| Background The survival rate of extremely premature infants/extremely low birth weight infants has increased dramatically as our country’s neonatal treatment has improved,and the incidence of bronchopulmonary dysplasia(BPD)is increasing yearly.BPD is the most prevalent respiratory problem of preterm infants,and the lower the birth weight and younger the gestational age,the greater the morbidity and death.In the future,the children may suffer from respiratory and neurological system sequelae to varying degrees,posing a significant burden on the family and society.The etiology and pathogenesis of BPD are yet unknown.Traditional therapies such as glucocorticoids,caffeine citrate,and bronchodilators ease symptoms but do not encourage alveolar growth and development.Mesenchymal stem cell treatment has emerged as a promising supplementary therapy for BPD.Mesenchymal stem cells(MSCs)from umbilical cord blood have been demonstrated to be safe and effective in treating BPD,although they do not change the course of the condition in phase Ⅰ and Ⅱ clinical trials.This may be the case since cord blood mostly contains hematopoietic stem cells,which are employed to treat immunological and hematologic illnesses.Clinical specimens abandoned after prenatal diagnosis or cesarean section are the source of amniotic fluid MSCs.They comprise more primitive cells from the fetus’s many tissues and organs.They are especially well suited for autologous cell transplantation in newborn disorders due to their high ability for proliferation and differentiation.However,more research is needed to determine whether MSCs from amniotic fluid are beneficial for treating BPD.Recent research has revealed that MSCs primarily function biologically through paracrine effects,including as anti-inflammatory,immunological modulation,and angiogenesis stimulation.By transporting a range of bioactive substances from their parent stem cells,extracellular vesicles(EVs)produced from stem cells perform a biological function that is similar to that of those stem cells.MSC-EVs have the benefits of adaptable use,minimal immunogenicity,and simple storage and transportation,which can reduce the danger of tumorigenesis from surviving cells and the negative effects of rapid cell death after direct transplantation of MSCs.Ultracentrifugation is the traditional technique for separating EVs;however,it takes a long time and requires heavy equipment.The polyethylene glycol precipitation method is another common EV enrichment technique that is appropriate for clinical use due to its low cost and straightforward operation.However,the physical and biological features of MSC-EVs in amniotic fluid derived from these two approaches have not been examined and compared in research.Objective The current research aimed to examine the therapeutic effects of amniotic fluid MSCs for the treatment of hyperoxia-induced BPD model by isolating MSCs from amniotic fluid and optimizing the extraction of EVs.To provide a more efficient cell-free treatment technique for children with BPD.Methods1.MSCs were isolated,cultured and identified from mid-pregnancy amniotic fluid,including observation of morphological properties of MSCs cultured in vitro,flow cytometry detection of the expression of specific surface markers of the obtained AFMSCs,and in vitro induced differentiation into osteoblasts,adipocytes and chondrocytes.2.To compare the therapeutic effects of intratracheal administration of MSCs from different sources(AF-and BM-MSCs)interfering with hyperoxia-induced BPD rat model;including the establishment of an animal model of BPD and analysis of lung histology,inflammatory cell levels,cell mortality and vascular neogenesis.3.To compare the efficiency and biological properties of MSC-EVs from amniotic fluid extracted by different methods(UC-and PEG-);including observation of the morphology of EVs under electron microscopy,analysis of particle size distribution and number,RNA level and protein expression,and comparison of the biological effects of both EVs on target cells by lipopolysaccharide(LPS)-induced inflammation model of THP-1 cells.Results1.The AF-MSCs were successfully isolated and cultured.2.The rat model of BPD was successfully constructed.MSCs were safe for intratracheal administration,no experimental animal death,and no abnormalities in other histopathology of various organs;and MSCs could inhibit alveolar dilation,reduce inflammatory factors,induce angiogenesis and reduce cell mortality.AF-MSCs have biological effects similar to those of BM-MSCs.3.There was no significant difference in morphology,size and positive rate of specific markers between PEG-EVs and UC-EVs,but PEG-EVs had more particles,proteins and RNA than UC-EVs.In the LPS-induced THP-1 inflammation model,MSC-EVs did not result in changes in protein expression but inhibited the LPS-induced increase in cytokine secretion;UCE-EVs inhibited TNF-α more,whereas PEG-EVs inhibited IL-10 more.Conclusion This study showed that endotracheal implantation of mesenchymal stem cells in rats with hyperoxia-induced bronchopulmonary dysplasia was both safe and effective.Furthermore,immunosuppression and angiogenesis stimulation are key components of the therapeutic approach.The potential of amniotic fluid MSCs to encourage angiogenesis was more significant than that of bone marrow MSCs.The PEG precipitation approach can generate and use extracellular vesicles with comparable biological capabilities in clinical therapy. |
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