| Research Background:1.The oral health is considered to be the important symbol of the processing of society civilization,and an absolutely indispensible part of improving life and live quality.However,we are undergoing a serious problem of tooth loss in our country.The third investigation of The national oral health epidemiological demonstrated that,there are 86.1% tooth loss patients in 65-74 years of aged people,10% of this issue was lacking of full dentition,it had the negative impact on exerting oral function and systemic healthy.Some datas had shown that,the number of population of aged people has been reached 144 million,accouted for 11% of population in the whole country.Till the middle age of 21 st century,it would be reached 30%.The social ageing issue highlights the severity and urgency of tooth loss.Tissue engneering tooth is thought to be the ideal means to solve this problem,and is the most animate research part of stomatology.Especially the rapid improvement of stem cells biology,it laid the research foundation of tissue engneering tooth.The ectomesenchymal stem cells are originated from cranial neural crest,and considered to be the formation cells of tooth tissue except tooth enamel,termed as the primitive cells of dental origin cells.P75NTR(p75 neurotrophin receptor)is considered to be the specific cell surface marker of neural crest origining cells,it had been used to select neural crest originated cells of ectomesenchymal stem cells.Some studies suggested that p75 NTR involved in morphogenesis and development of mutiple tissue(adipogenesis,liver,muscle and teeth).However,the important functions of p75 NTR in nervous system were fully discussed,the study on regulated functions in adipogenesis,liver,muscle and teeth is at the initial stage.Especially,the study on the morphogenesis and development of teeth was rarely reported.This study focused on the regulated funtion of mineralized differentiation of p75 NTR in ectomesenchymal stem cells,combined its binding partner Mage-D1,the NGF high affinity receptor Trk A,and its ligand NGF to discuss the detailed mechanism of the mineralized regulation effect in ectomesenchymal stem cells by p75 NTR.To provide the theoretical basis for realizing the mechanism of tooth origin and development,and to provide the experimental basis for further improving the tooth tissue engneering.Material and methods:1.The extraction,in vitro culture,and detection of biological characteristics of different embryonic stage ectomesenchymal stem cellsThe extraction,in vitro culture,and detection of biological characteristics of E12.5d EMSCs group(the Embryo 12.5 day Sprague Dawley rattus norregicus ectomesenchymal stem cell group)and E19.5d EMSCs group(the Embryo 19.5 day Sprague Dawley rattus norregicus ectomesenchymal stem cell group):When the vaginal plug had been detected by the day,to define the noon to be the embryonic 0.5d.Wait until the noon of 12 days of embryo,injected 2% sodium pentobarbital at the concentration of 40mg/kg for anesthesia in rat,then exert the abdominal surgery.There were 3 to 5 embryos to extract,and then straticulate saturation the surgery wound at abdomen.After the pregnant rat was waking up,continued to feed.The tissue of maxillofacial process was dissected,rinsed by PBS,cut into pieces by eye scissors,digested by ?type collagenase,neutralized and centrifuged at 1000r/min,3min.The precipitation of tissue were evenly plated at 6cm cells petri dishes,added 4-5ml 100ml/L FBS DMEM-F12 culture medium and cultured in 5% CO2 cell incubator at 37?.E19.5d EMSCs group(the Embryo 19.5 day Sprague Dawley rattus norregicus ectomesenchymal stem cell group):The abdonminal surgery pregnent SD rat continued to feed to E19.5d.At the noon of E19.5d,injected 2% sodium pentobarbital at the concentration of 40mg/kg for anesthesia in rat,then exert the abdominal surgery.There were 3 to 5 embryos to extract.The mandibular bone and maxillary bone were dissected into two separate sections,and extracted the teeth germ by eye forcep under the stereo microscope,digested by ?type collagenase,neutralized and centrifuged at 1000r/min,3min.The precipitation of tissue were evenly plated at 6cm cells petri dishes,added 4-5ml 100ml/L FBS DMEM-F12 culture medium and cultured in 5% CO2 cell incubator at 37?.To identify and detect these two kinds of EMSCs by cell morphology,cell proliferation,surface marker of flow cytometry.2.The spatial and temporal expression of p75 NTR and mineralization related gene Runx2 detected by immunohistochemical method.To detect the spatial and temporal expression of p75 NTR and mineralization related