| BackgroundTissue regeneration requires the implantation of cellular adaptations to a wounded environment featuring a lesion.Because of their high proliferative,multi-differentiation potential and low immunogenicity,hDPCs are considered to represent a promising source of stem cells for regenerative medicine and engineering tissue.However,stem cells become aged during in-vitro expansion and the transplantation environment can often be extremely harsh for such cells,thus representing a significant challenge for regenerative medicine.An increasing number of studies pertaining to pretreatment strategies have attempted to improve the ex-vivo expanded microenvironment and thus facilitate implantation,homing,and survivability.Hypoxic preconditioning(HP)is a direct and effective strategy which to promote the in vivo survival and differentiation of transplanted stem cells;this is a vital factor for the success of stem cell-based tissue regeneration.In recent years,hypoxia has also been found to play an important role in a number of physiological processes.Different levels of hypoxia are associated with various physiological behaviors,and can regulate signal transduction in stem cell proliferation and differentiation during embryogenesis.One of the advantages of living in a hypoxic niche is that stem cells can maintain a slowly circulating proliferation rate while avoiding tissue-related oxidative stress.Essentially,all cells that undergo aerobic metabolism can produce ROS.This oxide can destroy DNA stability and induce oxidative stress in cells.The response of cells to ROS is highly dependent on other factors such as cell phenotype,cell differentiation status,or another stimulus state.For example,hematopoietic stem cells(HSCs)have lower levels of ROS than differentiated hematopoietic cells.There is a fine balance between ROS and hypoxia.Physiological oxygen tension varies from 1%to 14%,and is much lower when culturing in vitro.Although there is no definitive experimental data for the measurement of physiological oxygen concentration in human dental pulp,a previous study using a rat animal model found that the oxygen concentration of incisor pulp tissue was approximately 3%.The present literature relating to the effect of hypoxia on phenotypic changes in hDPCs show that different oxygen-dependent concentrations are used for the treatment of hDPCs,and that such changes are variable,especially in terms of cell differentiation and proliferation.However,experiments have shown that the angiogenic ability of dental pulp cells after HP is significantly higher than that under normoxic conditions.In addition,HP-treated cells exhibit increased levels of exosome secretion,which are effective in enhancing effector cell osteogenic and angiogenic capacity.Contradictory reports of hypoxic preconditioning may be due to various conditions such as the degree of hypoxia,different cell lines,culture time,and donor's age.To test the hypothesis that HP within the physiological range of oxygen concentration can maintain cell stermness by reducing oxidative stress in hDPCs,we investigated the effect of HP on the properties of oxidative stress and stemness.Our findings reveal that HP may effectively modulate oxidative stress in hDPCs via the PI3K/Akt pathway.Chapter I The isolation,culture and identification of human dental pulp cellsObjective:To isolate and culture hDPCs in vitro and identify the source of the cells.Methods:The primary cells of human dental pulp tissue were isolated and cultured by tissue method.The tissue source and stem cell characteristics of the cultured cells were identified by observing the morphology of the cultured cells,detecting cell surface markers and observing the ability of the cells to multi-directionally differentiate(osteogenesis,cartilage,and adipogenesis).Results:On the 7th day of culture,it was observed under the inverted microscope that hDPCs climbed out from the edge of the dental pulp tissue.A ring growth ring was formed after about 14 days.Individual hDPC exhibit typical fibroblast-like cell morphology,star or long fusiform,rich in cytoplasm,central nucleus elliptical,and clearly visible nucleoli.After the cells growing out of adjacent tissue blocks are converged,they are digested and passaged.The hDPCs after passage showed continuous growth characteristics through continuous culture.The surface markers of cells detected by flow cytometry showed that the expression rates ofmesenchymal stem cell markers CD 105 and CD44 were 84.31%and 87.08%,respectively,and the expression rates of hematopoietic stem cell markers CD34 and CD45 were 0.01%and 0.02%,respectively.After multi-directional induction culture,the cells differentiate into osteogenesis,cartilage and adipogenesis,and the related markers