| Stem cells are capable of self-renewal and multi-directional differentiation,and have great potential application value in disease therapy,such as treating cardiovascular diseases,nervous system diseases,and autoimmune diseases.The clinical application of stem cells first requires a sufficient number of cells,but the number of stem cells in human tissues is limited,so in vitro expansion is required,however stem cells can easily differentiate when in vitro expansion,thus it is an urgent problem need to be solved that how to maintain the undifferentiated state while massively expansion of stem cells in vitro.Nanog is a key factor in maintaining self-renewal and pluripotency of stem cells,and plays an important role in the maintenance of undifferentiated state of stem cells.In this study,Nanog promoter was used as a target to screen small molecule compounds that activate Nanog promoter,and then explored the roles of target compounds in maintaining the stemness of stem cells and the underlying mechanisms of target compounds promote the expression of Nanog gene.1.Screening and identification of compounds that promote Nanog expressionIn order to screen for compounds that can maintain stem cell properties,the p GL3-Nanogp plasmid was transfected into HEK293 cells to establish a luciferase reporter gene screening model targeting Nanog promoter.The compounds were added to the cell culture system respectively and the activator of Nanog promoter was screened from 480FDA-approved drug by detecting its effect on luciferase activity,and then detected its effect on the expression of Nanog in both m RNA and protein levels by semi-quantitative RT-PCR and Western blot in P19 cells.The results showed that compounds A4-46,A6-55 and A6-72 could activate the Nanog promoter activity and increase the expression of Nanog at both m RNA and protein levels.2.The role of the target compounds in maintaining the stemness of P19 cellsIn order to test the role of compounds A4-46,A6-55 and A6-72 in the maintenance of stemness,P19 cells were first treated with the target compound.After 48 hours,the protein was extracted and Western blot was used to detect the expression levels of stem cell transcription factors Oct4 and Sox2.The results showed that compounds A4-46,A6-55 and A6-72 were able to up-regulate the expression of Oct4 and Sox2.The effect of the target compound on the stemness of P19 cells was then further examined by embryoid body formation experiments.The P19 cells were cultured in suspension,and the embryoid bodies were obtained after 4 days,and subjected to alkaline phosphatase staining and counting.The results showed that the cells treated with compounds A6-55 or A6-72 highly expressed alkaline phosphatase and promoted P19 cells form more embryoid bodies,which was statistically significantly different from that in control group.While there was no change in the number of embryoid bodies when treatment with compound A4-46.In order to further detect the effects of compounds on the expression of the stem cell transcription factors Oct4,Sox2 and Nanog in the cell mass of pre-embryonic formation,IVimmunofluorescence staining and Western blot were performed on the cell mass.And the results showed that the expression level of stemness factors were increased after treatment with compounds A6-55 or A6-72,while compound A4-46 has no this effect when compared with the control group.The above experiments showed that the compounds A6-55 and A6-72 could promote the stemness maintenance of P19 cells,while the compound A4-46 did not have this function,so the compound A4-46 was excluded in the subsequent study.A natural small molecule compound T2-78 that promotes the expression of Oct4 has been screened in the early stage of the laboratory.Therefore,we considered that the compounds A6-55 and A6-72 which promote the expression of Nanog can be combined with the compound T2-78 which promotes the expression of Oct4,respectively,to investigate the effects of the combination on the stemness maintenance of P19 cells by detecting the formation of embryoid body and the expression of the stemness factors.The results showed that the number of embryoid bodies and the expression of stemness factors Nanog,Oct4,Sox2 were further improved in the combined treatment group.Suggesting that the combination of the compounds A6-55 or A6-72 with the compound T2-78,could further promote the stemness maintenance of P19 cells.Then,the effect of the target compound on the pluripotency of P19 cells was further examined by the nude mouse teratoma experiment.The P19 cells were treated with the target compounds for 7 days respectively,and then the cells were subcutaneously injected into nude mice,and the volume and weight of the teratoma were measured after the formation of the teratoma.The expression of AFP(endodermal marker),c Tn T(mesoderm marker)and ?3-tublin(ectoderm marker)were detected by Western blot.It was found that the weight of teratoma in the treatment group of A6-72 combined with T2-78 was increased and statistically significant.Western blot results showed that the expression of AFP,c Tn T and ?3-tublin in the teratoma of the treatment group of A6-72 were higher than that of the control group,and the expression levels of the three germ layer markers were more higher in the treatment group of A6-72 combined with T2-78.The above results indicate that the compound A6-72 can promote the pluripotency of P19 cells,and the A6-72 in combination with T2-78 is more effective.3.Identification the effects of target compounds on mouse embryonic stem cells with Oct4-GFP reporter geneSince P19 cells are not true stem cells,it is necessary to identify the effects of compounds A6-55 and A6-72 on the real stem cells.The cell line used here is embryonic stem cells derived from Oct4-GFP transgenic OG2 mice(OG2-m ES cells).OG2-MES cells were treated with compounds A6-55 and A6-72,respectively.On the 6th day,cell morphology and GFP expression were analyzed.The results showed that compounds A6-55 and A6-72 were able to maintain tight colony morphology and GFP expression in OG2-MES cells,indicating that compounds A6-55 and A6-72 can promote the stemness maintenance of the mouse embryo stem cells.4.Molecular mechanisms of target compounds promote the expression of Nanog geneTo elucidate the molecular mechanisms by which compounds A6-55 and A6-72 promote the expression of Nanog gene,P19 cells were treated with the target compounds and the proteins were extracted after 15 min,30min and 60 min,respectively.And Western blot was used to detect the changes of stem cell-related signaling pathways NF-?B,PI3K/Akt and Wnt/?-catenin.The results showed that compound A6-55 could increase the level of p-I?B in the cytoplasm of P19 cells and promoted the nuclear translocation of p50 and p65,indicated that A6-55 activated the NF-?B signaling pathway.While PDTC,a inhibitor of NF-?B signaling pathway could return the Nanog protein level induced by A6-55 to normal level,suggest that compound A6-55 promoted the expression of Nanog by activating the NF-?B signaling pathway.Compound A6-72 could also activate the NF-?B signaling pathway and increased the level of p-Akt in cells,indicate A6-72 activated the PI3K/Akt signaling pathway.While both of PDTC and LY294002,a inhibitor of PI3K/Akt signaling pathway could return the increased Nanog level induced by A6-72 to normal level,suggest that compound A6-72 promoted the expression of Nanog by activating the NF-?B signaling pathway and PI3K/Akt signaling pathway.In summary,this study targeted Nanog promoter and screened out two small molecule compounds that promoted the expression of Nanog gene and promoted the stemness maintenance of mouse embryo stem cells.And then we focus on the Wnt/?-catenin,PI3K/Akt and NF-?B signal pathways to explore the molecular mechanisms of target compounds promote the expression of Nanog gene.This study not only lays the experimental foundation for the expression regulation of Nanog gene,but also provides new clues for the stemness maintenance of embryo stem cells. |
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