The Repair Effect And Mechanism Of Human Umbilical Cord Mesenchymal Stem Cells Mediated Radiation Induced Lung Injury

Objective:1.Establish a model of human umbilical cord mesenchymal stem cells to treat radiation induced lung injury in mice,and explore the effect of human umbilical cord mesenchymal stem cells on radiation-induced lung injury.2.Screening and verifying differential miRNAs through miRNA-seq,bioinformatics analysis of the sequencing results,and preliminary exploration of the mechanism of hUCMSCs on the repair of RILI.Methods:1.Establishment of RILI mouse model:8 C57 male mice from 5-7 weeks were randomly divided into 4 experimental groups and 4 control groups.The two groups of mice were fixed with a mouse holder,irradiated with 14GY of the chest,and the experimental group 'was irradiated.After 24 hours,the tail vein was injected with human mesenchymal stem cells 1.5×10^6 cells/ml,100ul injection,and the third day after irradiation,the tail vein was injected with human mesenchymal stem cells 1.0×10^6 cells/ml,100ul injection,and the control group was not treated..1 week,3 weeks,8 weeks,and 12 weeks after irradiation,1 mouse was sacrificed in each group.Lung tissue was taken for pathological section,and HE and MASSON staining were performed,respectively.Immunohistochemistry was used to detect the radioactive pulmonary fibrosis-related factor collagen l.a-SMA,Vimentin,immunofluorescence measurement of radiation-induced lung injury-related factor a-SMA,one-third of each mouse lung tissue was saved for miRNA-seq sequencing.2.Through miRNA-seq sequencing,we screened and verified the targets of hUCMSCs for RILI repair.After the extracted total RNA samples were subjected to agarose electrophoresis and Nanodrop quality inspection and quantification,the library was constructed.The Agilent 2100 Bionalyzer was used to determine the library quality.The sequencing libraries of the mixed samples were denatured by 0.1M NaOH to generate single-stranded DNA.In Illumina 51-cycle sequencing on the NextSeq 500 sequencer.Based on the sequencing results,the 13 MiRNAs with the highest expression differences were selected,and RT-PCR was used to verify whether the sequencing results were reliable.Through bioinformatics analysis of miRNA-seq sequencing results,preliminary exploration of the mechanism of hUCMSCs on RILI repair.Grouped according to 12-week sequencing results:stem cell group compared with control group,and differential miRNA analysis was performed on the standardized data.Based on the miRWalk2.0 database,miRWalk,targetscan,miranda,RNAhybrid and other 4 tools respectively predict the target genes of different miRNAs.Use the cluster profiler tool of R software to predict the KEGG pathway enrichment results of key miRNAs,and obtain the pathways regulated by each miRNA.Then build the miRNA-target relationship and use the cytoscape 3.6.1 tool to make the map.Find key pathways and build a pathway map.Use the KEGG mapper tool based on the KEGG database to construct a path mapping map for key pathways.Initially explore the mechanism of hUCMSCs repairing RILI.Results:1.In the RILI mouse model,the mice in the stem cell group had a more stable body weight and the general condition was better than the control group.The pathological HE and Masson staining in the lung tissue of the mouse showed that the inflammation of collagen fibers and tissues in the stem cell group was reduced compared with the control group.Immunohistochemistry and immunofluorescence showed that the stem cell group had fewer radiation-induced lung injury related factors(collagenl,Fibronectin,a-SMA)than the control group.2.The miRNA-seq test found that compared with the control group,there were 102 differentially expressed miRNAs in the stem cell treatment group at week 12,and the results of the first 10 miRNAs with the highest differential expression by RT-PCR were consistent with the miRNA-seq test results.The four tools including miRWalk,targetscan,miranda,and RNAhybrid respectively predict the target genes of differential miRNA.The clusterProfiler tool of R software is used to predict the KEGG pathway enrichment results of key miRNAs,and the pathways and pathway maps regulated by each miRNA are obtained.Conclusions:1.In the RILI mouse model,stem cell treatment can improve the general condition of mice in the stem cell treatment group and reduce radiation lung injury,suggesting that stem cell treatment can alleviate radiation lung injury.2.MiRNA-seq results showed that the differentially expressed genes in the stem cell treatment group were up-regulated,and mmu-miR-466i-3p,mmu-let-7f-1-3p,mmu-miR-450a-2-3p had the deepest correlation with radiation lung injury,They can inhibit fibrosis related pathways through Prkcb,Prkca,and smad2 proteins.