Study On The Role And Mechanism Of Human Umbilical Cord Mesenchymal Stem Cells And Their Exosomes In The Treatment Of Emphysema

Objective: Chronic obstructive pulmonary disease(COPD),as a common and frequently occurring disease,has a high mortality rate and poses a major threat to human health.In addition,patients need to bear severe economic burden in the treatment process,so it has become a public health problem of widespread concern in the society.However,the current clinical treatment is limited to the alleviation of symptoms,there is no method to completely reverse the development and deterioration of the disease,so it is urgent to find new effective methods to solve this problem.Existing studies have shown that mesenchymal stem cells(MSCs)have the ability to repair damaged tissues.Compared with other MSCs,umbilical cord derived MSCs(human umbilical cords mesenchymal stem cell,hUC-MSCs)have many advantages in clinical application.However,bone marrow derived MSCs are more widely studied in the field of COPD and emphysema,while hUC-MSCs are rarely studied.The existing view is that MSCs play a role mainly through the paracrine pathway,so we intend to explore the role and mechanism of hUC-MSCs and their exosomes in COPD and emphysema,which is conducive to find new ideas for disease treatment.Methods:Part 1: Acquisition and identification of hUC-MSCs and their exosomes(1)The obtained fresh umbilical cords were immediately isolated and cultured by tissue block adhesion method.The morphology of hUC-MSCs was observed under a microscope,and the surface markers were identified by flow cytometry.The multidirectional differentiation potential was identified by osteogenic and adipogenic differentiation experiments in vitro;(2)hUC-MSCs exosomes were extracted,and their morphological characteristics was observed by transmission electron microscope,their particle size was analyzed by nanoparticle tracking analysis technology(NTA),and the CD63 and HSP70 markers of exosomes were detected by western blot.Part 2: Experimental study on the effect of hUC-MSCs on SU5416-induced emphysema(1)After SU5416-induced emphysema was successfully established,hUC-MSCs were injected into the tail vein,and the curative effect was observed 2 weeks later.HE staining was used to evaluate the therapeutic effect of hUC-MSCs on emphysema;(2)ELISA and western blot were used to detect the changes of VEGF expression in alveolar lavage fluid and lung tissue,TUNEL and western blot were used to detect lung tissue apoptosis and active caspase-3 expression,and western blot was used to detect apoptosis-related proteins BAX and Bcl2;(3)Western blot was used to detect VEGF,p-VEGFR2,VEGFR2,p-AKT,AKT proteins of lung tissue to evaluate the changes of VEGF signal pathway;(4)We used SU5416 to treat endothelial cells to observe the effect of SU5416 on endothelial cells in vitro;(5)SU5416 was used to treat endothelial cells,and hUC-MSCs were co-cultured with endothelial cells,the effect of hUC-MSCs on endothelial cells was observed,and the content of VEGF in the co-culture supernatant was detected;(6)Western blot was used to detect Bcl2,BAX,caspase-3 proteins of co-cultured endothelial cells to evaluate their apoptotic status,VEGF,p-VEGFR2,VEGFR2,pAKT,AKT proteins were detected to evaluate the changes of VEGF signal pathway in co-cultured endothelial cells.Part 3: Study on the effect of hUC-MSCs and their exosomes on papaininduced emphysema(1)We used papain by intratracheal instillation to establish a rat emphysema model.HE staining and mean linear intercept(MLI)analysis were used to evaluate whether the modeling was successful;(2)After successfully modeling,hUC-MSCs and their exosomes were injected into rats through the tail vein,and different time and dose groups were used to evaluate the difference in treatment effect.HE staining and MLI were used to evaluate the therapeutic effect of hUC-MSCs and their exosomes on emphysema;(3)The changes of inflammatory cytokines IL-1β,IL-6 and TNFα in bronchoalveolar lavage fluid and lung tissue were detected by ELISA and western blot,respectively;(4)In vivo imaging was used to observe whether the fluorescent labeled exosomes can reach the lungs,and fluorescence imaging under a microscope was used to observe the uptake of hUC-MSCs exosomes by epithelial cells and endothelial cells;(5)High-throughput sequencing was used to analyze the possible