| BackgroundWith the aging of the population,the incidence of osteoporosis has increased year by year.Aging results in decreased bone marrow mesenchymal stem cells differentiation to osteogenic and is closely related to the onset of osteoporosis.At present,it is generally believed that the pathophysiology of osteoporosis lies in the imbalance of osteoblast bone formation and osteoclast bone resorption.Therefore,the treatment of osteoporosis is mainly based on drugs.The commonly used bone resorption inhibitors in clinical practice include bisphosphonates,selective estrogen receptor modulators,calcitonin,and estrogen.Although various types of anti-osteoporosis drugs have achieved certain effects in clinical treatment,there are still no clear targets for the treatment of osteoporosis,and there is no definite mechanism and precise treatment.Osteoporosis in patients with senile estrogen deficiency can be treated with oral DHEA.Clinically,DHEA has been shown to improve osteoporotic symptoms and increase bone mineral density.DHEA is mainly secreted by the adrenal glands,but also secreted by the gonads and brain.It can act in the peripheral tissues by transforming androgens or estrogens.However,so far,no basic research has been reported on DHEA's protein molecules that osteogenic differentiation being promote to mesenchymal stem cells.Various types of drugs containing DHEA anti-osteoporosis still have no clear target in clinical treatment.There is no clear mechanism and precise treatment.In this project,± DHEA of hMSCs osteogenic induction system was adopted to collect the proteins of the drug treatment group and the control group at different time points.Firstly,it was confirmed that DHEA could determine osteogenic differentiation of BMSCs and analyze the expression of related genes.Quantitative proteomics technology creates differential protein expression profiles in drug-treated and control groups.Through the analysis of relevant protein components and possible signal pathways involved in differential protein molecules,selecting potential molecular markers as targets,RT-PCR and Western-Blotting were used to verify the important differential molecules screened,and analysis of the molecules in DHEA promotion was conducted.The role of mesenchymal stem cells in osteogenic differentiation provides a theoretical basis and experimental basis for further exploration of the potential application value of this protein in improving osteogenic differentiation of stem cells.ObjectiveThere is a close relationship between DHEA and osteogenic differentiation of bone marrow mesenchymal stem cells.Through the study of DHEA to promote the differentiation of bone marrow mesenchymal stem cells into osteoblasts,contrast the DHEA,osteogenic inducing system and the presence of DHEA osteogenic inducing system for the influence of stem cell osteogenic differentiation ability,and in-depth study of the process of changes in the type and quantity of proteins,whether there is a characteristic difference protein in the entire MSCs to osteoblast differentiation process gradually increased or decreased,can be used as MSCs to promote osteogenic differentiation drug therapy targets,and the mechanism of action of characteristic differential protein molecules and osteogenic differentiation of MSCs,clinical treatment of osteoporosis for DHEA.Methods(1)Extraction and drug treatment of human bone marrow mesenchymal stem cells: Healthy adult human bone marrow mesenchymal stem cells were extracted and cultured to the third generation,to induce osteogenic differentiation of BMSCs by DHEA± osteogenic inducing system.The expressions of ALP activity and related osteogenic genes(ALP\BGLAP\BMP2\RUNX2\COL1A1\SPP1)in osteoblasts were measured at day 4?7 ?14 and alkaline phosphatase and alizarin red staining were performed to observe the degree of osteogenic differentiation.(2)Mass spectrometry analysis Determine the types and amounts of relevant differential proteins: Extract total protein from each group,determine protein concentration,reductive alkylation,then proteolysis,label all peptides with TMT reagents,and then conduct tandem mass spectrometry analysis.Screening for differential proteins that promote stem cell-derived osteoblasts osteoblasts by DHEA.Verification of differential protein expression: RT-PCR technique was used to validate that DHEA promoted the expression of differential protein CHAD at the mRNA level in bone marrow mesenchymal stem cells to osteoblast differentiation at different time points.Western-Blotting technique was used to verify the expression of differential protein at the protein level in DHEA-promoted bone marrow mesenchymal stem cells to osteoblast differentiation at different time points.Results(1)To investigate the ability of bone marrow mesenchymal stem cells to differentiate by osteogenic differentiation: In each subgroup,DHEA+ osteogenic inducing system was most effective Increases the rate of differentiation of stem cells to osteogenic differentiation,showing the alkaline phosphatase activity of osteoblasts.The best effect was detected,followed by the osteogenic induction group,and the performance of the simple drug group was the lowest.Alizarin red staining and alkaline phosphatase staining showed that the DHEA+ osteogenic inducing system group had the best induction effect in each subgroup;among the related osteogenic genes detected by RT-PCR,compared with the blank control group,all the other groups had the osteogenic gene.Up-regulation,which DHEA + induction group was more obvious upside,compared to the effect of the simple induction group and drug group was the second.