The Research On The Mechanism Of PIP Affecting Osteogenic Differentiation Of Periodontal Ligament Stem Cells Through Akt Signal Pathway

Background and objectivesBecause there are some problems that are difficult to overcome in clinical practice of bone defects,such as donor site morbidity,limited availability,immune rejection and pathogen transfer,so the bone tissue engineering research focuses on finding the best alternative treatment to solve these problems.In recent years,scholars have strong interest in bone tissue engineering,and great progress has been made in this field.Over the last 20 years there has great improvement in bone tissue engineering research.Like other tissue and organ engineering,it needs the integration of many disciplines,such as cell biology,stem cells,molecular biology,biomechanics,biomaterials,immunology and transplantation.Bone tissue engineering is mainly composed of three parts:seed cells,cytokines and scaffold materials.Among them,the selection of seed cells is most important.Mesenchymal stem cells(MSCs)have been considered as an important research tool in tissue engineering and regenerative medicine.Oral tissue-derived stem cells are a type of MSCs.Recent studies have shown that oral tissue-derived stem cells have the characteristics of strong renewal ability and great differentiation potential.Oral tissue-derived stem cells also have the advantage of easy access and no ethical disputes.These characteristics make oral tissue-derived stem cells have broad prospective applications in tissue engineering and regenerative medicine.Among a variety of oral tissue-derived stem cells,considering the osteogenic differentiation,source accessibility,proliferation and anti-apoptosis,periodontal ligament stem cells(PDLSCs)are considered as a good potential substitute for MSCs in bone tissue engineering.In vitro culture,PDLSCs have strong osteogenic differentiation ability,and their osteogenic ability can be regulated by a variety of growth factors.It can be used as a potential cell source for alveolar bone regeneration.With the continuous development of the technical and theory of PDLSCs research.PDLSCs will bring revolutionary progress to medical theoretical research and clinical application.In order to improve the application of PDLSCs in bone regeneration research,it is very important to study the intrinsic molecular regulation mechanism of PDLSCs osteogenic differentiation more comprehensively.After years of research,the regulation of some classical factors related to osteogenic differentiation has been understood,but the regulating network of stem cell osteogenic differentiation is still to be clarified.Scholars gradually focus on more and more new mRNA.microRNA,LncRNA and protein,attempting to further improve the regulation of osteogenic differentiation of stem cells.Therefore,in the early stage of this study,the gene chip technology was used to obtain the differentially expressed genes before and after the osteogenic differentiation of PDLSCs,and the bioinformatics analysis was used for comprehensive analysis and comparison,in order to discover new genes related to the osteogenic differentiation of PDLSCs.After comparing all differentially expressed genes,The change of Prolactin Induced Protein(PIP)was most before and after PDLSCs osteogenic induction.Further qRT-PCR experiments with different cell samples and different culture times showed that PIP had a significant and relatively stable increase,so we speculated that it may have a regulatory role in the process of PDLSCs osteogenic differentiation.PIP is a 17 kDa glycoprotein.It is an aspartate-type protease.The substrate of PIP protease activity is fibronectin.Fibronectin is an adhesive glycoprotein,which plays an important role by inducing specific cell morphology and activating intracellular signaling pathways.Molecular studies have shown that fibronectin has a profound effect on osteogenesis and is necessary for normal osteogenic differentiation.We speculate that PIP degrades fibronectin through its protease activity,which has certain effect on osteogenesis.It has been reported that PIP is involved in the Erk and Akt signaling pathways,and affects cellular biological processes such as cell proliferation and invasion by regulating the phosphorylation of Erk and Akt.Erk and Akt signaling pathways are also associated with stem cell osteogenesis differentiation.Some studies have shown that activating the Erk and Akt signaling pathways can promote the osteogenic differentiation of PDLSCs.Could PIP also affects the osteogenic differentiation of PDLSCs through signaling pathways such as Erk and Akt?At present,studies on PIP rarely focus on the function of PIP in the process of osteogenic differentiation of stem cells,especially periodontal ligament stem cells.In view of the great significance of stem cells in bone tissue engineering,the search for key genes that regulates the osteogenic differentiation of stem cells is also the focus of future research.Based on previous gene chip results and the above facts,this study speculated that PIP might be related to the osteogenic differentiation of PDLSCs.We will further explore and verify this problem.Methods1.Bioinformatics analysis of differentially expressed genes in osteogenic differentiation of periodontal membrane stem cellsPrimary PDLSCs were obtained through enzyme digestion and tissue blocks method.PDLSCs were obtained by limited dilution method,and the immunophenotype was used to detect the stemness of PDLSCs.The multidirectional differentiation ability of PDLSCs was detected by osteogenic induction,adipogenic induction and chondrogenic induction.PDLSCs cultured in common medium and osteogenic induction medium for 7 days were collected for gene chip detection;qRT-PCR experiments were used to