| Part?Establishment of Hyperoxia induced BPD Model and Extraction of hucMSC-sEVsOBJECTIVE:To establish a hyperoxia induced BPD model in neonatal rats,to extract and to identify hucMSC-sEVs.METHODS:Neonatal SD rats were put in the chamber for continuous hyperoxia(Fi O2=85%),dams were alternated every 48h from hyperoxia to normoxia to minimize oxygen toxicity.Paraffin embedded sections of the left lung were collected on P3,P7,P14,and P21.H&E staining was used to observe the alveolar structure.The mean linear intercept(MLI)was calculated by Plus Pro Plus software(IPP)to assess the alveolar diameter.huc MSCs of P4-P6 were cultured in medium without small extracellular vesicles(sEVs)after 60-70%growth for 48 h.The cell supernatant was collected and hucMSC-sEVs were extracted and purified by ultracentrifugation.The morphology and size of hucMSC-sEVs were detected by transmission electron microscope(TEM),the surface markers Alix and CD63 were detected by Western blot,and the particle size distribution of hucMSC-sEVs was detected by nanoparticle tracking analysis(NTA).RESULTS:1.Compared with the room air group,the alveoli were simplified,the secondary ridges were atypical or even disappeared,and the alveolar size of P7(65.30±2.77 vs.55.70±1.40?m,P(27)0.05),P14(82.74±3.70 vs.58.64±1.96?m,P(27)0.01),and P21(92.08±2.52 vs.59.28±1.84?m,P(27)0.001)in hyperoxia group were significantly increased.2.hucMSC-sEVs showed biconcave disc shape with a diameter of about30-150nm under TEM;and low-density transparent area could be seen in the capsule;hucMSC-sEVs positive-expressed the exosome surface marker Alix and CD63;NTA showed that most of the particles were concentrated in 70-300nm,with a peak value of 175nm.CONCLUSION:The typical pathological manifestations of BPD(alveolar simplification)were found in neonatal rats after 14-day exposure to hyperoxia;hucMSC-sEVs mainly containing exosomes could be extracted by ultracentrifugation.Part ? Study on the Effectiveness of hucMSC-sEVs in the Treatment of Hyperoxia BPD ModelOBJECTIVE: To observe the therapeutic effect of human umbilical cord mesenchymal stem cells derived small extracellular vesicles(hucMSC-sEVs)on hyperoxia induced BPD in neonatal rats.METHODS: Newborn rat pups were immediately randomly divided into three groups:(1)Room air group(RA+PBS): normoxia(21% O2)plus PBS was administrated by intratracheal route on postnatal 7(P7).(2)BPD group(BPD+PBS): hyperoxia plus PBS was administrated by intratracheal route on P7.(3)hucMSC-sEVs group(BPD+hucMSC-sEVs): hyperoxia plus hucMSC-sEVs was administrated by intratracheal route on P7.hucMSC-sEVs were labeled with PKH26,then intratracheal administrated on P7 in BPD model.Lungs were harvested at 0,2,12,24 and 72 h after hucMSC-sEVs transplantation.To explore the therapeutic effect of hucMSC-sEVs in studies,their lungs were harvested at P14,then,hematoxylin and eosin(H&E)staining was used to observe the morphological structure of lung tissues,the body weight gains ratio were calculated,pulmonary hypertension related indexes were calculated(Fulton's index and right heart to weight ratio),bronchoalveolar lavage fluid(BALF)were harvested for the detection of total cell number,protein concentration,concentration of IL-6 and concentration of IL-10.RESULTS: 1.Immunofluorescence tracking showed that PKH26 labeled hucMSC-sEVs concentrated in trachea at the beginning after intratracheal administration,then progressively infiltrated the whole lung parenchyma at 2h post-injection,reaching the peak at 12 h and lasted to 72 h.2.Hyperoxia-induced increase in MLI(98.93±7.27 vs.61.34±3.99 mm,P<0.001)was significantly ameliorated by huc MSC-s EV treatment(67.26±5.32 mm,P<0.01 vs.BPD).3.The body weights of neonatal rats were measured at birth(BW)and on P14(W),and the W-BW to BW ratio was calculated as weight-gain rate,which was decreased in the BPD group compared with the RA group(1.69±0.09 vs.2.77±0.05,P<0.001),administration of hucMSC-sEVs significantly improved the weight-gain rate relative to the BPD group(2.09±0.11,P<0.05 vs.BPD).4.Fulton's index(0.38±0.02 vs.0.28±0.01,P<0.001)and right heart to weight ratio(1.17±0.06 vs.0.83±0.03,P<0.001)were significantly elevated in the BPD group compared with the RA group,and that hucMSC-sEVs treatment attenuated the Fulton's index(0.27±0.16,P<0.001 vs.BPD)and right heart to weight ratio(0.89±0.03,P<0.05 vs.BPD)compared with BPD group.5.LR(1.23±0.05 vs.0.49±0.04,P<0.001)was significantly increased and Cdyn was significantly decreased(0.02±0.002 vs.0.05±0.004,P<0.001)in the BPD group compared with RA group,and intratracheal administration of hucMSC-sEVs significantly reversed the changes in LR(0.65±0.03, P<0.001 vs.BPD)and Cdyn(0.03±0.002,P<0.05 vs.BPD)compared with the BPD group.6.The cell count(101.70±11.09 vs.34.92±2.06,P<0.001),total protein concentration(8.54±0.86 vs.2.09±0.22,P<0.001)and IL-6 concentration(49.59±6.85 vs.20.19±1.82,P<0.01)of BALF in BPD