Effect Of Down-regulate Of Hepatocyte Growth Factor Secreted By Human Placenta-derived Mesenchymal Stem Cells On Lung Vascular Endothelial Repair In Acute Lung Injury

Objective To construct human placental MSC cell line with down-expression of hepatocyte growth factor(HGF)after isolating and culturing human mesenchymal stem cells(MSC),and to act on the cells and animal models of acute lung injury(ALI)about vascular endothelial injury,to explore the effect of down-regulation of HGF secreted by human placental mesenchymal stem cells on vascular endothelial repair of acute lung injury,and improve the damage repair mechanism of human placenta MSC.Methods(1)(1)Human placenta MSC was isolated by enzymatic digestion.(2)The morphology and growth of the cells were observed under light microscope.The cells were induced to differentiate into fat,bone and chondrocytes under specific culture conditions.The cell phenotype was detected by flow cytometry,and the isolated cells were identified to have the general characteristics of MSC.(3)Compare the secretion level of HGF in human placenta,bone marrow and adipose-derived mesenchymal stem cells.(2)(1)Detecting the effect of human placenta MSC on pulmonary vascular endothelial permeability and HGF secretion level: constructing human placental MSC and human pulmonary microvascular endothelial cell(HPMVEC)in Transwell co-culture system,setting normal control group(Control group)),ALI model group(LPS group),human placenta MSC group(LPS+MSC group).Transwell co-culture system has upper and lower two culture chambers,respectively,in each group of Transwell upper chamber culture HPMVEC,LPS group and LPS+MSC The HPMVEC in the upper chamber was treated with 200 ng/mL lipopolysaccharide(LPS)for 6 h,and the LPS+MSC group was implanted with placentalMSC in the lower chamber.The permeability test was used to determine the permeability change of HPMVEC,and the concentration of HGF in the lower layer culture solution was determined by Enzyme-linked-immunosorbent assay(ELISA).(2)Construction and identification of human placental MSCs with low expression of HGF: Human placental MSCs were transfected with the lentivirus of interfering HGF(SiHGF)and normal control(SiNC),respectively,to construct the human placenta MSC cell line(MSC-SiHGF)of low HGF and the human placenta MSC cell line(MSC-SiNC)of interfered with the control sequence,and the transfection efficiency of SiHGF lentivirus and SiNC lentivirus was observed under a fluorescence microscope.Western blot(WB)and ELISA were used to detect the expression and secretion of HGF in MSC-SiNC and MSC-SiHGF cell lines,and the HGF gene interference effect of human placenta MSC was verified.(3)To detect the effect of HGF down-regulation of human placenta mesenchymal stem cells on the permeability of pulmonary vascular endothelial cells: setting ALI model group(LPS group),human placenta MSC group(LPS+MSC group),transfection control lentiviral human placenta MSC group(LPS+MSC-SiNC group),transfected HGF-interfering lentivirus human placenta MSC group(LPS+MSC-SiHGF group),respectively,in each group of Transwell upper chamber cultured HPMVEC was terated with LPS for 6 h,LPS+MSC group,LPS+ MSCs,MSC-SiNC,MSC-SiHGF cells were cultured in the lower chamber of MSC group,MSC-SiNC group and LPS+MSC-SiHGF group.Permeability assay was used to determine the permeability of pulmonary vascular endothelial cells after down-regulation of HGF in human placenta mesenchymal stem cells..(3)(1)Modeling and grouping: 36 healthy male C57bl/6 mice aged 6-8 weeks were randomly divided into 4 groups(n=9),namely: normal control group(NC group)and ALI model group(LPS group),human placenta MSC treatment group transfected with control lentivirus(MSC-SiNC group),human placenta MSC treatment group transfected with HGF interference lentivirus(MSC-SiHGF group).The NC group was intratracheally instilled with0.1 mL of normal saline.The ALI model group,the MSC-SiNC group and the MSC-SiHGF group were intraperitoneally instilled with 0.1 mL of 2.5 mg/kg LPS normal saline to establish an ALI mouse model.After 12 h of modeling,the NC group and the ALI model group were injected with 0.3 ml Dulbecco's minimum essential medium(DMEM),and the MSC-SiNC group and MSC-SiHGF group were injected 0.3 mL DMEM with 1×10^6 cells.(2)Specimen collection: 24 hours after injection of treated human placenta MSCs into the tail vein,mice in each group were sacrificed,and lung tissue and Bronchoalveolar lavage fluid(BALF)were collected.(3)Index determination: Calculate the wet/dry mass ratio(W/D)of lung tissue,detect the permeability of pulmonary vascular endothelium by Evans blue tail leak test,and determine the pro-inflammatory factor tumor necrosis factor-?(TNF-?),interleukin-1(IL-1),IL-6 and anti-inflammatory factor IL-10 in BALF by ELISA,detect expression of Vascular Endothelial-cadherin(VE-cadherin)by Western blot analysis,Hematoxylin Eosin(HE)staining was used to detect pathological changes in lung tissue.Results(1)Human placenta tissue cells isolated by enzymatic digestion showed long fusiform and spiral growth,strong proliferation ability,differentiation into fat,bone and chondrocytes,high expression of CD73,CD90 and CD105,low expression of CD14,CD34,CD45.The expression conforms to the antigenic phenotypic characteristics of typical MSCs and has tissue-specific specificity.It can secrete HGF at a higher level,and its secretion is significantly higher than that of human bone marrow and adipose-derived MSCs(P<0.05).(2)Compared with the Control group,the fluorescence intensity of the LPS group was significantly increased(P<0.05),and the HGF secretion level was not significantly different(P>0.05).Compared with the LPS group,the fluorescence intensity of the LPS+MSC group was significantly higher decreased(P<0.05),HGF secretion levels were significantly increased(P<0.05).The transfection efficiency of SiNC lentivirus and SiHGF lentivirus to human placenta MSC was 96.75% and 95.48%,respectively.Compared with the LPS group,the fluorescence intensity of the LPS+MSC group was significantly lower than that of theLPS group(P<0.05).There was no significant difference in the fluorescence intensity between the LPS+MSC group and the LPS+MSC-SiNC group(P>0.05).Compared with the LPS+MSC-SiNC group,the fluorescence intensity of the LPS+MSC-SiHGF group was significantly higher(P<0.05).(3)Compared with the NC group,the W/D,Evans blue leakage,pro-inflammatory factors TNF-?,IL-1,IL-6 expression in the ALI model group were significantly increased(P<0.05).The secretion of anti-inflammatory factor IL-10,the expression of VE-cadherin protein in lung tissue decreased(P<0.05),and the injury of lung tissue was severe.Compared with the ALI model group,the W/D,Evans blue leakage,proinflammatory factors TNF-?,IL-1,IL-6 expression in the MSC-SiNC group were significantly reduced(P<0.05).The secretion of anti-inflammatory factor IL-10 and the expression of VE-cadherin protein in lung tissue increased(P<0.05),and the pathological damage of lung tissue was significantly relieved.Compared with the MSC-SiNC group,the W/D,Evans blue leakage,proinflammatory factors TNF-?,IL-1,IL-6 expression in the MSC-SiHGF group were significantly increased(P<0.05).The secretion of anti-inflammatory factor IL-10,the expression of VE-cadherin protein in lung tissue decreased(P<0.05),and the pathological damage of lung tissue was severer.Conclusion: After the level of human placental mesenchymal stem cell paracrine hepatocyte growth factor is down-regulated,the effect of human placental mesenchymal stem cells on the repair of lung vascular endothelial cell injury in acute lung injury is relatively weak.