| Part I Establishment and Identification of Hypoxia-Preconditioned h U-MSCs culture systemOBJECTIVE: To establish a hypoxic pretreated human umbilical cord mesenchymal stem cell model and explore the appropriate preconditioning conditions.METHODS: Passage 4-5 of h U-MSCs were cultured till the cell were grown to 70-80% confluence,the original medium were removed,then 10 ml of hypoxia induction medium containing different concentrations of deferoxamine(DFO)was replaced for 48 h.CCK8 was used to detect the proliferation of h U-MSCs treated with different concentrations of DFO under normal or hydrogen peroxide stimulation;the cell protein was extracted from h U-MSCs treated with different concentrations of DFO,and the expression of HIF-1α was detected by Western Blot.The morphology of hypoxic pretreated stem cells(HPMSC)was recorded under a light microscope;CD45,HLA-DR,CD73,CD105 surface markers were detected by flow cytometry;HPMSC was induced to differentiate for 14 days using a specific external induction culture system.Alizarin red S staining was used to observe the osteogenic differentiation ability,and oil red staining was used to observe the adipogenic differentiation ability.RESULTS: 1.DFO-induced hypoxia preconditioned h U-MSCs culture system was successfully established.2.The results of CCK8 test indicated that DFO pretreatment improved the proliferation ability of h U-MSCs under oxidative stress injury,cell proliferation enhanced significantly especially in the 50μM and 100μM groups.Western Blot showed that the expression of HIF-1α in 100μM,150μM and 200μM DFO groups were significantly improved than that in the control group(1.78±0.13 vs.2.13±0.27 vs 2.50±0.38,p<0.05).3.The expression rates of HPMSC surface markers CD45 and HLA-DR were 1.39% and 1.93%,respectively,which were negative;the expression rates of surface markers CD73 and CD105 were 98.8% and 99.0%,respectively,which were positive.HPMSCs had the ability to differentiate into osteogenic and adipose tissues after 14 days of induction.CONCLUSION: The HPMSC culture system was successfully established,cells culturing with 100 μM DFO for 48 h was selected as a appropriate hypoxic preconditioning strategy for h U-MSCs.The morphology,surface markers,and differentiation ability of HPMSC was consistent with the identification of human umbilical cord mesenchymal stem cells.Part II Efficacy of Hypoxia-Preconditioned h U-MSCs on A BPD Rat ModelOBJECTIVE: To establish a rat model of h U-MSCs transplantation for the treatment of BPD,observe the therapeutic effect of hypoxia-preconditioned human umbilical cord mesenchymal stem cells(HPMSC)on the BPD model.METHODS: Neonatal SD rats were divided into 4 groups: normoxia control group(RA+NS),BPD group(BPD+NS),NMSC treatment group(BPD+NMSC),HPMSC treatment group(BPD+HPMSC).On the PN7,airway perfusion of saline or MSC was administered by group.We used PKH-26-labeled NMSC and HPMSC to trace in the lung tissue of BPD rats,and observed their survival on the 1st,3rd,5th,and 7th days after transplantation.In addition,rat samples from each group were collected on the 14 th day,and the survival curve,pathological structure of lung tissue,pulmonary hypertension-related indicators(RV/LV+S,RV/BW),pulmonary function(LR/Cydn),lung vascular immunofluorescence and vascular marker protein quantification were used to evaluate the therapeutic effect of HPMSCs on BPD neonatal rats.RESULTS: 1.A h U-MSCs airway transplantation model for BPD was successfully constructed.2.After the 3rd,5th,and 7th days after cell transplantation,the number of stem cells in the HPMSCs group was significantly higher than that in the NMSCs group(17.60±3.17 vs.29.60±1.69,12.20±0.80 vs.21.20±1.50,12.00±2.07 vs 18.80±1.91).3.The MLI in the HPMSC treatment group was significantly lower than that in the NMSC group(59.44±2.06 vs.75.75±1.95 μm,p<0.001),and the right ventricular index in the HPMSC treatment group was significantly lower than that in the NMSC group(0.28±0.01 vs.0.35±0.01,p<0.01).Compared with the NMSC treatment group,the ratio of LR/Cydn in the HPMSC treatment group was more significant than that in the NMSC treatment group(23.39±2.52 vs.36.44±3.70,p<0.05).4.The v WF immunofluorescence of lung tissue in the HPMSC treatment