Effects Of Notch Signaling Pathway On Human Dental Pulp Stem Cells’ Proliferation And Differentiation In The Hypoxia Environment

Human dental pulp stem cells(HDPSCs) are isolated from human dental pulp tissues and have highly proliferative ability and powerful multi-directional potential. HDPSCs have been regarded as important seed cells for tissue engineering because of its characters such as convenient and low immunogenicity.Hypoxia has effects on the proliferation, differentiation,apoptosis and damage repair of adult stem cells. Dental pulp is often exposed to ischemia and hypoxia, for the reason, that its anatomical location is susceptible to stimuli or injury. Researches on the effects of hypoxia in DPSCs have proved the role of hypoxia in the regulation of dental pulp stem cell‘s proliferation and differentiation.The Notch signaling pathway is an evolutionarily conserved signal transductional mechanism. Notch signaling pathways play an important role in cell proliferation, differentiation, apoptosis, etc. Our team have found that low oxygen environment can cause the activity increase of Notch signaling pathway in glioma stem cells(GSCs), and thus enhanced the proliferation of GSCs and self-renewal ability, hints that the effect of hypoxia on the GSCs probably through activation of Notch signaling pathway to achieve. For HDPSCs, is there the same role of the Notch pathway under hypoxia environment? By MTT cell proliferation experiment, the clone formation, alizarin red staining, ALP activity detection, oil red O staining, determination of related gene expression level and flow cytometry methods, we observed the effects of nomoxic and hypoxic environment on HDPSCs’ ability of proliferation and differentiation.Here we emphasized the effects of hypoxia on the proliferation and differentiation of HDPSCs, and the role of Notch signal pathway that involved in these processes, which may provide experimental evidence for further research of tooth regeneration research.Methods: 1. The isolation, culture and identification of HDPSCs: Enzyme digestion and limited dilution were used to isolate and purify the HDPSCs. The cells were then identified for cell surface marker by immumofluorescence and flow cytometry(FCM) methods. Induction of cell multi-directional differentiation was also applied.2. The effects of Notch signaling pathway on HDPSCs ability of proliferation and differentiation: GSI were joined in culture system to block Notch signal pathway, MTT, colony formation, mineral and adipogenic differentiation, alizarin red staining, ALP activity detection, oil red O staining, reverse transcription polymerase chain reaction(RT-PCR) and FCM were used to evaluate and compare the effects of Notch signal pathway on HDPSCs.3. The influence of simulated hypoxia environment on HDPSCs: Co Cl2 was joined in culture system to simulate hypoxic environment. By MTT cell proliferation experiment,,mineral and adipogenic differentiation,alizarin red staining, ALP activity detection, oil red O staining, determination of related gene expression level and flow cytometry methods, we observed the effects of nomoxic environment and hypoxic environment on HDPSCs’ ability of proliferation and differentiation.4. The influence of simulated hypoxia environment and Notch signaling pathway on HDPSCs: By MTT cell proliferation experiment, mineral and adipogenic differentiation, alizarin red staining, ALP activity detection, oil red O staining, determination of related gene expression level and flow cytometry methods, we observed the effects of Notch signal pathway on HDPSCs’ ability of proliferation and differentiation under hypoxic environment and the role of Notch signal pathway that involved in these processes.Results: 1. HDPSCs were isolated and cultured in vitro successful, fiber cell clones were visible under microscope. Immunofluorescence experimental results show that nestin and GFAP positive expressed; flow cytometry experimental results show that stem cell markers such as Stro-1, CD90, CD105 and vascular endothelial cell marker CD146 were highly expressed in the HDPSCs, and hematopoietic system marker CD45 and endothelial marker CD31 were low expressed.After osteogenic induction the HDPSCs stimulation resulted in mineralized differentiation as shown by alizarin red staining, and alkaline phosphatase( ALP) activity also increased with the increase of induced time. Oil Red O staining of the HDPSCs after 2 weeks of culture demonstrated numerous intracellular lipid droplets. The above results show that the HDPSCs obtained here has a good ability of proliferation and multi-directional differentiation potential.2. The effects of Notch signaling pathway on HDPSCs ability of proliferation and differentiation:(1)Notch signal pathway was blocked in HDPSCs after GSI treated:The expression level of Notch downstream target gene Hes1 in HDPSCs was significantly lower after GSI joined in culture system to block Notch signal pathway, which state that Notch signal pathway was blocked in HDPSCs after GSI treated.