The Effect Of Knocking Out P75NTR On Mineralization Of Mice Ectomesenchymal Stem Cells

Research background:Missing tooth is a common problem in human life.Tooth loss can not only influence one's physiological health,but also influence psychological health.The traditional repair method is dental restoration,which are not comparable to natural teeth in terms of function and sensation.Therefore,tissue engineered teeth are considered to be the best way to solve the problem of missing teeth.Ectomesenchymal stem cells(EMSCs)originated from cranial neural crest are considered the primogenitor cells of all the dental tissues except tooth enamel.In our previous studies,we found that EMSCs at the bell stage of tooth germ had better ability of mineral ization in vitro.p75 neurotrophin receptor(p75NTR)is the low affinity neurotrophin receptor.It is not only the classical surface marker of neural crest derived mesenchymal stem cells,but also involved in the regulation of many biological processes,su ch as cell proliferation,migration,differentiation,survival and apoptosis.The role of p75 NTR in the development of nervous system has been fully explored,but their participation in other systems(such as the liver,muscle,fat,teeth etc.)occur in the course of the development of research is still unclear,especially in the tooth development.In this study,we used p75 NTR knockout mice from the US Jackson Laboratoryt to compare the mineralization difference between E18.5d p75NTR(-/-)EMSCs and p75NTR(+/+)EMSCs,which are at bell stage of tooth germ.We will explore influence of knocking out p75 NTR on mineralization of mice ectomesenchymal stem cells..This will provide basis for realizing the role of p75 NTR in tooth origin and development mechanism.Material and methods:1.The acquisition,culture,gene identification and biological characteristics of E18.5d EMSCsThe p75NTR(-/+)female mice and p75NTR(-/+)male mice were paired breeding conventionally.The mice were seen as 0.5d pregnant from the midday of the female suppository,at the E18.5d,they were injured with 2% pentobarbital sodium(50mg/kg)for anesthesia,then exert the abdominal surgery 5-10 minutes later.Extracted the embryos and cut the facial processes into 0.1 mm * 0.1 mm * 0.1 mm of tissue block under sterile conditions.Then digest the tissue with 1% type I collagenase,neutralized and centrifuged.The tissue were resuspended in six well plates with 2ml DMEM-F12 medium which containing 10% fetal bovine serum and 1%penicillin,streptomycin.Later they were cultured in incubation box containing 5%CO2 at 37 degrees.Cut the remaining limb tissues for genotyping.Identification of fetal rat tail and limb tissues by genotyping.Two types of EMSCs were detected and identified by cell morphology,flow cytometry and cell cycle.2.The comparation of the mineralization ability between E18.5d p75NTR(-/-)EMSCs and p75NTR(+/+)EMSCsThe p75NTR(-/-)and p75NTR(+/+)EMSCs were cultured with mineralization induction culture medium in vitro.The culture medium containing 10 % FBS ?-MEM,50mg/m L ascorbic acid,10 mmol/L ?-glycerol phosphate and 10-8M dexamethasone were changed every 3 days.ALP staining was exerted at 7th day and alizarin red staini ng was acted at the 21 th day.And cells were collected after mineralized induction for 7 days,14 days,and 21 days to detect changes of mineralization related genes ALP and Col I.3.The detection of bone mineralization in p75NTR(-/-)and p75NTR(+/+)miceSelect p75NTR(-/-)and p75NTR(+/+)male mice which were conventional breeded to 4 months ? Injected 2% 50mg/kg pentobarbital sodium intraperitoneally and then killed mice off.After peeling off the left femur,fixing them in 4% paraformaldehyde,and then scanning by Micro-CT,then analyse three-dimensional image reconstruction and related data ?Select p75NTR(-/-)and p75NTR(+/+)male mice which were conventional breeded to 40 days?Injected calcein at concentration of 25mg/kg every 5 days for 4 times.And killed mice off 2 days after the last injection.Stripping the left femur of mice for hard tissue slices,and then calculate the distance between the observed fluorescence band,so we can calculate the mineral apposition rate(MAR:um/d)respectively.Extracted total RNA and protein respectively of 4 week old p75NTR(-/-)and p75NTR(+ / +)mice,then expression level of mineralization related genes ALP and Runx2 were detected through RT-PCR and Western blot.Results1.