| Part Ⅰ Establishment of Antenatal Lipopolysaccharide Induced BPD Model and Study on the Effectiveness of hUCMSCs in the Treatment of Antenatal Lipopolysaccharide Induced BPD ModelOBJECTIVE:To establish an antenatal lipopolysaccharide induced BPD model in neonatal rats,and to observe the effectiveness of hUCMSCs on this antenatal BPD model.METHODS:Pregnant female Sprague-Dawley rats received intra-amniotic injections of LPS on day 20.5 of embryo(E20.5)under general anesthesia with isoflurane inhalation(2%).Cesarean section was performed on E22.5,and all the rat pups in the injected amniotic sacs were delivered and then placed with foster mother rats.The left lungs were harvested on PN1,PN7 and PN14.H&E staining was used to observe the alveolar structure.The mean linear intercept(MLI)and secondary septa were calculated.The treatment groups received 40 μL hUCMSCs of P4-P6(1×10^6 cells per pup)or MVs by intratracheal route,while the control groups received 40 μL of normal saline(NS).Lung function was measured and the left lungs were harvested on PN14.Comparison of the morphological structure of lung tissues(MLI and radial alveolar counts(RAC)),weight gain,small vessels(<100 μm),lung function and right ventricular index(RVI)among the groups were conducted to assess the effectiveness of hUCMSCs on the rat model of BPD induced by intra-amniotic injections of LPS(IA-LPS BPD model).RESULTS:1.Compared with the control group,the MLI of the LPS group were significantly increased on PN7 and PN14,while RAC of the LPS group were decreased on PN1,PN7 and PN14,shown as increased alveolar space,simplified alveolarization and reduction of secondary septal.2.Compared with the IA-LPS BPD group(LPS+NS group),both of MLI and RAC of the hUCMSCs treatment group returned to the normal level.3.Rats exposed to LPS had a slow weight gain,but there was a catch-up growth period after transtracheal administration of hUCMSCs.4.Small vessels(<100 μm)in rats exposed to LPS were significantly less abundant than those in the NS control group,but the abundance of small vessels increased after hUCMSCs treatment,compared with the LPS group.5.Compared with the control group,LPS increased lung resistance and reduced Cdyn,which reversed by hUCMSCs treatment.6.hUCMSCs treatment decreased RVI as compared with rats exposed to LPS alone.CONCLUSION:1.IA-LPS injection successfully induced BPD-like lung injury in newborn rats.2.hUCMSCs treatment has improvements on alveolarization,improved weight gain,restored small vessels,improved lung function and decreased pulmonary hypertension.Part Ⅱ Extraction and identification of MVs derived from hUCMSCs and Study on the Effectiveness of MVs in the Treatment of IA-LPS BPD ModelOBJECTIVE:To extract and to identify MVs derived from hUCMSCs,to observe the effectiveness of MVs on the IA-LPS BPD model.METHODS:hUCMSCs were grown to 60-70%and then cultured in medium without exosomes after 60-70%growth for 48h.MVs were extracted and purified by ultracentrifugation.The morphology of MVs was detected by transmission electron microscope(TEM),particle size distribution was measured by nanoparticle tracking analysis(NTA),and the surface markers,CD9,CD63,CD81,TSG101 and Alix were detected by Western blot.Newborn rats were randomly divided into four groups:(1)Normal control group(NS+NS):NS was injected through amniotic on E20.5 and pups received NS on PN7.(2)normal treatment group(NS+MVs):NS was injected through amniotic on E20.5 and pups received MVs on PN7.(3)IA-LPS BPD model group(LPS+NS):LPS was injected through amniotic on E20.5 and pups received NS on PN7.(4)MVs treatment group(LPS+MVs):LPS was injected through amniotic on E20.5 and pups received MVs on PN7.Firstly,a dose-dependent assay was performed in LPS+NS group.Doses ranged from 0 to 80 μg/pup.Lung were harvested on PN14 to compare the MLI among the four groups and evaluate effective dose.Furthermore,MVs were pre-labeled by DiO and intratracheally administrated on PN7 in LPS+MVs group.Lungs were harvested at 0,12,24,48,72 and 96 h after MVs administration,to observe the duration of MVs in lung.Finally,to explore the therapeutic effect of MVs in IA-LPS BPD model,pups’lungs were harvested at PN14.Lung development,weight gain,small vessels,lung function and RVI among the four groups were compaired.RESULTS:1.MVs showed a double membrane