(2)The effects of Notch signaling pathway on HDPSCs:The Real-time quantitative PCR detection results showed that HDPSCs after Notch signal pathway blocked by GSI,the expression level of related gene Oct4 and C-Myc significantly lower than the control group(P< 0.05),hinted Notch signaling pathway blocked inhibits HDPSCs proliferation ability. MTT and colony formation assay showed that the expansion ability of HDPSCs decreased after Notch signal pathway blocked by GSI. Alizarin red staining results showed that after osteogenic induction the mineralized nodule of GSI treated HDPSCs significantly more than the control group(P< 0.05). ALP activity and the expression level of related genes including RUNX2, OCN and DSPP of GSI treated HDPSCs significantly higher than the control group(P< 0.05). Oil Red O staining of the adipogenic induced HDPSCs after GSI treated reacted positively, and the expression level of related gene PPARγ significantly higher than the control group(P< 0.05). The results mineralized and concomitant induction showed that HDPSCs after GSI joined characterized by enhancer differentiation ability.3. The influence of simulated hypoxia environment on HDPSCs: The expression level of hypoxia inducible factor-1α(HIF-1α) increased as Co Cl2 concentration increases, indicated that can be effectively simulated hypoxia environment with dose effect. The Real-time quantitative PCR detection results also showed that HDPSCs in hypoxia expressed higher Oct4 and C-Myc than which in normoxia(P< 0.05);The results of MTT showed that hypoxia can promote cell proliferation of HDPSCs. Flow cytometry test results showed that hypoxia can arrest G1 phase, but has no influence on the apoptosis of HDPSCs. The results mineralized and concomitant induction showed that HDPSCs in hypoxia conditions characterized by weaker differentiation ability. The above results suggest hypoxia can promote dental pulp stem cell proliferation and inhibit the differentiation of HDPSCs.4. The influence of simulated hypoxia environment when Notch signaling pathway blocked on HDPSCs: The expression level of Notch downstream target gene Hes1 increased as Co Cl2 concentration increases,and the Notch signaling pathway may be activated by Co Cl2.The results of MTT showed that when Notch signal pathway blocked under hypoxia, the proliferation promotion ability of hypoxia on HDPSCs can be reduced. Flow cytometry test results showed that the trend of hypoxia arrest G1 phase of HDPSCs can be restrained after Notch signal pathway were blocked. Notch signal pathway may not influence the apoptosis of HDPSCs, displayed by the results of annexin V/PI double staining detection. Real-time quantitative PCR detection results also showed that HDPSCs in hypoxia expressed higher Oct4 and C-Myc,the results mineralized and concomitant induction also the weaker differentiation ability and under hypoxia conditions can be offset after Notch signal pathway were blocked. The above results show that the notch signaling pathway mediated hypoxia on the role of dental pulp stem cells.Conclusion: These data indicated that Notch signaling signal pathway can contribute to the proliferation and inhibit the differentiation of HDPSCs. Hypoxia could promote the proliferation and inhibite the differentiation ability of HDPSCs. Under hypoxia conditions, when Notch was blocked in HDPSCs, the effects of hypoxia on HDPSCs were inhibited,which hinted that hypoxia condition affects the ability of HDPSCs’ proliferation and differentiation through Notch signal pathway at least to some degree.For the detailed mechanism of hypoxia influence the proliferation and differentiation of HDPSCs, further research will be taken from the interaction of Hif-1α, Hif-2α with NICD, may draw the molecular mechanism of the activation of Notch signaling pathways under hypoxia, and study the applications of this regulation role in the field of tissue engineering.