group showed more complete vascular structure,and the ratio of the vascular area was significantly improved than that in the NMSC treatment group(4.51±0.80 vs.2.60±0.90,p<0.001).Western Blot results showed that the expressions of v WF,CD31,and VEGFA in HPMSC group were all higher than those in NMSC group.CONCLUSION: HPMSC retained more than NMSC in BPD lung tissue,and HPMSC transplantation had better therapeutic effect on alveolar structure,pulmonary hypertension,pulmonary function and pulmonary vascular repair than NMSC transplantation.Part III The Mechanism of Hypoxia-Preconditioned Mesenchymal Stem Cells in the treatment of BPD Rat Models via HIF-1α signalingOBJECTIVE: To investigate the protective effect of hypoxia preconditioning on h U-MSCs in vitro,and speculate the molecular mechanism of HPMSC in the treatment of BPD rat models.Methods: h U-MSCs were divided into four groups in vitro: control2O2),HPMSC oxidative damage group(HPMSC+ H2O2),and HIF-1α inhibitor group(HPMSC+ H +Oltipraz).Cells were treated with 500 μM H2 with or without 10 μM Oltipraz for 24 hours by groups,and the morphology was recorded under a light microscope.Flow cytometry was used to detect the apoptosis rate,cell cycle,and ROS generation in each group.Western Blot was used to detect the expression levels of caspase3 and HIF-1α in each group.VEGF concentrations in culture medium of NMSC and HPMSC were detected by ELISA;human umbilical vein endothelial cells(HUVEC)tube formation assay were divided into 4 groups: control group(HUVEC),oxidative damage group(HUVEC+ H NMSC),co-culture group(HUVEC+ HR2ROR2R+NMSC),HPMSC co-culture group(HUVEC+2 25/well,and NMSC or HPMSC(3×104+HPMSC),the HUVEC with or without oxidative damage were seeded on Matrigel of 24-well plate at/well)were seeded in the transwell chamber,then the angiogenesis of each group was evaluated under microscope after co-culturing for 5 hours,and the apoptosis rate of HUVEC was detected by flow cytometry.The m RNAs in the lung tissues of the control group and the BPD model group were sequenced,and the differentially expressed m RNA target genes were subjected to KEGG pathway enrichment analysis.Then the neonatal rats were divided into 5 groups: control group(RA+NS),BPD group(BPD+NS),NMSC treatment group(BPD+NMSC),HPMSC treatment group(BPD+HPMSC)and PI3K/AKT inhibitor group(BPD+HPMSC+LY294002),the lung tissues of each group were collected on the PN14,Western Blot was used to detect the expression of p-PI3K/PI3 K and p-AKT/AKT.RESULTS: 1.The apoptosis rate of HPMSCwas significantly lower2O2 was higher than that in the NMSC group,and the generation of ROS was significantly decreased than than that of NMSC under oxidative stress(8.42%±1.44% vs.22.36%±2.33%,p<0.001),and the expression of caspase3 was significantly reduced(0.46±0.03 vs.0.88±0.03,p<0.001),the expression of HIF-1α was significantly increased(0.85±0.13 vs.0.40±0.09,p<0.05),the proportion of cells entering the S phase in the HPMSC group after oxidative that in the NMSC group(18318 ± 2857 vs.31890 ± 5153,p < 0.001),while the HIF-1α inhibitor Oltipraz reversed these effects.2.The VEGF concentration of HPMSC culture medium was significantly higher than that of NMSC(28.20±38.09 vs.24.35±1.15 pg/ml,p<0.001).Compared with the NMSC co-culture group,the number of rings(6.60±1.17 vs.3.20±0.73,p<0.05)and the length of tubes(11495±914.4 vs.9072±661.6 mm,p<0.05)in the HPMSC co-culture group were significantly increased.RNA transcriptome analysis showed that compared with control group,there were 765 up-regulated genes and 473down-regulated genes in rat lung tissue in the BPD group(p<0.05),PI3K/AKT was one of the 20 most significantly enriched KEGG pathways,and the HPMSC treatment group had significantly higher protein phosphorylation expression levels of PI3K(1.35 ± 0.12 vs 0.97 ± 0.05,p <0.001)and AKT(1.37 ± 0.07 vs 0.89 ± 0.07,p <0.001)than that in NMSC treatment group.CONCLUSION: HIF-1α protected HPMSC against oxidative stress injury,enhanced the secretion of VEGF.HPMSC promoted angiogenesis and reduced cell apoptosis of HUVEC in oxidatively damage in vitro.HPMSC transplantation affected the changes of PI3K/AKT signaling pathway in lung tissue of BPD rats. |
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