structure and the diameter ranged from 150 to 900 nm,with the main peak at 255 nm.WB confirmed the expression of MVs markers,CD9,CD63,CD81,TSG101,and Alix.2.Dose-dependent assay show that the effective dose was 20μg/pup,and there was no significant difference in MLI among 20,40,or 80μg/pup of MVs.3.The DiO green fluorescence gradually increased and reached a maximum at 48 hours post-administration,then gradually decreased and finally disappeared after 96 hours.4.Compared with NS+NS group,the MLI increased and RAC decreased in LPS+NS group.After MVs treatment,both of MLI and RAC returned to the normal level in LPS+MVs group.6.MVs did not normalize the aberrant loss of small vessels caused by LPS.7.LR increased and Cdyn decreased in LPS+NS group,as compared with NS+NS group.MV treatment reversed the increased LR and decreased Cdyn.8.Increased RVH induced by LPS was also reversed by MVs.CONCLUSION:1.MVs derived from hUCMSCs successfully extracted by ultracentrifugation.2.The effective dose of MVs was 20 μg/pup.3.MVs gradually increased and reached a maximum at 48 hours,then gradually decreased and finally disappeared after 96 hours.4.MVs treatment improved alveolarization,weight gain and lung function,decresed pulmonary hypertension,but did not restored small vessels.Part Ⅲ Mechanism of MVs in the Treatment of IA-LPS BPD ModelOBJECTIVE:To observe the main target cells of MVs in lung,to explore the molecular mechanism of MVs in the treatment of IA-LPS BPD model.METHODS:MVs were pre-labeled with DiO green fluorescence in vitro.Co-localization of DiO green fluorescence with various lung cell markers were examined by immunofluorescence staining at 48 hours after MVs administration.The number of SP-C,Iba-1 positive cells in the frozen sections of P14 lung tissue in different groups were detected by immunofluorescence.The level of IL-6 and IL-10 in lung homogenate were detected by ELISA.MLE-12 cells were used for in vitro experiments,which were divided into four group:(1)Normal control group(PBS+PBS);(2)normal treatment group(PBS+MVs);(3)IA-LPS BPD model group(LPS+PBS);(4)MVs treatment group(LPS+MVs).The proliferation of MLE-12 cells was detected by Ki-67 immunofluorescence and CCK-8.The apoptosis of MLE-12 cells was measured by annexin V flow cytometry in the four groups.The expression of pulmonary surfactant(SP-A1,SP-B,SP-C and SP-D)in lung homogenate were detected by western blot.Single-cell sequencing were performed between LPS+NS group and LPS+MVs group,and top 10 functional pathways were analyzed using KEGG pathway enrichment analysis.The expression of PTEN,p-AKT/AKT,MAPKin lung homogenate were detected by western blot.RESULTS:1.Co-localization of DiO green fluorescence with various lung cell markers showed that DiO green fluorescence most frequently co-localized with SP-C-positive AT2(18.2%)and was also identified with AQP-1-positive AT1(4.5%),CD31-positive endothelial cells(7.6%),Iba-1-positive alveolar macrophages(14.0%),α-smooth muscle actin-positive smooth muscle cells(2.4%),and NG-2-positive pericytes(2.4%).2.Compared with NS+NS group,SP-C(+)cells significantly decreased and macrophage infiltration increased in LPS+NS group,whereas MVs restored the number of SP-C(+)cells and decreased macrophage infiltration.3.IL-6 increased and IL-10 decreased in LPS+NS group as compared with NS+NS group.MVs treatment restored the expression of IL-6 and IL-10.4.In vitro experiment showed that Ki67(+)MLE-12 cells decreased in LPS+NS group as compared with NS+NS group,which was restored in LPS+MVs group.The CCK-8 assay results showed that MV treatment improved the LPS-induced decrease in MLE-12 cells’ survival.5.LPS significantly inhibited the expression of SP-A1,SP-B,SP-C and SP-D,but only SP-C restored by MVs treatment.6.MAPK pathway was one of the top 10 functional pathways.7.MVs inhibited the expression of PTEN and MAPK pathway proteins(p-p38,p-JNK and p-ERK),increased the expression of p-AKT.CONCLUSION:1.MVs were mainly uptaken by AT2 cells(18.2%)and macrophages(14.0%).2.MVs protected AT2 cells from IA-LPS by improving AT2 cells proliferation and restoring SP-C expression.3.MVs reduced lung inflammation induced by IA-LPS.4.MVs improve alveolarization and attenuate lung inflammation associated with the PTEN/AKT and the MAPK pathways... |
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