Study On Tracheal Injection Of Human Amnion-derived Mesenchymal Stem Cells In The Treatment Of Neonatal Rat Lung Injury Induced By Hyperoxia

Background:Bronchopulmonary dysplasia(BPD)is a common lung disease in premature infants.Conventional treatment has limited effect on long-term lung development.There is an urgent need for a new treatment for BPD.Mesenchymal stem cells(MSCs)can improve the survival rate of rats with acute lung injury.Several clinical trials have also shown that MSCs can reduce the degree of pulmonary fibrosis in patients with BPD,but the therapeutic mechanism is not clear.Human amnion derived mesenchymal stem cells(hAD-MSCs)have the characteristics of rapid proliferation,high purity and low immunogenicity.hAD-MSCs also have good immune regulation ability.We treated the neonatal rat BPD model of hyperoxia induced lung injury by intratracheal injection of hAD-MSCs,conditioned medium(hAD-MSCs-CM)or exosomes(hAD-MSCs-EXOs).Finally,further explore the molecular mechanism of stem cells in the treatment of BPD.Objectives:hAD-MSCs were cultured and identified.Obtain hAD-MSCs-EXOs and hAD-MSCs-CM and study their basic characteristics.To compare and analyze the efficacy and mechanism of intratracheal injection of hAD-MSCs,hAD-MSCs-EXOs and hAD-MSCs-CM in the treatment of neonatal rats with hyperoxia induced lung injury.Methods:1.Isolation and identification of hAD-MSCs,hAD-MSCs-CM and hAD-MSCs-EXOs:(1)the amniotic membrane was digested with enzyme and adhered to culture hAD-MSCs cells.The cell phenotype was identified by flow cytometry.The multi-directional differentiation ability of hAD-MSCs was identified by inducing medium.(2)When the cell growth reached 80%fusion,the culture supernatant was collected and centrifuged to obtain hAD-MSCs-CM.After serum deprivation starvation,the cell culture supernatant was collected.hAD-MSCs-EXOs were obtained from the supernatant of ultra-high speed centrifugation.The ultrastructure of hAD-MSCs-EXOs was observed by transmission electron microscope(TEM).Western blot was used to identify the expression of CD63,CD9,CD81,and heat shock protein 70 in exosomes.2.Intratracheal injection of hAD-MSCs,hAD-MSCs-CM and hAD-MSCs-EXOs to treat hyperoxia induced lung injury in neonatal rats:(1)neonatal rats were divided into experimental group:normoxia control group(normoxia group),hyperoxia model group(hyperoxia group),hyperoxia model intratracheal injection of hAD-MSCs treatment group(hAD-MSCs group),hyperoxia model intratracheal injection of hAD-MSCs-CM treatment group(hAD-MSCs-CM group)and hyperoxia model intratracheal injection of hAD-MSCs-EXOs treatment group(hAD-MSCs-EXOs group).The hyperoxia group was lived with 80%oxygen.On the 7th day after birth,hAD-MSCs group,hAD-MSCs-CM group and hAD-MSCs-EXOs group were injected with hAD-MSCs(1×106 cells/50μL),hAD-MSC-CM(50 μL),and hAD-MSCs-EXOs(300ng/50 μL).In order to display the hAD-MSCs injected through the gas tube,PKH26 was incubated for in vivo tracing.Observe the body and activity of rats every day and draw the survival curve.(2)Detect the level of inflammatory factors in bronchoalveolar lavage fluid(BALF):collect BALF and lung tissue samples in each group.TNF-α,IL-6,IL-1β and MCP-1 concentration were analyzed by ELISA.MDA and SOD in BALF were also detected.(3)Analysis of lung histopathology and lung weight/body weight ratio:the degree of lung tissue injury of neonatal rats in each group was analyzed by pathological grade 5 scoring system.Standardized analysis was performed using mean linear intercept(mLI).(4)Microarray analysis of signal transduction pathways and differentially expressed genes:total lung RNA was extracted for RNA hybridization to screen differentially expressed genes.qPCR was used to verify the genes with significant differential expression in the microarray results.The differential protein expression was detected by Western blot.3.Statistical analysis:The results are expressed as mean values ± SEM of three separate experiments.Student’s t-test was used to compare two groups.Results with P values less than 0.05 were considered significant.GraphPad Prism 8.0.1(GraphPad Software,Inc,La Jolla,CA,USA)was used for statistical analysis.Results1.Acquisition and biological characteristics of hAD-MSCs,hAD-MSC-CM,and hAD-MSC-EXOs.(1)Culture and identification of hAD-MSCs.The adherent cells in primary culture showed typical fibroblast morphology These cells expressed MSCs markers CD90,CD105,CD44,CD29 and CD73.At the same time,these cells negatively expressed endothelial cell markers CD31 and hematopoietic markers CD34 and CD45.These cells can be induced into adipocytes,chondrocytes and osteoblasts.