An Experiment Study On Effects Of Proliferation And Differentiation Of Human Dental Pulp Stem Cells By Over-expression NICD

Dental Pulp Stem Cells (DPSCs) was first proposed in2000by Shi S and Gronthos S.They are the undifferentiated mesenchymal cells with stem cell properties existing in thedental pulp tissue. They have been considered as the precursors of mature dental pulp tissue,and have been found to be capable of differentiating into odontoblasts under physiologicalor pathological conditions and then forming reparative dentin. As with most other stem cells,DPSCs can also encounter self-differentiation in the process of vitro cultivation andgradually lose the self-renewal capability. Therefore, DPSCs, as an important source of theseed cells used in dental tissue engineering, how to maintain their sternness and avoidself-differentiation in the process of vitro cultivation is the imperative problem needs to besolved urgently.Notch signaling pathway regulates cellular differentiation, proliferation and apoptosis.Its most fundamental and most important role in Notch-mediated lateral inhibition mayblock the cellular differentiation-related specific gene expression, and thus maintain cells ina primitively undifferentiated state. Some scholars put forward to using Notch pathway’scharacteristic of "differentiation and inhibition” to maintain the self-renewal capacity of thestem cells in the process of vitro cultivation. Through the over-expressed Notch receptors inhematopoietic stem cells, use this concept to make the hematopoietic stem cells cultured invitro successfully obtain the "eternal life".Many studies showed that NICD (Notch1intracellular domain) is exposed because ofthe combination of Notch’s ligands and receptors located on the cell membrane of adjacentcells, and then the activated NICD transfers to the nucleus to activate Notch pathway’starget genes, as a result, Notch signaling is activated.Scholars have demonstrated thatexogenous NICD can activate the genes of downstream pathway of Notch signaling. Theregulation of exogenous NICD to target genes is as same as the approach that the combination of Notch ligands and receptors induce the activation of Notch signaling.Therefore, the way of over-expressing NICD in cells to simulate the process of Notchpathway up-regulation is widely applied to the study of Notch pathway. In this study,lentiviral vector-mediated NICD was designed to transfect human DPSCs to up regulateNICD expression in DPSCs, then research its impact on the proliferation and differentiationof DPSCs, and preliminarily explore the mechanisms of Notch pathway on the proliferationand differentiation of DPSCs.Objectives: dental pulp stem cells(DPSCs) which is over-expressed of NICD is built;Observe the impact of over-expressed NICD on the proliferation and differentiation ofDPSCs; Research the mechanisms of Notch pathway on the proliferation and differentiationof DPSCs.Methods:1. Over-expressed of NICD in DPSCs was built: revive human DPSCs in previousexperimental groups, observe the cell morphology under the microscope,make sure the cellbe in good condition; transfect the exogenous over-expressed NICD lentiviral vector, anduse puromycin to select the stably expressed cell strains;Western Blot technology andRT-PCR technology was used to verify the uprising expression level of post-transfectedNICD and the cell immunofluorescence technique was used to observe the changes in theexpression level and expression location of NICD. The experiment is divided into DPSCs/NICD group, DPSCs/vector group, and DPSCs/wt group.2. Observe the impact of over-expressed NICD on the proliferation, migration anddifferentiation of DPSCs: observe the effect of exogenous NICD on the proliferation ofDPSCs by means of CCK-8method and flow cytometry detecting the changes of S phase;induce the cellular differentiation and detect the expression of specific protein DSPPdifferentiated by DPSCs through Western Blot; Transwell detects the migration capacity ofthese three cell groups.The experiment is divided into DPSCs/NICD group, DPSCs/vector group, and DPSCs/wt group.3. The mechanism study of over-expressed NICD on the proliferation anddifferentiation of DPSCs: Western Blot and RT-PCR technology was used to observe thealtered expression of Hes1, Runx2,The experiment is divided into DPSCs/NICD group,DPSCs/vector group; observe the altered expression of Runx2and DSPP after inhibiting the Hes1expression through siRNA-Hes1; explore the mechanisms of Notch pathwayregulating the proliferation and differentiation of DPSCs. The experiment is divided into:DPSCs/vector group, DPSCs/NICD group, DPSCs/vector/siRNA-Hes1group andDPSCs/NICD/siRNA-Hes1group.Results:1. DPSCs which is over-expressed of NICD was successfully built. NICD expressionin DPSCs/NICD group is significantly higher than that in DPSCs/vector group and inDPSCs/wt group, and its nuclear expression is accelerated, while there is no significantdifference in expression between the latter groups.2. Compare to DPSCs/vector group and DPSCs/wt group, the proliferation capacityof DPSCs/NICD group is significantly enhanced; S phase fraction rises; the expressionlevel of DSPP significantly reduce; the cellular migration capacity is enhanced, while thereis no significant difference between the former groups.3. Compare to DPSCs/vector group, the Hes1expression of DPSCs/NICD significantlyascends while Runx2expression level significantly descends. Compare with control groupafter siRNA-Hes1, the expression of Hes1descends with varying degrees both in mRNAand protein,the expression of Hes1significantly descends in DPSCs/vector/siRNA-Hes1group.After siRNA-Hes1,the mRNA and protein both in Runx2and DSPP ascend,comparewith DPSCs/vector group,Runx2and DSPP significantly ascend in DPSCs/vector/siRNA-Hes1group.The results indicate that Runx2and DSPP are positive correlated.