| Objectives:Induced pluripotent stem cells(i PSCs)have great potential in regenerative medicine,but their efficient and safe clinical application still faces great challenges,which limits their application prospects.This study aims to find a safe and effective method to promote the generation of i PSCs,and to provide an experimental basis for obtaining clinical-grade stem cells and treatment options using stem cells.Methods:1.Screening signaling pathways: m RNA-Seq transcriptomics was used to screen out the genes with large differences between mouse embryonic fibroblasts(MEFs)and embryonic stem cells(ESCs),and the related signaling pathways were further screened by GO and KEGG enrichment analysis.RT-q PCR was used to further verify the screened differential genes and determine the final research object.2.Induction of mouse somatic cell reprogramming: Sox2,Oct4,and Klf4 were transcribed into MEFs with retroviruses to induce their reprogramming.Inverted fluorescence microscopy was used to observe the cell aggregation state and GFP-positive clones to determine whether the induction process was successful.3.Evaluation of the effect of GSH: In the process of somatic cell reprogramming,different concentrations of exogenous GSH are added,and the results of GFP-positive clones and alkaline phosphatase(AP)staining are counted to determine whether exogenous GSH can promote somatic cell reprogramming and the optimal concentration of its effect.4.Evaluation of the role of GSTs: Construct related plasmids,overexpress related GSTs plasmids during mouse somatic cell reprogramming and observe them,count GFP positive clones and AP staining results,and compare their roles in mouse somatic cell reprogramming.5.Pluripotency verification: i PSCs were established,and immunofluorescence staining and RT-q PCR were used to verify whether the generated i PSCs had pluripotency.Results:1.GO enrichment analysis showed that 5 of the genes significantly up-regulated in m ESCs were genes related to the activity of GSTs.KEGG enrichment analysis showed that GSH metabolism was an important pathway in m ESCs.Because GSTs are a part of GSH metabolism,it is inferred that GSH metabolism is related to the pluripotency of m ESCs,and it is speculated that exogenous GSH can promote somatic cell reprogramming.RT-q PCR was used to compare the expression of GSTs in MEFs,m ESCs,and i PSCs,and it was found that the differences between these five GSTs in MEFs,i PSCs,and m ESCs were statistically significant.It is speculated that GSTs may play a role in promoting the reprogramming process.2.Observe the cells during the reprogramming process of the three-factor induction system by inverted fluorescence microscopy.We found that during this process,the cells showed an aggregation trend and generated GFP-positive clones,marking the success of reprogramming.3.By adding different concentrations of GSH in the early stage of reprogramming and comparing their reprogramming effects,we found that GSH has the best effect on promoting somatic cell reprogramming at a concentration of 0.5 mg/m L,which can improve the efficiency by about 3 times.4.By constructing plasmids,using viral packaging plasmids,and using packaged viruses to infect MEFs,we overexpressed these five GSTs during the reprogramming process,and compared the clone generation.We found that overexpression of Gstp3,Gsta4,and Gstm6 in GSTs can promote mouse somatic cell reprogramming and improve efficiency by about 2-5times.5.The pluripotency factor in induced i PSCs was verified by immunofluorescence staining and RT-q PCR,which proved that it has pluripotency.Conclusions: GSH metabolism is related to the pluripotency of m ESCs;exogenous GSH can improve the efficiency of mouse somatic cells reprogramming into induced pluripotent stem cells;Gstp3,Gsta4,and Gstm6 can improve the efficiency of mouse somatic cells reprogramming into induced pluripotent stem cells;experimentally generated i PSCs have pluripotency. |
PDF Full Text Request