Study On Expression Of P75 Neurotrophin Receptor During The Mineralization Of Ectomesenchymal Stem Cells In Vitro

BackbroundMissing teeth is a problem that almost everybody must face in the life,especially the society gradually entered the aging of the population today.This problem is especially common,and the harm to human?s health and quality of life is becoming more and more prominent.Now the main means to solve this problem is clinically the denture,but whether in function or feeling,it can not be compared with a natural tooth.So we need a kind of natural tooth that is the most similar or identical function restoration.Tissue engineered tooth is undoubtedly ideal choice,but due to the precise mechanism of the tooth development is not yet clear,which restricts the development of tissue engineered tooth.And the cell reunion technology that simulates the tooth development between embryonic cells and mesenchymal epithelial function theory provides a new idea for the development of tissue engineered tooth.Asthe primogenitor of dental stem cells--the cranial neural crest EMSCs is the ideal seed cells to obtain regeneration teeth by reunited cell technology.But now the differentiation fate in vitro of cranial neural crest EMSC and the influencing factors is not clear.Therefore,the aim of this research is to understand its differentiation fate in vitro by discussing mineralization ability of mesenchymal stem cells from different embryonic age,which help to promote the tooth tissue engineering process.p75 neurotrophin receptor(p75NTR)is the low affinity receptors neurotrophic factor,mainly expresses in the early development nerve cells.It is considered as a cranial neural crest classic marker.It involve in cell survival,migration,proliferation,differentiation and apoptosis,etc..At present,the regulation function of p75 NTR in development and regeneration of nervous system has been fully descripted,but its role in the other organizations(such as fat,liver,muscle,teeth,etc.)is still unclear,especially in the development of tooth.Our previous studies have confirmed that the cranial neural crest cells in rat E11.5 d moved to facial processes,tooth development has not yet started at E12.5 d.Based on the previous research,we will explore the role of p75 NTRin the process of mineralization of ectomesenchymal stem cells by comparing the expression of p75 NTR in ectomesenchymal stem cells of different embryonic age of rats in vitro.This will provide the experimental basis for revealing molecular biology mechanism of tooth development,and searching for a good seed cells for tissue engineered tooth.Content1.Haematoxylin-eosin(HE)staining to observe the morphology of tooth germ of different embryonic age of rat2 percent sodium pentobarbital(40 mg/kg)were injected into the abdominal cavity of the embryo 12.5 d?13.5 d?15.5 d and 18.5 d sprague dawley(SD)rats.After waiting for about 5-10 min,the embryo was taken out by abdominal surgery,and then fixed in the 4% paraformaldehyde for 24 h?embedding?slicing the tissue in 8 um thickness by sagittal direction,HE staining,in the last observe under the microscope and taking pictures.2.Culture and identify the ectomesenchymal stem cells of E12.5 d ?E13.5 d?E15.5d and E18.5 d SD rats in the vitro2 percent sodium pentobarbital(40 mg/kg)were injected into the abdominal cavity of the embryo 12.5 d?13.5 d?15.5 d and 18.5 d sprague dawley(SD)rats.After waiting for about 5-10 min,the embryo was taken out by abdominal surgery,and cut the facial processes of the embryo into 0.1 mm * 0.1 mm * 0.1 mm of tissue block.After pancreatic enzyme digestion,centrifugal and filtering by the 100 mesh stainless steel filter of the tissue,the tissue were respectively treated with culture medium of fetal bovine serum,then put into 37 ?,5% CO2 incubation cultivation box.The biology identification of E12.5d ? E13.5d ? E15.5d ? E18.5d EMSCs were detected respectively by the cell morphology,cell proliferation,cell immunofluorescence and flow cytometry technique to detect cell surface antigen of3.Mineralized induced E12.5 d?E13.5d?E15.5 d and 18.5 d EMSCs in vitroAfter mineralized induction for 0 d,7 d and 14 d and 21 d of E12.5 d?E13.5 d ?E15.5 d and E18.5d EMSCs,respectively to observe the cell morphology,ability of ALP activity and the expression of mineral-associated gene and the p75 NTR.Results1.The tooth germ of E12.5 d rat