verify the results of chip analysis;GO analysis,KEGG analysis and other bioinformatics analysis methods were used to predict potential functions of the differentially expressed genes;Based on the above results,candidate genes that may have the function of regulating osteogenic differentiation were screened out.2.Exploring the regulation of PIP on the proliferation,apoptosis and osteogenic differentiation of PDLSCs in vitroPIP was knocked down in PDLSCs by lentivirus infection technology,and the knockdown efficiency was detected;The changes of PDLSCs proliferation activity after PIP knockdown were compared by CCK-8 experiment and cell cycle analysis.The apoptosis of PDLSCs after PIP knockdown was detected by flow cytometry.The osteogenic differentiation ability of PDLSCs was analyzed by ALP activity analysis,alizarin red staining,ALP staining,coll and ALP protein expression.3.The regulation of PIP on PDLSCs-based bone regeneration in vivoPDLSCs with PIP reduction were mixed with BIO-OSS and transplanted subcutaneously into the back of nude mice.After 6 weeks,HE staining and masson staining were used to analyze the morphological characteristics of the sample sections.The expressions of PIP,ALP and RUNX2 were analyzed by immunohistochemical staining..4.Exploring the molecular mechanism of knockdown PIP promoting osteogenic differentiation of PDLSCsTo explore the potential molecular mechanism of PIP regulating osteogenic differentiation of PDLSCs,the study will include two parts.Part Ⅰ:This part mainly studied the activation of PIP in the process of osteogenic differentiation of PDLSCs.Through the separate culture experiment of each component of osteogenic induction solution,the effect of each component on PIP in the process of osteogenic differentiation of PDLSCs was observed.After inhibiting glucocorticoid receptor,the changes of PIP expression were observed in order to understand the activation mode of PIP.Part Ⅱ:This part mainly studies the effect of PIP on the osteogenic differentiation signal pathway of PDLSCs.In order to explore the molecular mechanism of PIP regulating osteogenic differentiation,Western Blot was used to detect the expression of key factors of Erk pathway,Wnt pathway and Akt pathway after PIP gene knockdown.Then.PIP knockdown PDLSCs were treated with Akt signaling pathway inhibitor LY294002.The changes of osteogenic differentiation potential were detected by Western Blot,alizarin red staining,ALP staining,ALP activity detection,qRT-PCR and so on.Results1.Bioinformatics analysis of osteogenic differentiation regulatory genes of PDLSCs.The cell morphology,growth characteristics,surface markers and biological characteristics of the periodontal ligament stem cells obtained in the experiment were consistent with those reported in previous literature.They had good cell activity,strong subculture ability,proliferation ability and multidirectional differentiation ability.They can be our experimental cell model.PDLSCs were successfully isolated,cultured and identified.246 differentially expressed genes in PDLSCs before and after osteogenic differentiation were obtained by microarray analysis.The results of early microarray analysis were confirmed by qRT-PCR experiment.The genes with different expression before and after osteogenic differentiation of PDLSCs were analyzed by GO and KEGG analysis.Combined with the gene chip results and literature review results,PIP was determined as a candidate research object.2.Regulation of PIP on proliferation,apoptosis and differentiation of PDLSCs in vitroPIP was successfully knocked down in PDLSCs by lentivirus infection experiments.The results showed that knockdown of PIP had no significant effect on the proliferation and apoptosis of PDLSCs.Knockdown of PIP can promote the osteogenic differentiation of PDLSCs.3.The effect of PIP on PDLSCs-based bone regeneration in vivoThe subcutaneous transplantation model of nude mice was successfully constructed,and the sample tissues were made into paraffin sections for HE staining,Masson staining and immunohistochemical staining of PIP,RUNX2 and ALP.The results showed that there were new collagen fibers in the subcutaneous graft of nude mice,and the arrangement of new collagen fibers was more compact and orderly in the PIP knockout group.4.Study on the activation mode of PIP in osteogenic inductionCulture with dexamethasone alone can promote the expression of PIP.However,interference with glucocorticoid receptor can not block the induced effect of dexamethasone on PIP.The induction of PIP by dexamethasone may not be related to glucocorticoid receptor.5.Knockdown PIP promotes osteogenic differentiation of PDLSCs through Akt signaling pathwayThe activity of Akt signaling pathway increased after knockdown of PIP,suggesting that Akt signaling pathway may mediate the regulation of PIP on osteogenic differentiation.The cells were treated with LY294002,an inhibitor of Akt signaling pathway,and found that the ALP activity,alizarin red staining and the expression level of osteogenic related genes in shPIP+LY294002 group were lower than those in shPIP group.Our data indicated that after inhibiting the activity of Akt pathway,the enhancement of osteogenic differentiation caused by knocking down PIP was weakened,which confirmed that Akt signaling pathway mediated the regulation of PIP on osteogenic differentiation.Conclusions1.Knockdown of PIP does not affect the proliferation and apoptosis of PDLSCs,but can promote the osteogenic differentiation of PDLSCs in vitro.2.Knockdown of PIP can promote promote PDLSCs-based bone regeneration in vivo.3.Dexamethasone can activate the expression of PIP,which is not through the activation of glucocorticoid receptor.4.Knockdown of PIP may promote osteogenic differentiation of PDLSCs through Akt signaling pathway.