The Exploration Of The Effect Of Notch1 Signal Pathwaywhen Human Periodontal Ligament Stem Cellstransdifferentiate Into Osteoblast Under Dynamic Strain

The mankind periodontal ligament stem cells（PDLSC）is a special kind of mesenchymal stem cell locating in periodontal ligament tissues.Orthodontic dental movement will happen when the proper forces are seted to the dental structure by orthodontic instrument.This phenomenon was coused by the periodontal ligament through the alveolar-ridge’s re-absorption insqueezing side and sedimentation in extending side.Our Last study showed thatPDLSCwould differentiate intoosteoblastcells and response to the osteogenicprogresswhenthe expansion of periodontal ligament tissues was caused bydynamic mechanical stress,which means PDLSC is a essentialresource in the orthodontic teethmovement.It is universial believed that this osteoblastprogress under mechanical tensionsituation is complex and numbers of pathways are involved,like Wnt pathways and Hedgehog（Hh）pathways.Signale pathways are involved in this process and the detail of this mechanism,specially the early stage of PDLSC osteogenic progress,has not yet been clearly detected.Researches on mesenchymal stem cell in other tissues of human body showed that the Notch1 signal pathway has been confirmed as a important regulator in the multiplication anddifferentiation of mesenchymal stem cells.The Notch1 signal pathway are essential highly widely spearded in mammals.In mammals,the Notch gene has several homologs,but Notch1 signal pathway is the most important one in human body.Many researches confirmed that the Notch1 pathway play an leading role in the differentiate inductionof the mesenchymal cells into the osteoblast cells.In our previous wroks,we successfully built an experiment model including humanperiodontal ligament stem cells（PDLSC）separation and identification.And we confirmed that dynamic cyclic stress situationlead hPDLSCs to transform intoosteoblast cells.But the effect of Notch1 sinal pathway is still unclear.Beacuse the Notch1 signaling pathway has been shown to be adeterminerin the osteoblastdifferentiation progress in many cells,we assume that the Notch1 signaling pathway also plays an critical role in the osteoblast differentiation process of human PDLSC under dynamic tension condition.DPAT（γ-Secretase Inhibitor,a suppressor of Notch1 signal pathway）and human recombination Jagged1 protein（a Notch1 signalingpathway stimulator）were applied to hPDLSCs which will modify the Notch1 signal pathway.So that we canstudy its role in theosteoblast differentiation of human PDLSC in vitro.These study will provide anotherpassageand build up a new experimental model for furtherreseach into the molecularmechanism of orthodontic dental movement.Wewill further explore the mechanism of PDLSC differentiation from cellular andmolecular levels.And theseknowledges will benefit us a lot whenimproving clinical therapeutic process in orthodontics.ObjectiveTo explore the effect of Notch1 signal pathwaywhen human periodontal ligament stem cells（PDLSCs）transform into osteoblast cells under dynamic strain.MethodsPDLSCs were separatedfrom freshly extracted teeth then identified and prolifed.Notch1 signal pathway was regulated by chemicals.Dynamic strains were applied to PDLSCs with the Tension Plus System.Then Notch1 signal pathway key factor Notch intracellular domain（NICD）,osteoblastic related indexes Alkaline Phosphatase（ALP）and BoneMorphogeneticProteins2（BMP2）was detected by western blot examination.1 Separation and identification of human PDLSCs: extracting those premolars with good periodontal and dental healthy condition beasuce of orthodonticreasons.Theseteeth were taken from patients aged 14-20 years,at theorthodontic clinic of stomatology ofSouthwest Hospital.The pations was informedsufficiently before theextraction.Thesepremolars were transferedin phosphate buffered saline（PBS）added with 1% penicillin and streptomycin immediately.they should be transfered into a sterile bench no more than 20 minutes.Periodontal ligament tissues was attaching on the extracted teeth.They needto bescrathed from the middle third of the extracted premolar root gently.Afterthese processes,tissues werediposedinto type I collagenase solution for 45 minutes with the temperature of 37℃.Using centrifugation to collect the periodontal ligament tissues,a six welles culture plat were used to plated them.These tissues,adding with Alpha-minimal essential medium（α-MEM）and 15% fetal bovine serum（FBS）,were incubated at 37℃ with 5%CO2in a thermostat incubator for primary