Effect Of Radiation On Differentiation Of Embryonic Stem Cells And Its Mechanism

Objective:1.To study the effect of radiation on embryonic stem cells and its molecular mechanism;2.Develop a simple,efficient,safe,nontoxic,economical and environmentally friendly cell transportation method so that researchers can realize cell transportation under normal temperature conditions worldwide.Methods:1.In order to study the effects of radiation on embryonic stem cells,we first conducted a gradient experiment on radiation doses to compare the effects of exposure to 0.5 Gy,1 Gy,2 Gy,4 Gy,6 Gy,and 8 Gy X-ray irradiation on embryonic stem cells in mice.Using qPCR and Western Blots to detect changes in gene expression of major stemness and three germ layer marker genes,we found that X-ray irradiation of 8 Gy significantly increased the expression of three germ layer marker genes and lowered the protein levels of the main stem genes.Therefore,we chose this radiation dose for follow-up experiments.Our experimental results show that radiation will reduce the expression of major stem gene proteins and promote the differentiation of embryonic stem cells into the three germ layers.In order to study the molecular mechanism of radiation promoting the differentiation of embryonic stem cells into the three germ layers,we used RNA-seq to compare the differential expression of genes in the embryonic stem cells of the radiation group and the control group.We found that radiation caused upregulation of the expression of Yap and its target genes.We used qPCR was verified.By analyzing the ChIP-seq data,we used the ChIP-qPCR experiment and found that after radiation exposure,YAP binding to its target gene increased.Our research shows that radiation promotes the expression of Yap and its target genes.In order to explore whether radiation can promote the differentiation of embryonic stem cells into the three germ layers by up-regulating the expression of Yap,we overexpress Yap in embryonic stem cells.The results show that overexpression of Yap down-regulates the expression of major stem genes and promotes the expression of major differentiation genes in the three germ layers.It may indicate that radiation regulates the molecular mechanism of embryonic stem cell differentiation by regulating the expression of Yap gene.2.Digesting cells into single cells,and storing them in 100%Fetal bovine serum,FBS)for transportation.The new method was compared with the traditional dry ice transportation method for eight cell lines commonly used in laboratories:mES,Hela,MEF,MS1,HuH7,J774a.1 and 293T.After digestion,the cells were divided into four parts,three of which were stored separately in EP tubes equipped with 800 μl FBS by the new method,and the cells were stored at 4℃,20℃ and 30℃ for 3 days respectively.The other was stored in a medium containing 10%DMSO in dry ice for 3 days using conventional methods.The survival rate of cells was detected by MTT,and the proliferation ability of cells was detected by counting and plotting the growth curve of the resuscitated cells during the culture process,so as to compare the effects of different cell transportation conditions.Results:1.Radiation up-regulates the expression of the marker gene of the three germ layers,down-regulates the protein level of the main stem factors,and promotes the differentiation of embryonic stem cells into the three germ layers;2.Radiation up-regulates the expression of Yap gene,increases the accumulation of YAP on its target genes,and increases the expression of YAP target genes;3.Overexpression of Yap down-regulates the expression of major stem genes and up-regulates the expression of the three germ layer marker genes to promote embryonic stem cell differentiation;4.Radiation may promote the differentiation of embryonic stem cells into the three germ layers by up-regulating the expression of the Yap gene;5.Compared with the traditional method,the cells transported by the new method have no obvious differences in cell characteristics such as cell morphology,proliferative capacity,cell activity,stemness of embryonic stem cells and their differentiation ability.So we have developed a method for cell transport under normal temperature conditions.Conclusion:1.Irradiation promotes the differentiation of embryonic stem cells into the three germ layers by up-regulating the expression of Yap and its target genes.2.We have developed a method for cell transportation under normal temperature conditions,which does not require dry ice consumption,is simple,safe,efficient and economical and environmentally friendly.