorosis mice model with osteoporosis phenotype.3. The Realtime PCR testing found that Clcn7mRNA expression level in the treatmentgroup incisor of2months was downregulated by49.7%(p <0.05), the expression level in6monthsgroup did not show any significant difference (p>0.05); Treatment group femurClcn7mRNA expression level in2months was upregulated5.5-fold (p<0.01), there was nosignificant difference in6month groups (p>0.05); IHC staining found that mice incisors incontrol group had a higher ClC-7protein expression in proliferating phase, differential, andsecretory phase enameloblast. High expression was found in the junctional zone of enamelmatrix and secretory phase enameloblast, proliferating phase and secretory phaseenameloblast. In treatment group, the positive staining was found in proliferating phaseenameloblast which are arranged disorder. The secretory phase enameloblast were associatedwith apoptosis. ClC-7high expression was found in the crumb proliferation proliferatingphase enameloblast. The femur in treatment group showed high expression of ClC-7inmouse femoral epiphyseal plate and substantial accumulation of chondrocyte. Above resultsindicated that ClC-7is closely related with enamel fluorosis. Fluorosis related bonereconstruction speed was high and induced more osteoclasts and acid secretion. ClC-7isclosely related to bone resorption. On the other hand, chondrocyte under high fluoride gothigh expression of ClC-7. Thus ClC-7can be used as a biological indicator of fluorosis.4. The P1mice tooth germ Clcn7mRNA expression level was downregulated by72%(p <0.05) in2months treatment group to the control group; The P1mice tooth germ Clcn7mRNA expression level was downregulated by63%(p<0.05) in6months treatment groupto the control group. The P1mice tooth germ Clcn7mRNA expression level in2months and6months treatment group showed no significant difference (p>0.05). HE staining found thatthe treatment group showed tooth tip growth pleomorphism, especially in2months. Theameloblast in treatment group arranged irregular, orsparsely. The enamel became thicker, butless smooth, and enamel and ameloblast were seperated. Above results showed that the P1 mice tooth development will also be affected by parental fluorosis. Its ClC-7relevantbiological characteristics change may be caused by high fluoride.The above results showed that fluorosis mice and ClC-7related biologicalcharacteristics are changed, High fluoride stimulates changed the histological morphology inparental teeth and femoral tissue; ClC-7, as a biological indicator to detect the fluorosis, mayparticipate in the mechanism of fluorosis development.