gene Runx2 by immunohistochemical method in different embryonic stages.The embryonic facial processes were dissected from the E12.5d and E19.5d SD rats and fixed in 4 % paraformaldehyde,to obtain 6-?m sections of tissue specimens.Hematoxylin and eosin(H.E.)staining and immunostaining were performed using rabbit monoclonal p75NTR(1:1500,abcam,Cambridge,UK)by DAB Detection Kit Streptavidin-Biotin)(ZSGB,Beijing,CHN)according to the manufacturer's protocols,followed by visualization under phase contrast microscopy.3.The comparation of the differences of EMSCs derived in different embryoic stagesThe E12.5d and E19.5d EMSCs were regularly cultured with 10 % fetal bovine serum(FBS)DEME/F12.The third to sixth passages of E12.5d and E19.5d EMSCs were employed in this study.A 10 % FBS ?-MEM(containing 50mg/m L ascorbic acid,10 mmol/L b-glycerol phosphate and 10-8M dexamethasone)was considered as the mineralization induction culture medium.Cells were replenished every 3 days.The RNA of two groups were collected after mineralized induction for 7 days and reverse transcription to c DNA,then detected the mineralization related genes and ALP staining.After mineralized induction for 14 days,the proteins of two groups were extracted and then detected the related protein trends by western blot.The alizarin red staining was exerted after 28 days mineralization induction,to detect the formation of mineralized nodules.4.The detection of expression of p75 NTR and related genes during the process of mineralized inductionThe E19.5d ectomesenchymal stem cells were mineralized induction for 3,7,14,21 days In vitro,each groups were extracted RNA,collected proteins and cell culture medium.To detect the expression of related genes,proteins,and binding strength of p75 NTR and Mage-D1 by Real Time-PCR,western blot and co-immunoprecipitation.The autocrine secretion of NGF was detected by ELISA(enzyme-linked immuno sorbent assay)in different induction stage.5.The detection of mineralized capacity of ectomesenchymal stem cells regulated by p75NTRThe p75 NTR overexpression vector was packaged into lentivirus vector to infect E19.5d ectomesenchymal stem cells,in order to upregulate the expression of p75 NTR in target cells;The p75 NTR siRNA was used to transfect into ectomesenchymal stem cells to down regulate the expression of p75 NTR in target cells,divided into 4 research groups : p75 siRNA(transfection of p75 NTR siRNA group),NCon(Negative control of siRNA group),pLJM1-p75(the p75 NTR overexpression vector infection by lentivirus vector group),pLJM1(empty vector group).At the mineralized induction day of 7,The RNA of 4 groups were collected after mineralized induction for 7 days and reverse transcription to c DNA,then detected the mineralization related genes and ALP staining.After mineralized induction for 14 days,the proteins of two groups were extracted and then detected the related protein trends by western blot.The alizarin red staining was exerted after 28 days mineralization induction,to detect the formation of mineralized nodules.The proliferation capacity of pLJM1-P75 and pLJM1 were detected by CCK-8 kit(Cell Counting Kit-8).6.The effect on mineralized capacity of ectomesenchymal stem cells by Mage-D1 and NGFThe ectomesenchymal stem cells were transfected by Mage-D1 siRNA,in order to downregulate the expression of Mage-D1 in target cells;NGF is considered to be the upstream ligand and activator of p75 NTR,was used to treat target cells,in order to upregulate the expression of p75 NTR in the specific signaling pathway,divided into 3 groups: NCon(negative control of Mage-D1 siRNA),Mage-D1 siRNA(transfection of Mage-D1 siRNA group),100ng/ml NGF(the treatment of 100ng/ml NGF group).The RNA of 4 groups were collected after mineralized induction for 7 days and reverse transcription to c DNA,then detected the mineralization related genes and ALP staining.After mineralized induction for 14 days,the proteins of 3 groups were extracted and then detected the related protein trends by western blot.7.The culture,identification and Micro CT scaning of p75 NTR knockout experimental animal models.P75NTR knockout(p75NTR-/-)mice,p75 NTR heterozygous mice(p75NTR+/-),and wild type mice are all at the 129/sv background(derived from Jackson laboratory,USA).The copulation of p75NTR-/-,p75NTR+/-and wild type mice were employed to produce littermates.The tails of these mices were dissected into 2mm at the tenth day after birth,marked and extracted DNA according to the manufacturer's protocols.The identification of genotypes were detected by QIAGEN Master Mix.At the 60 th day after birth,The same littermate