Preliminary Study Of Human Amniotic Epithelial Cells Relieve Acute Lung Injury Induced By Lipopolysaccharide In Rats

ObjectiveClinically,acute lung injury/acute respiratory distress syndrome could be caused by infection,inhalation injury,severe burns,etc.And the bacterial infection was a major factor.To seperate and culture human Amniotic Epithelial Cells(hAECs);To identify the phenotype of hAECs.To build a model of acute lung injury induced by lipopolysaccharide,To intervene the acute lung injury model by hAECs;To observe the influence of hAECs on lipopolysaccharide induced acute lung injury about pathology,lung wet/dry weight,protein concentration in Bronchoalveolar Lavage Fluid and inflammatory factors(TNF-α、IL-6、IL-10).Methods1.Culture,digestion,freezing and identification of human amniotic epithelial cellPrimary human amniotic epithelial cells were isolated by collagenase/trypsin digestion;Human amniotic epithelial cells were digested by trypsin repeatedly when cells grew to 80%;Human amniotic epithelial cells were frozen by gradient cooling.hAECs morphology was observed under microscope;hAECs were detected by positive cytokeratin 19 immunofluorescence staining.Passage 2 hAECs were used to identify surface antigen,including CD34、CD45、CD90、CD105、HLA-DR、SSEA-4.2.The influence of hAECs on lipopolysaccharide induced acute lung injuryALI rat models were established by intratracheal instillation of LPS.Rats weighed 200±20g were randomly divided into 3 groups:fake injury group(n=12):200 μ L PBS were injected via tail vein after intratracheal instillation of 100 u L normal saline,LPS group(n=20):200 μL PBS were injected via tail vein after intratracheal instillation of 100 u L LPS,intervention group(n=20):5×105 cells in 200 μL PBS were injected via tail vein after intratracheal instillation of 100 μ L LPS.All rats in each group were sacrificed for lung tissue sample on 6,24,48,72h after intratracheal instillation.After observation of lung,rats’ lungs were divided into pieces;The wet/dry weight were assyed by a routing method;The lung hietopathological were observed under microscope by HE staining;The protein levels of BALF were determined by BCA method;The levels of TNF-α,IL-6,IL-10 in tissue homogenate supernatant were determined by ELISA method.Results1.Culture,digestion,freezing and identification of human amniotic epithelial cell:Human amniotic epithelial cells have flat cobblestone appearance under inverted microscope,P2 cells were in good conditon.CK-19 immunofluorescence staining was positive.Detection of stem cell surface molecule marker by the flow cytometry showed the expression of SSEA-4,CD90 on hAECs were positive,and the expression of CD105,CD34,CD45,HLA-DR on hAECs were negative.2.The intervention effect of hAECs on LPS induced ALI:The macroscopic observation and histopathology:The lung of fake injury group had normal color(smooth and ruddy),no bleeder and edema;The lung of LPS injuriy group had volume expansion edema,visible bleeder with the color of dark red;Compared with LPS injuriy group,The damage of lung of hAECs intervention group had improved.The lung tissues of fake injury group were found clear alveolar structure,there was no interstitial thickening,obvious exudation,edema or inflammation infiltration;The lung tissue of LPS injury group appeared interstitial thickening,exudate in alveolar space.There were occasional "horseshoe shape"inflammatory cells in alveolar.The damage degree of lungs increased with time prolonging;The lung injury degree of hAECs intervention group have improved at 48,72h,Interstitial thickness was reduced,degree of atrophied alveolar improved.But compared with fake injury group,lung injury of hAECs intervention group still existed.Wet dry weight ratio:Compared with the fake injury group,wet dry weight ratio of LPS injury group increased significantly at each time point(P<0.01);Compared with the injury group,wet dry weight ratio of hAECs intervention group reduced significantly at each time point(P<0.01).The protein concentrations of BALF:Compared with fake injury group,the protein concentration of BALF in LPS injury group increased significantly at each time point(P<0.05),the protein concentrations of BALF in hAECs intervention group increased significantly at 24,48,72 h(P<0.05);Compared with the LPS injury group,the protein concentrations of BALF in hAECs intervention group reduced at each time point than group and there was significant difference at 6,72 h(P<0.05).Inflammatory factors in tissue homogenate supernatant:Compared with fake injury group,the concentration of TNF-α,IL-6 and IL-10 in LPS injury group increased significantly at each point in time(P<0.05),the concentration of TNF-α,IL-6 and IL-10 in hAECs intervention group increased significantly at each time point(P<0.05);Compared with the LPS injury group,the concentration of TNF-α,IL-6 in hAECs intervention group decreased significantly at each time point(P<0.05),the concentration of IL-10 in hAECs intervention group increased significantly at each time point(P<0.05);The concentration of TNF-α,IL-6 and IL-10 in fake injury group held steady.The concentration peak of TNF-α,IL-6 in the LPS injury group appeared in 6 h,then reduced with time prolonging,the concentration peak of IL-10 in LPS injury group appeared in 24 h,then reduced with time prolonging.The concentration of TNF-α,IL-6 and IL-10 in hAECs intervention group changed the same with LPS injury group.ConclusionAmniotic epithelial cells(AECs)was obtained and purified by collagenase/trypsin and repeated trypsin digestion.The obtained cells confirmed human amniotic epithelial cells by morphology,CK19 immunofluorescence staining and Surface antigen identification by flow cytometry.Cells had high purity and good growth.This provided the guarantee for the establishment of the library of human amniotic cells and human amnion application research.HAECs can relieve inflammation and tissue edema of ALI caused by LPS.HAECs had certain therapeutic effects on ALI caused by LPS.