The Treatment And Initial Mechanisms Of Type ? Alveolar Epithelial Cells Differentiated From Human Umbilical Cord Mesenchymal Stem Cells In Vitro On Mice Pulmonary Fibrosis

PART ? INDUCTION OF HUMAN UMBILICAL CORD MESENCHYMAL STEM CELLS INTO TYPE ? ALVEOLAR EPITHELIAL CELLS IN VITROObjective: This study aimed at the directional differentiation of human umbilical cord mesenchymal stem cells(h UC-MSC)into type II alveolar epithelial cells(AEC2s)induced by a conditional medium with growth factors,so as to provide the foundation for further study on the therapeutic effects of h UC-MSCs-derived AEC2 s in pulmonary diseases.Methods: h UC-MSCs cultured in vitro were divided into a test group,in which cells were cultured in small airway epithelial cells growth basal medium for 2 days and then growth factors were added for the induction culture;and a control group,in which cells were cultured in DMEM/F12 culture medium containing 20% fetal bovine serum.14 days later,cellular morphology was observed and recorded with an inverted microscope.The protein level of pro-surfactant protein C(pro SP-C)was detected by Western blot,immumofluorescence method and flow cytometry,respectively.The content of surfactant protein C(SP-C)secreted by the cells in medium supernatant was detected by ELISA.The formation of lamellar body in cells was observed by a transmission electron microscope(TEM).Results: The results indicated that test group cells' shape transformed from long fusiform to polygon after the induction,while control group cells' shape changed little.Obvious protein level of pro SP-C was detectable in test group cells.Secreted SP-C was detectable in medium supernatant.Lamellar body formation was observed under TEM.The pro SP-C,SP-C secretion or lamellar body was not detectable in control group cells.Conclusion: It concluded that in vitro directional induction culture promoted the differentiation of h UC-MSCs into AEC2 s.Hopefully a large scale of h UC-MSCs-derived AEC2 s could be obtained through this method.PART II THE EFFECT AND MECHANISMS OF ALVEOLAR EPITHELIAL CELLS DERIVED FROM HUMAN UMBILICAL CORD MESENCHYMAL STEM CELLS ON PULMONARY FIBROSIS IN MICEObjective: this study aimed to establish the pulmonary fibrosis(PF)model of pulmonary fibrosis induced by Bleomycin(BLM),and to observe the therapeutic effect of induced type II alveolar epithelial cells(h UC-MSCs-derived AEC2s)on pulmonary fibrosis model mice,and discuss the possible mechanism.Methods: C57BL/6 mice were divided into blank control group,safety control group,pulmonary fibrosis model group and treatment group,8 mice in each group.10% chloral hydrate(3ml/kg)was for anesthesia before tracheal intubation.Bleomycin(3mg/kg)was injected into the pulmonary fibrosis model group and the treatment group,and normal saline(50?l)was injected into the blank control group and the safety control group.After 7d,the same method was used for anesthesia and endotracheal intubation.The h UC-MSCs-derived AEC2 s cell suspension were dripped into the treatment group and the safety control group,and the blank control group and the pulmonary fibrosis model group got the normal saline(40?l).The mice were killed and specimens were collected at 21 d.Statistical survival rate and Pathological changes of lung tissue were observed.Changes of collagen content in lung tissue and expression of related fibrotic proteins were analyzed.h UC-MSCs-derived AEC2 s and A549 were co cultured to explore the effect of h UC-MSCs-derived AEC2 s on A549.Results: In the pulmonary fibrosis model group,Compared with the blank control group,the H.E staining showed the thickening of the alveolar septum,and Masson stainingthe showed the increase of the fibrous cord.the increase of the collagen content and the expression of a-SMA and vimentin in the lung was observed,indicating that the mouse model of bleomycin was constructed successfully.In the treatment group,increased the survival rate and lung histopathology showing that the thickening of the pulmonary septum was improved.inflammatory cell aggregation in the lungs decreased.Collagen content decreased significantly,and the expression of a-SMA and vimentin decreased.The experiments in vitro showed that h UC-MSCs-derived AEC2 s could promote the proliferation of A549.Conclusion: h UC-MSCs-derived AEC2 s can alleviate the pulmonary fibrosis induced by bleomycin in mice,and may promote the proliferation of AEC2 s in mice with pulmonary fibrosis.