mices of p75NTR-/-,p75NTR+/-and wild type group were choosen random sexuality,then injected 2% sodium pentobarbital at the concentration of 50mg/kg for anesthesia in mice,and taken of the neck to death.The femur bone was clearly isolated and placed into 4% paraformaldehyde in order to fix tissue for 24 hours.The femur bones of mices in 3 groups were scanned by Micro CT(viva ct40 Scanco Medical AG;Brüttisellen,Switzerland),then analyzed the three-dimensional image reconstruction and collected and organized related data by viva ct40 software.Results:1.The primary cells of E12.5d and E19.5d were derived from E12.5d rat embryonic facial process and E19.5d rat tooth germ respectively,and E19.5d EMSCs exhibites more regular spindly fibroblast-like.The clonies number of E12.5d EMSCs is more than E19.5d EMSCs,and the clone formation size of E12.5d EMSCs is bigger than E19.5d EMSCs.The proliferation capacity of E19.5d EMSCs is lower than E12.5d EMSCs assessed by CCK-8.The flow cytometry analysis was employed to identify these two kinds of EMSCs.Ex vivo expanded E12.5d EMSCs expressed the cell surface marker molecules of MSC CD14(90.4%),CD29(79.5%),CD44(92.8%),CD90(99.03%),CD105(29.3%),CD146(98.1%)and CD166(99.45%)compared with these of E19.5d EMSCs CD14(90.46%),CD29(98.93%),CD44(98.26%),CD90(99.64%),CD105(25.97%),CD146(97.39%)and CD166(97.22%).They were all negative expressing hematopoietic marker CD45(3.34% and 2.08%,respectively).Notably,the cranial neural crest originated marker p75 NTR was expressed in E12.5d and E19.5d EMSCs,and the expression level of E19.5d EMSCs(92.89%)was much higher than that(23.1%)of E12.5d EMSCs.2.During the initiation of the tooth development(E12.5d),p75 NTR immunoreactivity was absent in the thickened oral epithelium,however faint staining was detected in subjacent mesenchyme(condensed mesenchyme),but the staining was detected in cells of mesenchyme(me)in future dental epithelial-mesenchymal interaction area.In the bell stage,p75 NTR immunoreactivity was detected in inner dental epithelial(ide),dental follicle(df)and dental papilla(p),which areas were about to be the mineralization zone.The expression of Runx2 were overlapped in some mineralized zone in future.3.EMSCs possesses the mineralized differentiation potential.ALP staining depth of E19.5d EMSCs was darker than E12.5d EMSCs after mineralized induction 7days.There were more and bigger mineralized nodules in E19.5d EMSCs.Col1 expressed higher suggest E19.5d EMSCs produced more mineralized extracellular matrices during mineralized induction.We determined the p75 NTR expression level by Real-time PCR and western blot,p75 NTR in E19.5d EMSCs was significant higher than E12.5d EMSCs.To futher compare the mineralized difference,we detected the mineralization-related genes Runx2,ALP and Osterix,and homeobox gene family Dlx5 and Msx2 related to the mineralization and odontogenesis,E19.5d EMSCs was higher than E12.5d EMSCs.There was no difference of Mage-D1 in E12.5d and E19.5d EMSCs.Interestingly,Trk A was not expressed in both kinds of EMSCs.4.During mineralized differentiation,p75 NTR was increasingly expressed assessed by Real-time PCR and western blot.The mineralization-related genes Runx2,ALP,Osterix and Col1 demonstrated the same trend with p75 NTR,as well as the homeobox gene family Dlx5 and Msx2.Trk A was also not expressed during mineralized differentiation of E19.5d EMSCs.Although Mage-D1 had no change in the process,its binding with p75 NTR gradually enhanced which detected by co-immunoprecipitation.The autocrine secretion of NGF was gradually increasing during mineralized differentiation.In order to investigate the endogenous NGF,we used the ELISA assay to detect that from a minimum of 31.3±4.4 pg/ml in 3days mineralized induction to a maximum of 378.3±10.9 pg/ml after 21 days mineralized induction5.It was reported that p75 NTR inhibits the mineralized differentiation of MSC.The intense staining of p75 NTR protein was observed in pLJM1-P75 group,Mage-D1 staining had no difference among the four groups by regulating p75 NTR.However,in this study,we forced expressed p75 NTR by pLJM1 vector,then promotes the expression levels of mineralization related-genes and homeobox genes.On the contrary,when transfected with p75 NTR siRNA,the mineralized capacity of E19.5d EMSCs was downregulated.ALP staining was applied to detect the secretion of alkaline phosphatase,which represents the marker of mineralized differentiation in early stage.In p75 NTR overexpression group(pLJM1-P75),the staining depth was darker than that of other groups,and the p75 siRNA group stained fainter.The formation of