ALP,Col III and LPL were positive for immunofluorescence staining(red).After staining with alizarin red,Alcian blue and oil red,it was observed that there were mineralized nodules of different sizes in the osteogenic induction group,and blue glycosaminoglycans were formed in the extracellular cartilage group.In the adipogenic induction group,it can be seen that there is a string or a droplet formation in the cytoplasm of the cell.Conclusion:This experiment demonstrates that the primary hDPCs can be effectively cultured in vitro by tissue culture.By observing the results of cell morphology,surface markers and multi-directional differentiation,it was proved that the cultured cells were derived from mesenchymal tissue and had certain ability to proliferate and multi-directionally differentiate.Chapter II Effect of hypoxic preconditioning on proliferation and migration of human dental pulp cellsObjective:To establish a hypoxic preconditioning model of hDPCs cultured in vitro and to investigate the effects of hypoxic preconditioning on the proliferation and migration of hDPCs.Methods:After culturing hDPCs with 3%hypoxia,Western-Blotting verified the expression of the key protein HIF-la.The cell proliferation ability of hypoxia group,normoxia group and reoxygenation group was detected by CCK-8 method;The cell cycle distribution of the three groups was detected by flow cytometry;The difference of migration ability between the three groups was detected by Transwell chamber.Result:The expression of HIF-la protein in the hDPCs increased gradually after hypoxia stimulation,reached the peak of expression at 24h time point,and then gradually decreased.Compared with the normoxia group and the reoxygenation group,the cell growth rate after hypoxia treatment was lower,and the growth trend showed cluster growth.In the early stage of hypoxic treatment(?6d),the difference of proliferation in the groups was not obvious.When the culture time was extended to 10d,the proliferation ability of the hypoxic group was lower than that of the normoxia and reoxygenation group,there was no significant difference between the normoxia and reoxygenation group.The proportion of S phase cells in the hypoxic group was higher than normoxia group.In the hypoxia group,the number of passing cells which through the transwell membrane was lower than other group and showed a tendency to aggregate.Conclusion:3%hypoxic treatment of hDPCs is a relatively stable and reliable precondition model,which can provide a good experimental basis for subsequent experiments.Based on the experimental results,it was found that HP can reduce the cells proliferation and migration ability,the cells are keep in a"quiescent" state.Moreover,the cell proliferation and migration ability of the reoxygenation group which was restored to normal oxygen culture was not affected.Chapter III Effect of hypoxic preconditioning on the characteristics of human dental pulp stem cellsObjective:To investigate the effect of hypoxic preconditioning on the characteristics of hDPCs stem cells.Methods:The cell clonality and morphology were identified after incubation with different oxygen concentrations;The changes of stem cell surface markers CD 133,CD 105,CD34 and OCT4 were detected by flow cytometry;Cell pellet formation was observed by serum-free conditioned culture.Result:The number of cell clones formed after hypoxia treatment was significantly higher than other group,the clones was hypoxia>reoxygenation>normoxia.Similarly,the number of cells in the hypoxic group was also significantly higher than other groups,the cell volume was smaller,the ratio of spindle cells was higher,and the culture area was clear.The expression of CD 133 was increased in stem cells in the hypoxia group,and there was no significant change in the expression of CD34,CD105 and OCT4.On the frist day of serum-free culture,a small amount of cell spheres could be formed in each group,but the volume and number of spheres did not increase significantly with the prolongation of culture time.Conclusion:The results of this experiment demonstrate that HP can significantly improve the colony forming ability of hDPCs and the expression of stem cell surface marker CD 133.and maintain the sternness characteristics of hDPCs cultured in vitro.Chapter IV Effect of hypoxic preconditioning on anti-oxidative stress of human dental pulp cellsObjective:To investigate the effect of hypoxic preconditioning on the anti-oxidative stress of hDPCs.Methods:Flow cytometry was used to detect the ratio of ROS production and apoptosis ratio after cultured at different oxygen concentrations;Elisa detected the expression of inflammatory factors IL-6 and IL-1? in each group supernatant;The expression of antioxidant enzymes SOD,CAT and GSH-Px in