was in a period of tooth board formation which is an epithelial structure formed by oral epithelium growed into the organization;E13.5d growed into the grey shape of tooth germ,that the tip of dental plate enlarged,and hyperplasia epithelial cells form ovoid or round epithelium buds,shaped like a bud.The tooth germ epithelial bud of E15.5 d rat continue to grow?increase into epithelial basel parts and inward concave,which shaped like hat,and cover the accumulation of embryo mesenchymal cells,which called a bonnet.The enamel orange is divided into three layers: the outer enamal epithelium,stellate reticulum and inner enamal epithelium.The cell clusters of the bottom of the enamal orange was called dental papilla,which will form dentin and dental pulp;and the embryo mesenchymal cells circumvolutioed by the enamel organ and dental papil was called gums,which form teeth support groups.The tooth germ of E18.5 d rat was in period of bell stage.The epithelium sag to deepen,and peripheral parts continue to grow.At this time enamal organ enter into the mature period,divided into four layer :outer enamal epithelium,stellate reticulum,stratum intermedium and inner enamal epithelium.The connection of inner enamal epithelium and outer enamel epithelium was called cervial loop.2.(1)E12.5 d ?E13.5 d? E15.5d and E18.5 d EMSCs were isolated and cultured in vitro,and they were all smiliar to fiber cell and they have plump cell body and stronger cell proliferation ability.(2)flow cytometry detection: The expression rate of CD14,CD29,CD90,CD146,CD166,p75 NTR of E12.5 dEMSCs were respectively 92.93%,98.39%,95.49%,92.86%,91.44%,22.53%.The expression rate of CD14,CD29,CD90,CD146,CD166,p75 NTR of E13.5 dEMSCs were respectively 97.01%,96.64%,97.70%,98.26%,97.15%,82.42%.The expression rate of CD14,CD29,CD90,CD146,CD166,p75 NTR of E15.5 dEMSCs were respectively 95.65%,98.50%,98.49%,97.90%,99.51%,85.09%.The expression rate of CD14,CD29,CD90,CD146,CD166,p75 NTR of E18.5 d EMSCs were respectively 95.79%,96.97%,91.28%,92.86%,97.92%,81.43%.The expression of CD45 of E12.5 d,E13.5 d,E15.5d and E18.5 d EMSCs were respectively 5.78%,5.24%,8.34%,8.51%.E12.5?E13.5 d?E15.5 d and E18.5 d EMSCs all positively expressed of CD14,CD29,CD90,CD146,CD166,p75 NTR,and expression od CD45 was negative.3.(1)Alkaline phosphatase staining :after mineralized induced for 7 d,the experimental group appeared deeper colour than the control group,and the colour of E18.5 dEMSCs experimental group were the deepest.(2)results of alizarin red staining: after mineralized induced for 21 days,each group had the mineralized nodule formation,the results are consistent with the ALP staining results,namely E18.5d EMSCs experimental group had the biggest mineralized nodule formation.(3)the results of RT-PCR:after mineralized induced for 7 days,the ALP expression level of experimental group was significantly higher than the control group,including E18.5 d EMSCs experimental group was highest(P ?0.05);after mineralized induced for 14 d,expression of Runx2,ColI and p75 NTR m RNA of the experimental group was higher than the control group,including E18.5 d EMSCs experimental group was highest(P?0.05);the expression of p75 NTR of control group did not have obvious changes(.4)Western blot : after mineralized induced for 14 d,expression of mineralization related protein Runx2,Col Iof E18.5 dEMSCs experimental group is higher,detecting of protein grey value was signally different(P?0.05);there was not obvious changes of p75 NTR in the crotol group,but the expression of the experimental group is significantly higher than the control group after 21 d,the difference of protein grey value to detect was statistically significant,(P?0.05).ConclusionThrough biological identification,mineralized induced in vitro,and detection of the mineralization related gene of ectomesenchymal stem cells of different embryonic age,we found that E12.5 d?E13.5 d? E15.5 d and E18.5d EMSC were all mesenchymal stem cells of neural crest origin,particularly proliferation ability and the mineralization ability of E18.5 d EMSCs were stronger,that suggested that E18.5 d EMSCs would be ideal seed cell;Expression of p75 NTR in E13.5 d had a significant rise,and maintain a high expression,as well as increased significantly after the mineralized induced expression,which speculated that p75 NTR may participate in the development of early teeth.