the extracellular matrices and mineralized nodules was detected more in pLJM1-P75 group.In marked contrast,transfection with p75 NTR siRNA leaded to the significant decrease of mineralized capacity in EMSCs.In consistent with cellular morphology of E12.5d EMSCs and E19.5d EMSCs,when p75 NTR expressed higher,the more regular spindly the EMSCs like.However,the proliferation capacity of EMSCs was decreased by transfection with pLJM1-P75 overexpression vector.6.To investigate whether the Mage-D1 and exogenous NGF could have effect on mineralized capacity of EMSCs,we employed Mage-D1 siRNA and rat recombination NGF to figure this out.The ALP staining depth of Mage D1 siRNA group was fainter than other groups.By contrast,when treated with 100ng/ml NGF,the ALP staining depth was darker than other groups.Accordingly,the mineralization-related genes Runx2,Col1,and homeobox genes Dlx5,Msx2 showed the same trends assessed by Real-time PCR and western blot.Mage-D1 had no change by treating with NGF.In order to investigate the binding strength of p75 NTR with Mage-D1,co-immunoprecipitation was used for determining the difference caused by Mage-D1 siRNA and NGF.The binding strength was stronger in NGF treatment group,when downregulated Mage-D1 by siRNA,its expression level was decreased.The same passage of EMSCs and the same conditions were used for this study.1.The identification of p75 NTR knockout mice: p75NTR-/-mice was demonstrated a 280 bp band,wild type was demonstrated a 345 bp band,and p75NTR+/-mice was demonstrated 280 bp and 345 bp bands.The same littermate mices of p75NTR-/-,p75NTR+/-and wild type group were choosen random sexuality,The femur bones of mices in 3 groups were scanned by Micro CT,then analyzed the three-dimensional image reconstruction and collected and organized related data by viva ct40 software: Trabecular bone: These data TRI-BV(Bone Volume),TRI-BV/TV(Bone Volume/Trabecular Volume),TRI-Tb.N(Trabecular Number),TRI-Tb.Th(Trabecular Thickness)in p75NTR-/-group are lower than p75NTR+/-and wild type group,but the TRI-BS/BV(Bone Surface/Bone Volume),TRI-Tb.Sp(Trabecular separation)in p75NTR-/-group are higher than p75NTR+/-and wild type group.Cortical bone:TRI-C.TBTh and TRI-C.BV,p75NTR-/-group are lower than p75NTR+/-and wild type group,but TRI-C.BS/BV in p75NTR-/-group are higher than p75NTR+/-and wild type group.Conclusion:1.We isolated and cultured the high purity E12.5d maxillofacial process originated ectomesenchymal stem cells and E19.5d tooth germ originated ectomesenchymal stem cells in the same pregnant SD rat,identified that they are all originated from mesenchyme.The proliferation capacity of E12.5d EMSCs is higher than E19.5d EMSCs,but the mineralization capacity of E12.5d EMSCs is lower than E19.5d EMSCs.The immunohistochemical staining demonstrated that the expression areas of p75 NTR and Runx2 were overlapped at E12.5d(the tooth initial development stage)and E19.5d(the bell stage),suggested that p75 NTR is involved in the maxillofacial and tooth development earlier stage.2.The increasing expression of p75 NTR during the mineralized differential induction period of EMSCs suggested that p75 NTR mediated the mineralized differential process of EMSCs.3.The fluctuation of mineralized capacity of EMSCs in accordance with the overexpression and interference of p75 NTR suggested that p75 NTR positively r.egulated the mineralized capacity of EMSCs4.P75 NTR promotes expression of mineralization and odontogenesis related genes and differential mineralization of rat ectomesenchymal stem cells via binding with Mage-D1.5.The Micro CT scanning of p75 NTR knockout mice suggested that the knockout of p75 NTR might have influence on the bone remodeling,verified the effect of p75 NTR on the mineralization in vivo.In conclusion,our findings suggest that p75 NTR promoted the differential mineralization capacity of rat EMSCs during the process of mineralized induction in vitro.The mechanisms underlying this phenomenon following NGF treatment and mineralized induction involve regulated intramembrane proteolysis of p75 NTR,which binds with MHD of Mage-D1 via p75NTR-ICD.The nuclear translocation of the binding complex and enhanced expression of mineralization-related genes including Dlx5,Msx2,Runx2,Col1 and Osterix,eventually increased the mineralization capacity of EMSCs.These results lay the foundation for deeply comprehending the mechanism of differential mineralization during tooth and maxillofacial development,and provide the theoretical basis for the further development of tooth tissue engineering. |
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