(2)Mass spectrometry analysis: In the 2 replicate experiments,the total number of MS spectra of the 1st mass spectrometry was 290495,the number of matches of the identified peptides was 123950,and the total number of identified peptides was 59073.The total number of unique peptides was 45,808,and the total number of identified proteins was 6,649.The total number of secondary mass spectrometry spectra of the second mass spectrum was 294,253.The number of matched peptides was 127,343 and the total number of identified peptides was 59,125.The total number of peptides was 45,975 and the total number of identified proteins was 6,699.Proteins that met the expression difference multiples greater than 1.2 fold(up and down)and P value Significance A)less than the 0.05 screening criteria were considered differentially expressed proteins.4 days of drug group compared with blank control group up-regulated 341 differentially expressed proteins,down-regulated 225 differentially expressed proteins,all differentially expressed proteins 566;4 days induced group up-regulated 321 differentially expressed proteins compared with blank control group,down-regulated differentially expressed protein 266 A total of 587 differentially expressed proteins were identified;281 differentially expressed proteins were up-regulated in the 4-day dehydroepiandrosterone and induction system groups relative to the control,and 178 differentially expressed proteins were decline,total 459 differentially proteins expressed;differences in up-regulation of the 7-day drug group compared to the group of control.362 proteins were expressed,244 proteins were differentially expressed,and 606 proteins were differentially expressed.In the 7-day induction group,405 differentially expressed proteins were rise.The number of 319 proteins were lower regulated,and observed that 724 differential expressed proteins;In the drug + induction group,302 differentially expressed proteins were up-regulated,222 differentially expressed proteins were down,discovery 524 proteins were differentially expressed;in the 14-day drug group,407 differentially expressed proteins were up-regulated compared to the blank control group,and 305 differentially expressed proteins were down-regulated,all differentially expressed proteins 712;14 days Compared with the blank control group,the guide group up-regulated 369 differentially expressed proteins,down-regulated 297 differentially expressed proteins,and all differentially expressed proteins 666;in the 14-day drug + induced group,317 differentially expressed proteins were up-regulated compared to the blank control group,and 230 differentially expressed proteins were down-regulated All of the 547 distinctive proteins.GO annotation of the target protein assembly can classify these proteins from three aspects: the involved biological processes,the molecular functions they have,and the cell components they are in.Significant enrichment analysis of the GO annotation was performed by the Fisher's Exact Test to evaluate the significance level of a certain GO term protein enrichment,using the KEGG pathway as a unit,all qualitative proteins as the background,and the Fisher exact test.(Fisher's Exact Test)to analyze the significance level of the protein richness of each pathway to determine the metabolic and signal transduction pathways that are significantly affected,and ultimately screen out the differential eggs of interest.The differential proteins that were continuously up-regulated in the subgroups were screened,and a database rich in leucine-rich chondroitin(CHD)was selected as a characteristic protein for further research.The CHAD in each group was verified by RT-PCR and Western-Blotting.The expression of CHAD in the DHEA+ induced group was the highest at the same time,and the highest expression was found on the 14 th day.ConclusionThe DHEA osteogenic induction system promoted osteogenic differentiation of bone marrow mesenchymal stem cells more significantly than other groups,and up-regulated osteogenic related genes.Statistical analysis was performed on the proteins that were continuously up-regulated in each group to screen possible DHEA-induced bone marrow mesenchymal disorders.The related protein molecules of stem cell osteogenic differentiation are rich in leucine chondroitin(Chondroadherin CHAD)and verified.The mechanism may be the activation of the CHAD/JAK/STAT signaling pathway,and activation of the expression of the downstream key gene IRF9,ultimately enabling the expression of DHEA to promote msc to the osteogenic and thereby participating in the process of inducing osteogenesis. |
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