| The theory of tooth movement during the process of orthodontics treatment lies in that persistent pressure is exerted on the teeth and the bone tissues adjacent to the teeth have reconstruction, resulting in the movement of teeth, and this is established on the biological reconstruction incurred after the periodontium stress including the movement and reconstruction of the teeth, the attached structure adjacent to them like cementum, parodontium, and alveolar bone. However, the movement of the teeth is not just simple mechanical movement, and during this process the joint participation and mutual coordination of the osteoblast and osteoclast could render the alveolar bone to realize selective absorption and formation, that is, the absorption of the bones on the side with pressure and formation of new bones on the tension side. In addition, the reconstruction of alveolar bone was mediated by periodontal membrane, so the movement of teeth is first manifested as periodontal membrane phenomenon.Periodontal ligament stem cells(PDLSCs) are originated from the stem cells having the abilities like multi-directional differentiation, self-renewal, and clone formation. It has been manifested by researches that under the function of stress the periodontal membrane stem cells could be differentiated into osteoblasts and chondrocytes, which are the key cells for the reconstruction of alveolar bone. The transformation of stem cells to osteoblasts is not a single process but the results of the joints functioning of a lot of growth factors and access, but the specific mechanism is not very clear.The Hedgehog signal has participated in the formation of various kinds of organs during the process of the embryonic development, and it especially plays an important role of regulating the process of the growth and development of bones. It has been shown according to researches that the Hedgehog signal routes had mediated the series of processes in which the mesenchymal stem cells(MSCs) was transformed into osteoblast and chondrocyte as well as endostosis. Many kinds of factors may directly or indirectly transform the mesenchymal stem cells to osteoblasts through the regulating the Hh signal route. This research group had proved that periodical stress may promote the PDLSCs to be differentiated into osteoblasts and verified through in vitro cell stress experiment that Hh signal route may have direct regulating effect on the PDLSCs stress osteogenesis.Glil(Gliloma-associated oncogene homoglog) is important transcription factors within the Hh signal routes and it is the most important marks of the activation of this route. There are few researches on how it affects the molecular mechanism of PDLSCs osteogenesis through regulating the Hh signal routes. Therefore, this experiment plans to construct overexpression Glil gene adenovirus vectors, transfect PDLSCs cells, and explore the effect of upgrading Glil gene on PDLSCs proliferation and osteogenesis under the effect of stress so as to lay experimental foundation for further understanding the mechanism of the construction of alveolar bone during the physiological shift of teeth under the effect of stress.Purpose:To upgrade the expression of Glil gene within periodontal ligament stem cells(PDLSCs) through the construction of Glil gene adenovirus vectors in vitro so as to explore its effect on human PDLSCs proliferation and stress osteogenesis differentiation.Methods:1. The separation, culturing and authentication of human periodontal ligament stem cells(PDLSCs)The teeth specimen came from the healthy fresh periodontal premolars extracted due to the clinical needs for correctionon those patients(aging from 12 to 24), which had been known and approved by the patients and their family members. The PBS containing 1% double-antibody was taken to washing the teeth repetitively, then periodontal membrane tissues were obtained through scrape the 1/3 middle part of fangs towards one direction, and the method of enzyme digestion combined with tissue block was taken to conduct primary culture.After 45 minutes of digestion with Type I collagenase, the suspension was centrifuged and then vaccinated into a six orifice plate. The first changing of the fluids was conducted after two days, and then the changing was done every three hours. Parodontium was obtained through separation, and PDLSCs was obtained through fluorescence-activated cell sorting. Cellular morphology and its clone formation ability were observed under microscope, the flow cytometry was adopted to analyze cell surface marker CD146 while the immunohistochemical method was taken to test the expression of waveform protein and keratin in addition to conducting the osteogenesis and adipogenesis induction and differentiation experiment to authenticate the features of stem cells.2 Adenovirus transfectionPDLSCs and Western blot transfected by adenovirus carrying Gli and having specific overexpress were taken to test the expression of Gil 1 in PDLSCs. The experiment was divided into three groups, that it, the normal cell contract group PDLSCs/normal group, empty adenovirus infection PDLSCs/empty carrying group, and the overexpression group of PDLSCs/Gli1 transfected by adenovirus carrying Gli and having