Study On The Therapeutic Effect And Mechanism Of Ren Umbilical Cord Mesenchymal Stem Cells On Lps-induced Acute Lung Injury

Objective:To explore the therapeutic effect of umbilical cord mesenchymal stem cells on LPS-induced acute lung injury.Methods:(1)Establishment of lung injury model in vivo: The animals were randomly divided into the following five groups according to the random counting method: Sham group,4-day ALI group,4-day ALI+h UC-MSCs group,14-day ALI group,14-day ALI+h UC-MSCs group.Five C57BL/6 mice were selected in each group for the experiment,a total of 25 mice.The ALI model group and ALI+h UC-MSCs treatment group were used to establish acute lung injury model by endotracheal infusion of LPS,In ALI+h UC-MSCs group,after intratracheal infusion of LPS2 h,h UC-MSCs(1 × 106/ml),and the same dose of saline was given to ALI model group at the same time.The mice were anesthetized 24 hours,4days and 14 days after the establishment of the model,and the lung tissue was taken after pulmonary circulation perfusion and stored at-80 ℃ for subsequent experiments.(2)Take three groups of mouse lung tissue,five samples in each group,and carry out HE staining of lung tissue to observe lung inflammation and pathological changes.According to the pathological staining results,select the mouse lung tissue for m RNA sequencing at the fourth day,and carry out KEGG and GO enrichment analysis on the results to explore the therapeutic effect and related mechanism of h UC-MSCs on LPS-induced acute lung injury in mice.(3)To verify the mechanism of h UC-MSCs in treating LPS-induced RAW264.7 cell injury in vitro: use mouse mononuclear macrophages to establish an in vitro acute lung injury model using LPS(100ng/ml),and randomly divide the cells in the6-well plate into Control group,LPS group,LPS+h UC-MSCs group,with 2 cells per well × 105 cells/ml,LPS+h UC-MSCS group was co-cultured with h UC-MSCs for 12 h,LPS group,LPS+h UC-MSCs were stimulated with LPS for 12 h,then cells were collected,and Trem2,m TOR,TLR4,NF of cells in each group were detected by western blotting and RT-PCR-κ Expression of B p65 and related inflammatory factors.(4)In vivo model to explore the mechanism of h UC-MSCs in treating LPS-induced acute lung injury in C57BL/6 mice: take the lung tissue of the two groups of mice on the 4th and14 th days,and use western blotting method to detect Trem2,m TOR,TLR4,NF in the lung tissue of the two groups of mice-κ B p65 expression.Results:(1)HE staining was performed on the lung tissue sections of mice in each group.The results showed that the lung tissue structure of Sham group mice was complete,the alveoli were not ruptured,the trachea was not collapsed,the alveolar septa were not edema and inflammatory cell infiltration;In ALI group,the alveoli fused into a mass,with a large number of inflammatory cells infiltrating inside,the trachea collapsed in a large area,and a large amount of exudation in the alveolar septa,especially on the 4th day;After intratracheal infusion of h UC-MSCs,the above pathological changes were significantly relieved.(2)According to the above HE staining results,we found that the degree of lung injury in mice was the most severe at the 4th day,and the effect was the best after treatment with h UC-MSCs.Therefore,we selected the 4d mouse acute lung injury model for transcriptome sequencing to quickly obtain the gene expression profile and analyze the differential genes.The results showed that the differential genes were mainly concentrated in the signal transduction,transmembrane protein transport,immune-related systems,and cell growth and death pathways.(3)Western blotting and RT-PCR of in vitro model showed that LPS could significantly reduce the expression of Trem2 in macrophages,while h UC-MSCs could significantly promote the activation of macrophages by increasing the expression of Trem2,thereby inhibiting TLR4/NF-κ The activation of B/m TOR inflammatory signal pathway reduces the secretion of inflammatory factors and reduces LPS-induced cell damage.(4)Western blotting of the in vivo model suggested that in the early stage of inflammation(4d),h UC-MSCs can significantly promote the expression of Trem2 in the lung tissue of mice,thereby promoting the activation of macrophages,and can inhibit TLR4/NF-κ B/m TOR inflammatory signal pathway,thus reducing the degree of lung injury.Conclusion:(1)h UC-MSCs have a therapeutic effect on LPS-induced acute lung injury in C57BL/6 mice;(2)h UC-MSCs can promote the expression of Trem2 in mouse lung tissues and RAW264.7 cells,induce the activation of macrophages,and inhibit TLR4/NF-κB/m TOR inflammatory signaling pathway;Reduce the release of inflammatory cytokines such as TNF-α,IL-6,CXCL9,IL-1β,etc.,and increase the release of IL-10 factors,thereby reducing the degree of LPS-induced acute lung injury in vitro and in vivo models,and preliminarily reveal the therapeutic mechanism of h UC-MSCs for acute lung injury.