culture.When the cells wereobtained from the cultured periodontal ligament tissues,digested the cells into single-cell turbid liquid and cultured in a 96-well plate.Then located the sole colony andsubcultured to obtain the PDSLC colony.After the colony-forming process,immunofluorescence examination for the expression vimentin and pan-cytokeratin,osteoblast and adipogenicinduction examination were applied to identify PDLSC.2 To studythe effect ofNotch1 when the PDLSC differentiated into a osteoblast.Witha stretching rate form 0 to 12% and frequency of 0.1Hz,dynamic mechanical extendedstress wasset upto PDLSC in osteoblast induction environment with Flexercell-4000 T tension Plus device for0 h,6h,12 h and 24 h.PDLSC were passaged into special Bio Flex 6-well plates.This special sinica-membrane BioFlex culture plates arecovered with rat tail collagen.Then the protein expressions of ALP,BMP2 and NICD were detected by western blot examination.3 In order to confirm the regulatory character of the Notch1 signaling pathway when PDLSC differentiated into a osteoblast cellthrough cyclic tention stress.Strain,extended rate from 0 to 12% and 0.1Hz,were set up to PDLSC in osteogenicinduction conditions with Flexercell-4000 T Tension Plus facilityfor 0h,6h,12 h and 24 h.PDLSC weresubcultured into special Bio Flex 6-well plates added with Notch1 pathway suppressor DAPTand stimulator Jagged1.Control groups were added DMSO of10^-2Min tension osteogenic induction environment.Then the protein expressions of ALP,BMP2 and NICD were detected by western blot examination.Results1 Through single-clony collection,we successfully separated and passaged PDLSC.In vitro,the shape of PDLSC mostly likesa long rhomboid.It is a classical shape,similars to the otherperiodontal ligament tissues but different in size.PDSLCs were smaller.It wasshowed by theimmunofluorescence exmination that PDLSC were highly positive for vimentin expressionbut negativefor pan-cytokeratin expression.under the sight of microscope,Calcium deposits could be findthrough alizarin redstaining.These confirming showed that PSLSC transformed into osteoblast cell,so as adipoblast cell,because theoil-redO could find lipid droplets.2The protein expressions of ALP and BMP2 had asignificantly up-regulated tendency（P < 0.05）during the 24 h loading.But after a 24 h loading of cyclic strain down-regulated tendency could be found in NICD expression（P<0.05）.As we can see,the Notch1 signaling pathway was involvedwhen the PDLSC differentiated into osteoblast cellthroughstress.3 To further study the effect of Notch1 when PDLSC was differentiating.PDLSC weresubcultured into special Bio Flex 6-well plates added with Notch1 pathway suppressor DAPTand stimulator Jagged1.Control groups were added DMSO of10^-2Min tension osteogenic induction environment.After 24 h tension loading,the protein expressions of BMP2,ALP in the DPATgroup were enhanced（P < 0.05）while the Jagged1 group expression decreased.As what our study showed,for the first time we confirmed that,under dynamic stress in vitro,thedifferentiation of PDLSC will be suppressed while Notch1 signale is activating.Conclusions1 The absence of PDLSC for is still a bottleneck in experiment study,however,the limiting dilution singl-cloning process,enhanced by our early working,has been proved to be a highly effective method to obtained PDLSC in vitro culture environment.2 This reaserch result indicated that the Notch1 signaling pathway was activated in the early stageof the osteogenic differentiation progress of PDLSC indynamic tension ebvironment.When the Notch1 signaling pathway was suppressed,the osteogenic differentiation wouldbe enhanced;while the early stage differentiation would be weakened as the Notch1 signaling pathway was activated.As all the results above proved that the Notch1 pathwaywill stop PDLSC differentiating into an osteoblast cell under dynamic tension.In our early experiment,we successfully separated PDLSC form human tissues.And we had confirmed that Hh signal wasinvolved in PDLSC differentiation.The purpose of this experiment was to further detecte the relationshipbetween thedifferentiation of PDLSC caused by dynamicstress and the Notch1 pathway.These results can help us enhancing the effect ofbone-repair therapy.Thus,the therapyof periodontal diseases will bebenefited a lot as well as the orthodontic clinical therapy.But mang signals are involved in the differentiation of PDLSC.Signalmolecularsmight corss-talk with each other.So,the accurate molecular mechanismof Notch1 signal in thedifferentiation of PDLSC still need more study.