(1)Using remaining limb tissue to identify the genotype of the EMSCs,a single band at 280 bp was p75NTR(-/-),a single band at 345 bp was p75NTR(+/+),280 bp and 345 bp both detected strip was p75NTR(-/+).(2)Successfully got E18.5d p75NTR(-/-)and p75NTR(+/+)EMSCs respectively from the corresponding p75NTR(-/-)and p75NTR(+/+)embryos,two genotypes of EMSCs both were long spindle shaped fibroblasts morphology in cell morphology.(3)Flow cytometry which detected cell surface antigen detection showed: the markers of MSCs in p75NTR(-/-)EMSCs CD14(99.07%),CD146(95.79%),CD166(95.79%)compared with those of p75NTR(+/+)EMSCs CD14(97.57%),CD146(96.32%),CD166(97.90%).two types of cells was both negative expressed hematopoietic marker CD45(p75NTR(-/-):0.28% and p75NTR(+/+): 0.50%).(4)Cell cycle test results show that in p75NTR(-/-)EMSCs G1(77.65%),G2(17.01%),S(7.56%);p75NTR(+/+)EMSCs G1(79.25%),G2(15.27%),S(8.90%),there was no significant difference between them.2.(1)Alkaline phosphatase staining results: mineralization induction of 7d showed the staining of p75NTR(+/+)EMSCs group were deeper than that of p75NTR(-/-)EMSCs group.(2)Alizarin red staining results: mineralization induction after 21 days,there were mineralized nodules in both groups,and the staining of p75NTR(+/+)EMSCs group were deeper than that of p75NTR(-/-)EMSCs group,besides there were more amount of mineralized nodules in p75NTR(+/+)EMSCs group.(3)RT-PCR results: After mineralization inductionn of 7 days,14 days and 21 days,the total RNA was extracted for reverse transcription and afterwards PCR reaction.The results showed that after mineralization induction of 7 days,14 days and 21 days,ALP expression level in p75NTR(+/+)EMSCs group was significantly higher than that in p75NTR(-/-)EMSCs group(P<0.05);Col I test showed the same results(P<0.05)3.(1)Left femur bones of 4 month old p75NTR(-/-)and p75NTR(+/+)mice were scaned by Micro-CT,then after scanning three dimensional reconstruction and data results were analyzed,including :bone value(BV),bone volume fraction(BV/TV),Trabecular Number(Tb.N),Trabecular Thickness(Tb.Th).All these data showed lower level in p75NTR(-/-)mice,but the bone surface / bone volume(BS/BV)and trabecular separation(Tb.Sp),were higher in p75NTR(-/-)mice than those of p75NTR(+/+)mice.The femur cortical bone analysis results showed cortical bone thickness(Ct.TBTh)and Ct.BV(cortical bone mass)were lower in p75NTR(-/-)mice than those in p75NTR(+/+)mice,but bone surface/ bone volume(Ct.BS/BV)was lower in p75NTR(-/-)mice than that in p75NTR(+/+)mice.(2)The detection results by fluorescence labeled calcein showed that the MAR in p75NTR(-/-)was significantly lower than that in p75NTR(+/+)mice(P<0.01);(3)PCR and Western blot results showed that both ALP and Runx2 gene expression level in p75NTR(-/-)in mice were significantly higher than those in p75NTR(+ / +)mice(p<0.01).Conclusion1.Through the cultivation and mineralization assay of two genot ypes of EMSCs,we found that p75 NTR involved in the regulation of mice EMSCs mineralization,and knocking out p75 NTR has negative effect on EMSCs mineralization differentiation.There was no significant difference in the cell cycle between the two genotype s EMSCs,which indicated that the negative regulation of EMSCs in mineralization after knocking out p75 NTR was not related to cell proliferation and apoptosis.2.Micro-CT analysis,calcein labeling experiments,RT-PCR and Western blot detected that p75NTR(-/-)mice femur mineralization formation ability was significantly lower than that of p75NTR(+/+)mice,while p75 NTR knockout mice did not express p75 NTR,which binds to NGF,so we initially hypothesis that p75 NTR regulate mice bone mineralization ability by combining to NGF.