downstream pathways of hUC-MSCs exosomes repairing papain-induced emphysema;(6)TUNEL was used to detect the apoptosis of lung tissues,and western blot was used to detect the protein expressions of VEGF,p-VEGFR2,VEGFR2,p-AKT,AKT,p-MEK,MEK,p-ERK and ERK in lung tissues to evaluate the changes of apoptosisrelated signaling pathways.ResultsPart 1: Successful acquisition and identification of hUC-MSCs and their exosomes(1)It was noticed that hUC-MSCs were fibroblast-like and growing in a whirlpool shape.Flow cytometry analysis found that the four positive markers CD29,CD44,CD73 and CD90 were all highly expressed,and the expression of the four negative markers CD19,CD11 b,CD34 and CD45 were negative;osteogenic differentiation and adipogenic differentiation experiments showed that hUC-MSCs had the potential to differentiate into osteogenic and adipogenic cells,which is in line with the general characteristics of MSCs;(2)The transmission electron microscopy showed that exosomes had a typical round or elliptical cup-shaped structure.NTA results showed that the average diameter of exosomes was 134.5 nm.Western blot results showed that exosomes expressed the marker proteins CD63 and HSP70,which is in line with the general characteristics of exosomes.Part 2: hUC-MSCs repaired the emphysema injury caused by SU5416(1)HE staining showed that hUC-MSCs obviously repaired emphysema caused by SU5416;(2)hUC-MSCs rescued the expression of VEGF in the treatment group.Compared with the SU5416 group,the apoptosis and active caspase-3 expression of lung tissues in the treatment group were significantly reduced,the expression of pro-apoptotic protein BAX decreased,and the expression of anti-apoptotic protein Bcl2 increased;(3)Compared with the SU5416 group,VEGF、p-VEGFR2、p-AKT expressions of lung tissue in the treatment group were significantly increased;(4)SU5416 could cause a dose-dependent death of endothelial cells;(5)When hUC-MSCs were co-cultured with endothelial cells under SU5416 treatment,the mortality of endothelial cells decreased,and the VEGF content in the co-culture supernatant was significantly increased;(6)In co-cultured endothelial cells,Bcl2 was significantly increased,and BAX and active caspase-3 were significantly reduced;(7)In co-cultured endothelial cells,VEGF,p-VEGFR2 and p-AKT were significantly up-regulated.Part 3: hUC-MSCs and their exosomes repaired emphysema caused by papain(1)Using intratracheal instillation of papain,we successfully established an emphysema model;(2)Both HE staining and MLI results showed that hUC-MSCs and their exosomes effectively repaired emphysema caused by papain.The effect of hUC-MSCs for 1week was better than 2 weeks,and the effect of 200 μg exosomes was better than 400μg;(3)The results of ELISA and western blot analysis showed that compared with the Papain group,the expression of inflammatory cytokines in the alveolar lavage fluid and lung tissue of the hUC-MSCs and exosomes group was significantly reduced;(4)In vivo imaging showed that fluorescently labeled exosomes could reach the lungs;also,in vitro,we observed that hUC-MSCs exosomes could be taken up by lung epithelial cells and endothelial cells;(5)High-throughput sequencing analysis showed that the pathways that are highly regulated after hUC-MSCs exosomes treatment mainly involves the immune system,PI3K-AKT signaling pathway and MAPK signaling pathway;(6)TUNEL analysis showed that in the Papain group,the level of apoptosis of lung tissues was significantly increased,and the apoptosis of endothelial cells and other types of cells was increased.hUC-MSCs exosomes reduced the degree of apoptosis of lung tissue,and the apoptosis of endothelial cells and other types of cells was significantly reduced;(7)Compared with the Papain group,hUC-MSCs exosomes treatment activated VEGF-VEGFR2-mediated AKT pathway and MAPK/ERK pathway in lung tissue,hUC-MSCs exosomes contain VEGF,p-AKT,p-MEK and p-ERK active proteins.Conclusions:(1)hUC-MSCs can significantly repair emphysema damage caused by SU5416.They might up-regulate the VEGF-VEGFR2-AKT pathway signal and fight against apoptosis;(2)hUC-MSCs and their exosomes could effectively repair emphysema damage caused by papain,and both had obvious immunomodulatory effects;(3)hUC-MSCs exosomes might activate VEGF-VEGFR2-mediated AKT pathway and MAPK/ERK pathway to inhibit cell apoptosis,thereby reversing papain-induced emphysema.