(2)Characteristics of hAD-MSCs EXOs:We quantified the exosomes by measuring the protein concentration.The results showed that 150 ng of exosomes could be obtained by collecting an average of 100 mL of culture medium.The average diameter of these vesicles is 30～150 nm.The exosomes we obtained expressed CD9,CD63,CD81 and HSP-70 positively.2.hAD-MSCs,hAD-MSC-CM and hAD-MSC-EXOs in the treatment of hypoxic-induced lung injury in neonatal rats.(1)Analysis of general condition,weight and survival rate:Compared with normoxia group,the vitality of neonatal rats in hyperoxia group was significantly decreased,and showed signs of depression,reduced activity and diet intake.Their fur becomes dim and their weight loses.At the end of the experiment,the survival rate of rats in hyperoxia group was significantly lower than that in normoxia group(P<0.05).14 days after birth,the survival rate of hAD-MSCs group was significantly increased(P<0.05),and there was no further death in neonatal rats.The survival rate of hAD-MSC-EXOs group and hAD-MSC-CM group was higher than that of hyperoxia group,but there was no statistical significance(P>0.05).hAD-MSCs,hAD-MSC-EXOs and hAD-MSC-CM could not increase the body weight of neonatal rats in the experimental group(P>0.05),but all the three groups decreased the ratio of lung weight to body weight(P<0.05).After fluorescence labeling of had MSCs,fluorescence in vivo tracing was performed after tracheal injection.It can be seen that the cells were mainly concentrated in the lungs at 12h,and gradually diffused to the whole body with the chest as the center after 24h,mostly in the head and trunk.After 48h,the distribution range was reduced,but the chest was still the main concentration area of stem cells.(2)Detection of BALF inflammatory cytokines.The levels of inflammatory cytokines TNF-α,IL-1β,L-6 and MCP-1 in BALF of neonatal rats in hyperoxia group were significantly increased(P<0.05).Compared with the hyperoxia group,the levels of the above four inflammatory factors in the hAD-MSCs group,hAD-MSC-EXOs group and hAD-MSC-CM group were significantly lower than those in the hyperoxia group(P<0.05).The number of neutrophils,MDA concentration and total protein in BALF also reflected the same trend(P<0.05).Among them,the decrease in hAD-MSCs group was the largest(P<0.05).The expression level of SOD in BALF of hyperoxia group was significantly lower than that of hyperoxia group(P<0.05).Both hAD-MSCs and hAD-MSC-EXOs could significantly increase the expression of SOD(P<0.05),while hAD-MSC-CM could not(P>0.05).(3)Pathological analysis of lung tissue.Compared with normoxic group,inflammatory cell infiltration,thickening of alveolar septum and structural destruction of pulmonary parenchyma were observed in neonatal mice in hyperoxia group.The lung injury grade 5 score and mLI value in hyperoxia group were significantly higher than those in normal group(P<0.05),and decreased significantly in hAD-MSCs group,hAD-MSC-EXOs group and hAD-MSC-CM group(P<0.05),especially in hAD-MSCs group.(4)Analysis of microarray signal pathway and differentially expressed genes.After hyperoxia injury,the expression of a total of four genes in lung tissue was significantly reduced,and the expression of six genes was highly up-regulated after hyperoxia injury.The expression of these genes has changed more than threefold.The differentially expressed genes in the above microarray analysis results were verified by RT-qPCR.The detected gene expression changes were consistent with the results of gene chip analysis.The expression level of APEH gene changed most significantly(P<0.01).Therefore,APEH gene was selected as the further detection target.The gray-scale quantitative data of APEH protein detected by Western blotting showed that the protein expression of APEH was significantly decreased after high oxygen injury(P<0.05),and the expression of APEH was significantly increased after hAD-MSCs treatment(P<0.01).Conclusions:1.The cultured hAD-MSCs have strong proliferation ability and meet the cell phenotype and multidirectional differentiation criteria of MSCs.2.The separation technology system of EXOs obtained by starvation and highspeed centrifugation was established.The size and expression markers of hAD-MSC-EXOs were determined.3.Compared with hAD-MSC-EXOs and hAD-MSC-CM,intratracheal instillation of hAD-MSCs can better improve the levels of lung inflammation and pulmonary edema in neonatal rats induced by hyperoxia.4.The expression of APEH was significantly changed before and after hAD-MSCs treatment.The expression of APEH may be closely related to hyperoxia-induced lung injury and the treatment of hAD-MSCs.