| [Backgrounds and objective]Acute lung injury (ALI) is common in intensive care unit,which is has abruptly onset, rapidly progress, high mortality, poor prognosis, and complex mechanism, there are Presently no proven,effective pharmacological therapies to treat ALI and prevent its associated complications.Alveolar epithelial cell lost and apoptosis is a hallmark event in the pathogenesis of ALI. Alveolar epithelial cell apoptosis is considered as the key step in trigerring, development and prognosis of ALI. Alveolar epithelial cell apoptosis contributes to the onset and progression of ALI, which plays the important role in the development of ALI.Therefore, alveolar epithelial cell apoptosis has been suggested as an inmportant potential therapeutic target. It urges some more effective novel therapeutic strategies to reduce the severity of lung injury and derease mortality.Bone marrow mesenchymal stem cells (BMMSC) are multipotent stem cells capable of self-renewal and differentiating into a variety of lineages under suitable conditions. BMMSC are now extensively applied in the fields of regenerative medicine, gene therapy and tissue repair. Therefore, BMMSC are thought to be an idea candidate seeding cell for cell therapy. This novel cell therapy represents an excellent clinical implication prospect. Recent studies have indicated that the effect of BMMSC therapy on acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and chronic disorders such as lung fibrosis and chronic obstructive pulmonary disease (COPD).Based on these data we hypothesize that BMMSC therapy to treat ALI may be related with effects of anti-inflammation, anti-edma and anti-apoptosis. BMMSC may act through paracrine effects to interfere significantly with the pathophysiological processes of ALI.[Methods]1. The reseach methods of BMMSC growing biological characterristics in vitro(1)Isolation and culture of BMMSC were using the whole bone marrow adherence method in vitro.(2)BMMSC morphology was observed under microscope; BMMSC multiple differatiation was detected by specific direction induced differatiation culture medium;The number of cultured BMMSC was counted by hemacytometer under microscope.(3)BMMSC surface markers and cell cycles were detected by flow cytometry.2. The reseach methods for effect of BMMSC on anti-inflammation,anti-lung edma and anti-apopttosis in vivo(1)ALI mice models were established by intratracheal instillation of LPS. ALI mice were randomly divided into3groups (n=18/group):①control group:100μl PBS were injected via tail vein30min after intratracheal instillation of PBS;②injury group:100μl PBS were injected via tail vein30min after intratracheal instillation of LPS;③herapy group:5×106BMMSC in100μl PBS were injected via tail vein30min after intratracheal instillation of LPS.(2) Gustavo Matute-Bello lung histopathological injury score were evaluated under microscope by HE staining.(3)The level of arterial blood PaO2were assayed by blood gas analyzer.(4)BMMSC engraftment in injury lung were observed under flueomicroscope;(5)The ratio of wet to dry lung weight were assayed by a routing method.(6) The percentage of apoptotic alveolar epithelial was assayed by TUNEL.(7)The levels of MPO in lung tissue homogenizing supernatant and levels of IL-6,TNF-a and IL-10in BALF were determined by ELISA method.(8)The levels of protein in BALF were determined by the Coomassie blue dye binding (CBB) method.(9) The levels of PMN in BALF were counted by hemotology analyzer. (10)The levels of caspase3,bcl-2and bax expression were assayed by immunohistochemisty.(11)Alveolar type Ⅱ epithelial cells apoptosis were observed under electron microscopy.3. The reseach methods for effect of BMMSC on anti-apoptosis of alveolar type II epithilial cell mechanism in vitro(1) Human alveolar type Ⅱ epithelial cell (A549) and BMMSC were cocultured on cell-cell noncontact transwell system in vitro. Cell experiments grouping:(6cells/group,5×106/cell):①negative control group, PBS+A549;②LPS-induced injury group, LPS+A549;③BMMSC control group PBS+A549+BMMSC;④BMMSC intervene group, LPS+A549+BMMSC(2) The percentage of apoptotic A549was quantified by flow cytometry staining with annexin V/PI.(3) Caspase3,bcl-2and bax protein expression in A549was assayed by Western blotting.(4) A549apoptosis feature were identified under electron microscopy.(5) The content of KGF and HGF in coculture medium was measured by ELISA method.(6) KGF and HGF mRNA expression in BMMSC was assayed by fluorescent quantitative RT-PCR.[Results]1. BMMSC growing biological characteristics in vitro(1) The BMMSC of4-6weeks mice cultured in BMEM with5%-10%fetal bovine serum(FBS) and seeding density of5-8×10/L,which could harvest sufficient quantities, strong activity and rapid proliferation of BMMSC is an optimal cultured condition for get high amount, high purity and high activity BMMSC.(2) Under the optimal tissue culture conditions, BMMSC have the clonogenic capacity. The cell cycle of most of BMMS are at G0/G1. They are spindle-shaped, like fibroblast. They are CD14-,CD34-,CD45-and CD90+, CD105+ CD106+.They have plastic adherent and multidirectional differentiation capacity. All this features are fit for the MSC growing biology characters.2. The effect of BMMSC on anti-inflammation, anti-lung edma and anti-apopttosis in vivo(1) BMMSC can engraft into injury lung tissue, ameliate lung histopathological injury degree, and increase the level of arterial blood PaO2.(2) BMMSC can decrease the concentration of MPO in lung tissue,the quangtities of PMN,the levels of IL-6and TNF-a, increase the level of IL-10in LPS induced ALL(3) BMMSC can decrease the lung W/D ratio and the concentration of protein in BALF.(4) BMMSC can decrease the percentage of alveolar epithelial cell apoptosis, the level of pro-apoptosis protein caspase3and bax expression, and increase the level of anti-apoptosis protein bcl-2expression.3. The effect of BMMSC on anti-apoptosis of alveolar type Ⅱ epithelial cells in vitro(1) LPS can induce A549apoptosis in vitro.The optimal condition is lOμg/ml LPS induced A54924h.(2) LPS can increase the expression level of pro-apoptosis protein caspase3and bax, decrease the expression level of anti-apoptosis protein bcl-2.(3) BMMSC can increase A549the expression level of anti-apoptosis protein bcl-2,decrease the expression level of pro-apoptosis protein caspase3and bax.(4) Cocultured with A549, BMMSC can paracrine growth factor KGF and HGF, increase KGF and HGF mRN A expression.[Conclusions]3.4-6weeks mice BMMSC cultured in BMEM with5%-10%fetal bovine serum (FBS) and seeding density of5-8×107/L,which is an optimal cultured condition, express CD14-,CD34-,CD45-and CD90+,CD105+CD106+.They have plastic adherent and multidirectional differentiation capacity. All this features are fit for the mesenchymal stem cell growing biology characters.2. BMMSC can engraft into injury lung tissue, attenuate LPS induced ALI mouse lung histopathological injury degree, and ameliorate lung function.3. BMMSC reduce lung inflammation and lung edma, and inhibit alveolar type Ⅱ epithelial cell apoptosis.4. Cocultured with A549cells, BMMSC can increase the expression level of anti-apoptosis protein bcl-2and decrease the level of pro-apoptosis protein caspase3and bax in A549cells.5. Cocultured with A549cells, BMMSC can paracrine growth factor KGF and HGF, increase KGF and HGF mRNA expression.6. BMMSC can paracrine growth factor KGF and HGF, which is one of mechanisms of BMMSC anti-apoptosis effect.
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