the supernatant of each group was detected by WST-1 method.Result:The activity of ROS and proportion of apoptosis were lowest in the hypoxic group,followed by the reoxygenation group,and the highest in the normoxia group.There was no statistically significant difference in the expression of inflammatory cytokines IL-6 and IL-1? in each groups.There was no significant difference in the expression of antioxidant enzymes CAT and SOD in each group,but the expression of GSH-Px was significantly different.Among them,the hypoxia group GSH-Px was significantly higher than other group.Conclusion:The experimental results in this chapter show that 3%HP hDPCs can reduce intracellular ROS release and the ratio of apoptosis,increase the expression of antioxidant enzyme GSH-Px,and thus effectively reduce cell-induced oxidative stress.Chapter V Effect of hypoxic preconditioning on oxidative stress in human dental pulp cellsObjective:To explore the mechanism of hypoxic preconditioning to regulate oxidative stress in hDPCs.Methods:RNA-seq analysis of differential gene expression profiles in the experimental group after 48 h of different oxygen treatments,screening for enrichment pathways that may regulate oxidative stress;Western-Blotting verified the expression of p-PI3K.CCK-8 was used to detect the proliferation of cells in each group;Flow cytometry was used to detect the expression of ROS and the proportion of apoptosis;Elisa was used to detect the expression of inflammatory factors IL-6 and IL-l0 in the supernatant of each group;The expression of antioxidant enzymes SOD,CAT and GSH-Px in the supernatant of each group was detected by WST-1 method;Western-Blotting verified the expression of p-PI3K,p-Akt,HIF-la,Caspase 3 and FOXO1 in each group.Result:The results of the whole transcriptome RNA-seq analysis showed that 1,271 genes were up-regulated and 559 genes were down-regulated.After cluster analysis,differential gene enrichment is related to biological processes,cellular components,and molecular functions,including increased glycolysis in cell metabolism,increased transmembrane transporter activity,opened cell membrane ion channels and so on.Further analysis using the ToppGene tool revealed that genes degraded in hypoxia were enriched in pathways including protein synthesis and metabolism.The P13K/Akt signaling pathway is also activated by 32 genes and 12 genes downregulated during HP.WB results verified that the expression of phosphorylated Akt protein in cells after hypoxia treatment was indeed higher than that in the normoxic group and the control group.In the functional experiment,the cell proliferation activity of the hypoxia+LY294002 experimental group was significantly reduced.The expression of ROS in the two experimental groups was lower than that in the normoxic group under hypoxic conditions.The ROS expression in the hypoxic control group was the lowest,and there was no difference in the normoxic group.The percentage of apoptosis in the hypoxic control group was the lowest,and the percentage of apoptosis in the normoxia+LY294002 experimental group was the highest.There was no statistically significant difference in the expression of IL-6 and IL-1? in the supernatants of each groups.The expressions of antioxidant enzymes CAT and SOD were not different but the expression of GSH-Px in the supernatant of hypoxic control group was higher than that in hypoxia+LY294002 group.The expression of phosphorylated PI3K,Akt and HIF-la was increased in the hypoxia group compared with the normoxia group.The expressions of NAC group and control group were consistent,and the expressions of H2O2 and LY294002 were consistent.The levels of phosphorylated PI3K and Akt in NAC group and control group were higher than those in H2O2 and LY294002 group.Caspase3 and FOXO1 are downstream proteins of the PI3K/Akt pathway,which are identical to the expression of HIF-la,but opposite to the expression of p-PI3K and p-Akt.Conclusion:The experimental results in this chapter demonstrate that the molecular mechanism of cells changes under hypoxia,a series of pathways related to hypoxia are activated,Increased expression of key proteins in the PI3K/Akt pathway associated with oxidative stress regulation.The pathway inhibitor LY294002 can reduce the proliferation of hDPCs,increased the release of intracellular ROS and the proportion of apoptotic cells,and reduced the expression of antioxidant enzyme GSH-Px.Increased PI3K/Akt signaling in HP hDPCs leads to down-regulation of downstream proteins FOXO1 and Caspase 3 expression,affecting intracellular ROS production.Hypoxia Activated PI3K/Akt Inhibits Oxidative Stress via the Regulation of Reactive Oxygen Species in Human Dental Pulp Cells... |
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