group on P14 were significantly increased compared with RA group,while IL-10 concentration(267.90±4.36 vs.326.30±5.95,P<0.001)was significantly decreased,hucMSC-sEVs treatment reduced the cell count(72.04±5.57,P<0.05 vs.BPD)total protein concentration(5.35±0.64,P<0.05 vs.BPD)and IL-6 concentration(29.75±2.53,P<0.05 vs.BPD),and promoted the secretion of IL-10 in BALF(292.6±6.11,P<0.05 vs.BPD).CONCLUSION:hucMSC-sEVs can be distributed in the lung at 2h after intratracheal injection,reaching the peak of uptake at 12 h and lasting to 72 h.Airway delivery of hucMSC-sEVs rescued the hyperoxia-induced model of BPD: improved the alveolar simplification,increased the weight gain rate,reduced pulmonary hypertension,improved lung function and attenuated the inflammatory indexes in BALF in BPD group.Part ? Mechanism of hucMSC-sEVs in the Treatment of Hyperoxia BPD ModelOBJECTIVE: To explore the affected cell population and molecular mechanism of hucMSC-sEVs in the treatment of neonatal rats with hyperoxia induced BPD,and to speculate key components of hucMSC-sEVs in the treatment of BPD.METHODS: The number of Ki-67,TUNEL,SPC positive cells and CD31 positive small vessels((27)100?m)in the frozen sections of P14 lung tissue in different treatment groups were detected by immunofluorescence.The uptake of PKH26 labeled hucMSC-sEVs was observed by immunofluorescence after co-culture with human umbilical vein endothelial cell line(HUVEC)or mouse type II lung epithelial cell line(MLE-12)for 0,2,12 and 24 hours.In vitro experiments,cells were divided into three groups: normal culture group(RA),90% hyperoxia group(HYP),hucMSC-sEVs treatment group(HYP+s EV).HUVEC cells were seeded on Matrigel after cultured under different conditions for 24 hours,then,angiogenesis was observed under light microscope after incubated for 5h.The proliferation of MLE-12 cells was detected by CCK-8 and Ki-67 immunofluorescence,and the apoptosis by annexin V flow cytometry and TUNEL immunofluorescence in different treatment groups.Western blot was used to detect the expression of PTEN,p-Akt/Akt,caspase3 and VEGFA in lung homogenate of neonatal rats in different groups.The mi RNA of hucMSC-sEVs was sequenced,and Top50-mi RNA target genes were analyzed by GO and KEGG pathways enrichment analysis.RESULTS: 1.Compared with RA group,the number of Ki67 positive cells in BPD model group decreased significantly(10.92 ± 0.46 vs.22.20 ± 3.27,P < 0.01),and the number of TUNEL positive cells increased significantly(2.91 ± 0.34 vs.0.19 ± 0.06,P < 0.001),administration of hucMSC-sEVs was reversed them(19.67 ± 1.24,P < 0.05;1.31 ± 0.25,P < 0.05 vs.BPD)respectively.2.Compared with RA group,the number of CD31 positive microvessels(< 100 mm)(4.20 ± 0.27 vs.12.23 ± 0.81,P < 0.001)and SPC positive cells(14.38 ± 1.24 vs.29.25 ± 0.61,P < 0.001)in BPD model group was significantly decreased,administration of hucMSC-sEVs reversed that(6.39 ± 0.32,P < 0.05;23.03 ± 1.50,P < 0.05 vs.BPD)respectively.3.PK26 labeled hucMSC-sEVs began to be absorbed by the cells after 2 hours,mainly located in the cytoplasm at 12 hours,and a few huc MSC-s EVS gathered on the nuclear membrane at 24 hours.4.The vascular length of HUVEC in hyperoxia group was significantly reduced compared with that in air control group(8.07 ± 0.27 vs.13.00 ± 0.44 mm,P < 0.01),hucMSC-sEVs significantly increased it(11.04 ± 1.05 mm,P < 0.05 vs.Hyp).5.hucMSC-sEVs significantly reduced the number of TUNEL positive MLE-12 cells in hyperoxia environment (0.44 ± 0.06 vs.2.21 ± 0.34,P < 0.01),and inhibited the apoptosis rate of mle-12 cells(8.93% ± 1.05% vs.12.12% ± 0.32%,P < 0.01).6.hucMSC-sEVs significantly inhibited the expression of PTEN(1.03 ± 0.14 vs.1.80 ± 0.20,P < 0.05)and Caspase3(0.69 ± 0.04 vs.1.08 ± 0.11,P < 0.01)in BPD group,and increased the expression of p Akt(0.49 ± 0.03 vs.0.33 ± 0.04,P < 0.05)and VEGFA(0.48 ± 0.05 vs.0.20 ± 0.03,P < 0.05).7.Mi R-21-5p was the most highly expressed mi RNA in hucMSC-sEVs,GO enrichment analysis of top 50 mi RNA was significantly correlated with the potential of regulating proliferation,apoptosis,angiogenesis and lung development;KEGG enrichment analysis was significantly correlated with PI3K-Akt,VEGF and RAS pathways.CONCLUSION:hucMSC-sEVs had effect on TIIAECs and PVECs of neonatal rats in BPD model;hucMSC-sEVs improved the survival of lung cells and the number of small blood vessels in BPD lung tissue,in addition,increased the survival of MLE-12 cells and promoted angiogenesis of HUVECs in hyperoxia in vitro;the mechanism of hucMSC-sEVs promoting alveolization and angiogenesis may related to the activation of PTEN/Akt;the highest expression of mi RNA in hucMSC-sEVs is mi R-21-5p;the GO enrichment analysis of top50-mi RNA was significantly related to proliferation,apoptosis,angiogenesis and lung development and KEGG enrichment analysis was to PI3K-Akt,VEGF and RAS pathways. |
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