The In Vivo Research Of Clc-7During Tooth Development

ClC-7has been closely related to tooth development in our previous clinical and basic research work. On one hand, osteopetrosis patients with CLCN7mutatio n have tooth disorders including root dysplasia, tooth impaction, enamel hypoplasi a, etc. CLC-7exists in tooth germs of embryonic and neonatal Pl mice. Therefor e, we predict that ClC-7plays an important role in tooth development. In order t o test this hypothesis, we chose chitosan as the delivery material of siRNA and e ncapsulate Clcn7-siRNA to protect siRNA from RNAase degradation and cellular uptake. Then we injected CS-Clcn7-siRNA nanoparticle solution to the local tooth germ sites and the surrounding tissues, and observed the developmental characteri stics of the tooth germs after a decrease of Clcn7expression levels.Methods:Experiment1:We chose the upper first molars of Kunming neonatal mice as objects of study, and injected siRNA encapsulated with chitosan, then compared th e effect of the local tooth germs after peritoneal injection to explore and make su re a local injection method exploring gene functions in tooth germs.Experiment2:CS-C/cn7-siRNA nanoparticle solution was locally injected in tooth germs. Then we observed the characteristics of tooth development, including certain cells and structures at different stages, such as ameloblast, odontoblast, enamel and dentin by H&E staining and Micro-CT scanning, and verified the role of Clcn7in tooth development.Experiment3:We compared CLC-7expression between control group and experiment group by IHC, then judged the silencing efficiency of CS-siRNA nanoparticle solution.Results:1. The fluorescence distribution and intensity of CS-siRNA nanoparticle solution injected at intraperitoneal injection sites:The fluorescence distribution was firstly extended then narrowed. The intensity was firstly strengthened, then weakened within the peritoneal cavity. But the fluorescence could not be tested in tooth germ sites all the time, even in the dissected tissues.2. We confirmed the foundation of local tooth germ injection method by three steps. Firstly, we dissected out the tooth germs in blue after local injections of blue dyestuffs in tooth germs of upper first molars. Secondly, the in vivo fluorescence imaging showed fluorescence distributions in injected tooth germ sites. Thirdly, frozen sections of tooth germs showed the same results, with the H&E staining showing tooth germ structures intact.3. Micro-CT and H&E staining of paraffin sections of P4, P7, P17experimental group mice showed that formations and structures of ameloblast, odontoblast, dental papilla, enamel and dentin were seriously affected, tooth eruption was blocked, tooth structures were disordered.4. In P4control group, CLC-7expressions of tooth germ were detected positive, with ameloblast ranked the first, odontoblast and dental papilla ranked the second. CLC-7expressions in experiment group were detected negative. Conclusions:1. Peritoneal injection method can not deliver CS-Clcn7-siRNA to neonatal mice tooth germs so far. Local injection method in mice upper first molar tooth germs is feasible.2. It is effective to silence Clcn7in tooth germs using local injection of CS-Clcn7-siRNA nanoparticles. It offered new ways to explore roles of other genes in mice tooth germs.3. Clcn7affects the development of enamel, dentin and tooth shapes, root formation and tooth eruption.In this research, for the first time, we applied CS-siRNA to the local tooth germs by the newly found local injection method, and observed the effect on tooth germs after local silencing of Clcn7genes. We believe that Clcn7may affect the formation of enamel and dentin by ameloblast and odontoblast, involve in tooth shape regulation by dental papilla, influence root development by tooth germ surrounding tissues and cells. In our experiment, we not only verified the involvement of ClC-7in tooth germs development process and explored its roles on tooth germs development, but also it offered new ways to explore functions of other tooth germ genes by siRNA local injection method we found.Although we have found that local injection of CS-Clcn7-siRNA may downregulate the expression of CLC-7and affect the cellular or tissue shape of tooth germ, the mechanism involved in this process has not been proved in our study, and we will go deeper to find the molecular mechanism in the future study.