Preventioprevention Of Bone Marrow Mesenchymal Stem Cell Transplantation Against Acute Lung Injury In Rabbits And Construction And Sequence Of Eukaryotic Expression Vector Carrying Human Angiopoietin-1

DESIGN, TIME AND SETTING: The randomized controlled animal studywas performed at the Central Laboratory of Tangdu Hospital from October 2007to January 2008.OBJECTIVE: To investigate the prevention of endotoxin-induced acute lunginjury in rabbit by BMSCs,and construct eukaryotic expression vectorpcDNA3.1+-hAng1 for human Ang1 (hAng1).METHODS: Rabbit BMSCs were isolated and cultured by the Ficoll method.At the third passage, BMSCs were harvested for use.In the acute lung injury andcell transplantation groups, endotoxin was infused into the trachea to establishmodels of acute lung injury/ARDS. Thirty minutes following modelestablishment, 2mL BMSC suspension (1×105) was infused into the right jugularvein in the cell transplantation group. An equal volume of saline was injectedinto the saline control and acute lung injury groups. The fragment of hAng1gene was amplified by nested polymerase chain reaction, the hAng-1 gene andpcDNA3.1+vector were digested respectively by HindⅢand XhoⅠ.The production digested was connected by T4 ligase and constructed the eukaryoticexpression vector pcDNA3.1+-hAng1.RESULTSRESULTS: ?The increase in wet to dry weight ratio indicated the existence ofpulmonary edema. The increase in neutrophilic granulocyte number suggestedsevere inflammatory reaction. The increased protein content showed the damageto lung alveolar-capillary membrane barrier. Following 48 hours oftransplantation, neutrophilic granulocyte number and protein content inbronchoalveolar lavage fluid was significantly increased (P <0.01), and wet todry weight ratio was significantly increased (P <0.01) in the acute lung injurygroup compared with the saline control group. Compared with the acute lunginjury group,neutrophilic granulocyte number and protein content wassignificantly diminished (P <0.01), and wet to dry weight ratio was significantlydiminished (P <0.01) in bronchoalveolar lavage fluid in the cell transplantationgroup. Hematoxylin-eosin staining suggested that pulmonary alveoli was normalin the saline control group, presented typical acute lung injury in the acute lunginjury group, and the pathological changes were mild in the cell transplantationgroup.?After the eukaryotic expression vector for hAng1, namely pcDNA3.1+-hAng1 was digested with HindⅢand XhoⅠ,electrophoresis revealed 1.5-kbfragment for hAng1 gene and 5.4kb fragment for pcDNA3.1+ vector. Thesequence analysis of eukaryotic expression vector was performed in BeijingAuGCT Biotechnology Co.,Ltd, results proved the resultant sequence wascorrect, which was in consistent with AY121504 in GenBank.CONCLUSION: BMSC transplantation can significantly reduce endotoxininducedacute lung injury.And The eukaryotic expression vector pcDNA3.1+-hAng1 can be constructed with gene recombination and digestion of HindIII andXhoI, and it will supply a foundation for studying its function and activity.