specific overexpress.3 Cell stress loadingThree groups of cells of the aforementioned ones from the fourth to the sixth generation were obtained respectively and then inoculated into a six-pore plate with silicone mold as the bottom plate for culturing specifically used for BioFlex. The FX-4000 T loading system, which had reached the level of international standard, was applied to load the dynamic tensile stress on the three groups of cells, the maximum deformation quantity 12% as well as the minimum 0 were taken as the sinusoid, which was conducted in the method of stressing of tensile stress with the frequency as 0.1Hz(that is, 5s stretching and 5s relaxation), and the thrust argmentattion was 0h(cultured still under the same conditions), 6h, 12 h, 24 h. Cells were collected, protein was extracted, and Western blot was taken to examine the changes of the expression of the osteogenesis markers Runx2 and ALP of the effect protein Gli1, PTCH1 and PDLSCs related to the Hh signal route.Results:1 The periodontal ligament cells obtained through primary culture are in the shapes of triangle or long spindle, and the two ends of some spindles had bumps and bifurcation, which could connect to neighbouring cell bumps, the positive rate of the PDLSCs obtained through fluorescence-activated cell sorting was 94.8%(the cell antibody is CD146). Compared to periodontal ligament cells, PDLSCs had smaller volumes with the cells arranged in shapes of radiation or swirling, and they were capable of clone formation; it had been manifested through immunohistochemical examination on PDLSCs that the expression of keratin was negative while that of the vimentin was positive, indicating that the cells were the sources of mesenchyme. Three weeks after adipogenesis induction, lipid droplets could be seen within the cytoplasm, the results of oil red o staining was positive.Three weeks after the osteogenic induction of PDLSCs, scattered mineralized nodules could be seen under the microscope and it was red after alizarin red staining. indicating that the obtained PDLSCs had the ability of multi-directional differentiation like osteogenesis and adipogenesis2 Western blot was taken to examine the changes of the transcriptional levels of the Gli 1 of the gene in PDLSCs having been transfected by Gli 1 adenovirus. It has been shown by the results that 48 hours after the transfection, the expression about of the Gli 1 protein was increased [the grey level was(0.041±0.024) and(0.148±0.017), P<0.05], indicating that the adenovirus vectors could effectively upgrade the expression of the protein Gli1.3 After stress loading was conducted on the three groups of PDLSCs in the same loading methods and different loading time, the protein was extracted and western blot was taken to examine the expression of related protein. Below are the results:(1) PDLSCs/the normal Group: After stressing, the expression of related osteogenesis markers Runx2 and ALP was increased compared to that of those not having stressing, indicating that the ability of the osteogenic differentiation of PDLSCs under stress stimulation was enhanced.(2) PDLSCs/ the normal Group: After stressing, the expression levels of the Ptch protein within the Hh route, the nuclear transcription factor Gli1, ALP and Runx2 which were the related osteogenesis markers were enhanced compared to those when not having stressing, which were also increased with the elapsing of time, indicating that the Hh signal route had participated in the process of PDLSCs stress osteogenesis.(3) After stressing of the PDLSCs/the normal group, the PDLSCs/empty loading group, and the PDLSCs/Gli1 overexpression group, the expression levels of Ptch, Gli1, Runx2 and ALP first upgraded but then lowered with the elapsing of time, and the difference just lied in their increasing degrees. Under the condition when the stressing times were the same, the expression levels of Runx2 and ALP in the PDLSCs/Gli1 overexpression/Gli1 group were more significant than those in the PDLSCs/normal group, indicating that Gli1 could promote the PDLSCs stress osteogenic differentiation.Conclusion:1 It had been proved that the human periodontal ligament stem cells cultivated in the methods of separation culture during the previous period by this researching group were capable of clone formation and the highly expressed stem cells markers had the potentiality of multi-directional differentiation under specific induction environment, verifying that this method(enzyme in combination with tissue block) is valid and feasible.2 After the overexpressed Gill adenovirus transfected PDLSCs, the proliferation rate of PDLSCs was reduced while the Glil gene could show stable high expression.3 The results of the related test after cell stress loading had indicated that the osteogenic capability of PDLSCs could first increase and then decrease with the elapsing of the stressing time, which showing that the dryness of PDLSCs was mitigated or even disappeared in later period. The PDLSCs with specific overexpressed Gli1 had stronger osteogenic capability, proving that Gli1 activated the Hh signal route and could directly regulate the process of the osteogenesis of PDLSCs. |
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