Construction Of Ang1 Adenovirus Vector And Its Effect On Inflammation Resolution Of ALI Induced By Lipopolysaccharide

Objective To construct the recombinant adenovirus carrying human Angl (hAngl) in order to explore the effect of Angl on the inflammation resolution of acute lung injury induced by lipopolysaccharide. Methods The cDNA of hAngl was cloned into adenovirus shuttle vector pDC315-EGFP by standard procedure to obtain the recombinant adenoviral plasmid pDC315-EGFP-Ang1. Genomic plasmid pBHG lox AE1,3 Cre and pDC315-EGFP-Angl were cotransfected into 293 cells by using site-specific recombinant system Cre/Lox. The Ad-EGFP-hAngl recombinant adenovirus vector bearing hAngl was then amplified in 293 cells and purified by cesium chloride. The hAngl protein in the transfected HEK 293 cells and the medium of transfented HEK293 cells were detected by western blot and Elisa respectively. Results The cloned hAngl cDNA was confirmed correction by sequencing and enzyme cutting. The titer of Ad-GFP-hAngl was 1.50×1012 PFU·mL-1. The hAngl protein was detected in the Ad-GFP-hAngl transfected HEK 293 cells and time-dependent expressed in the medium of Ad-GFP-hAngl transfented HEK293 cells. Conclusion The recombinant adenovirus carrying human hAngl was successfully constructed by this method and ex vivo assays suggested the expression of human Angl with high efficiency and specificity.Objective To establish a model of acute lung injury (AL1) in mice by LPS intratracheal instillation for studying the inflammation process. Methods Twenty male SPF BALB/c mice weighing 20-25g were randomly divided into two groups:Normal saline group (Control group) and lipopolysaccharide group(LPS group) with 10 mice in each group. Control group and LPS group received intratracheal injection of 2 mL·kg-1 normal saline or 3 mg·kg-1 popolysaccharide. All the mice were killed at 24 hours after injection. The wet-to-dry ratio and pathological change of lung were examined. In addition, the Leukocyte, PMN count and the total protein in BALwere analyzed with the incidence of mortality of each group observed. Results 3 mice died in the LPS group except one that was mis-intubated into the esophagus while no mouse died in the control group (P=0.001). The pathologic score of lung, the leukocyte, PMN and total protein in the BAL of the LPS group were significantly higher with the pathologic score of lung lower than that of the control group. Conclusion It is successful to imitate the development of ALI by injection of lipopolysaccharide. This is a suitable model for the following study of ALI in mice.Objective:To investigate the effect of angiopoietinl on the inflammation resolution in acute lung injury. Methods:Animals were randomly assigned to GFP/saline group (control group), Ad-GFP/LPS group (GFP group) and Ad-GFP-Ang1/LPS group (Angl group).3-4 mice in GFP or Angl group were killed at 4 h,12 h,24 h,48 h,72 h and 96 h respectively. The cells in the BAL were counted and differentiated. The inflammation resolution indices between the GFP group and the Angl group were analyzed. Apoptotic polymorphonuclear leucocyte and its phagocytosis by macrophage cells were determined by fluorescent activated cell sorter. The angiopoietin-1 in serum and the Granulocyte macrophage colony-stimulating factor levels in the BAL were assayed following the manufacturer's instructions. Results:ALI for 4 h,12 h,24 h,48 h,72 h and 96 h induced evident leukocytes infiltration into the lung tissues, with a maximal infiltration (13.5×106) at 48 h. Pretreatment with adenovirus-GFP-angiopoietinl markedly increased serum Angl, reduced the leukocytes and polymorphonuclear cells infiltration and shortened the time interval from the maximal PMN point to the 50% reduction point (Ri) in inflammation resolution compared with the GFP group. At the same time, pretreatment with Ad-GFP significantly promoted the apoptosis of PMN cells and its clearance by macrophage cells. GMCSF levels in the BAL were augmentated without altering the time course in Angl group. Conclusions:This study provides evidence, for the first time, that angiopoietin-1 pretreatment promotes inflammation resolution of endotoxin-induced ALI in mice. 1. It is successful to imitate the development of ALI by intratracheal injection of lipopolysaccharide at the dose of 3 mg·kg-1. This is a suitable model for the following study of ALI in mice.2. The PMN infiltration into the lung tissue in the ALI mice reached maximum at 48 hours after intratracheal injection of lipopolysaccharide at dose of 3 mg·kg-1. Then the inflammation resoluted and the PMN number in the BAL gradually reduced. Pretreatment with Ad-GFP-Angl significantly reduced the PMN infiltration and protein exudation by stabilizing the endothelium cells.3. Pretreatment with Ad-GFP-Angl promoted the inflammation resolution of ALI. The mechanism is that the Angl accelerated the apoptosis of PMN in the tissues and the phagocytosis of apoptotic PMN by macrophage cells.4. The endothelium cell takes part in the inflammation resolution.5. The endothelium cell is perhaps an important potential target in pretreatment and treatment of ALI.