| Chapter I The purification and identification of mouse bone marrow mesenchymal stem cells (MSCs) in vitroObjective To establish the method of separation, purification and identification of the murine bone marrow mesenchymal stem cells (MSCs) in vitro.Method Cultured mouse MSCs with the combination method of density gradient centrifugation separation and adherent cells separation, and observed cell morphology and growth; detected mouse MSCs surface antigen CD34, CD45, CD 105 and CD 106 expression and purity with flow cytometry.Results The cell growth of mouse MSCs:after primary culture for 24 hours, only few round adherent can be seen in cell culture flask, the number of adherent cells increased gradually after cultured for 72 hours, then some colony formation can be seen.the cell morphology is long spindle at this time. At the first 2-6th days, the cells growed very slower, cells proliferated faster in cultured 7-12 days, which were "whirlpool-like". Cultured for two weeks, cells closed to 80-90%of integration, cell morphology is more uniform. The passaged MSCs were spindle-shaped, about all cells adherent after passaged 24 hours, cells not grow as the cell colony that time, but evenly distribution. Cell viability is 92.6±1.8%. Flow cytometry results showed that the third passage mouse MSCs uniformity is more than 90%; CD34, CD45 is negatively expressed, CD105, CD106 is positively expressed.Conclusion The combination of density gradient centrifugation separation and adherent cells cultivation could successfully isolate mouse MSCs in vitro; the cell vitality and purity of mouse MSCs was high; Flow cytometry could identify the mouse MSCs in vitro. Chapter II Establishment an experiment model of co-culture lung tissue and bone marrow mesenchymal stem cells in vitroObjective Establishment a non-contact co-culture model in vitro to induce Bone Marrow Mesenchymal Stem Cells differentiate into alveolar epithelial cell, For studying the inflence of bone marrow mesenchymal stem cells differentiation in vitro by injuried lung tissue.Method Establishment an experiment model of bronchopulmonary dysplasi though feed newly born mouse at 60%oxygen concentration 21 days. after 21 days, took out lung of mouse, and grind, filtration, trypsinization under asepsis condition, made Single-cell suspension of lung tissue. We used the 3th passage stem cells cultured in vitro and inoculated in the bottom of 6-well plates, transwell clear inserts (the membrane with 0.4um pore size,) was inoculated with single-cell suspension of lung tissue, It was the co-culture model in which the lung tissue cells and MSCs were separated each other. We designed three groups:experimental group A:MSCs were cultured with injuried lung tissue single cell suspension; experimental group B:MSCs were cultured with normal tissue single cell suspension; control group:mice MSCs were cultured alone. After co-culture 6 days, we made experiment of Scarification and infestation, to observe the change of immigration ability of MSCs after co-culture. After co-culture 8 days, we made experiment of immunofluorescence, observed surfactant protein C (SP-C),aquaporin 5(AQP5) immune Fluorescence expression information though using laser scanning confocal microscopy. When them were co-cultured for 8 days, SP-C, AQP5, transforming growth factorβ1 (TGF-β1) mRNA was detected with real-time-PCR.Results After mouse being breed 21 days under 60%oxygen environment, we made a pathological section to observe the change of lung tissue. We found alveolus normal structures disappear, alveolar space expanded obviously, and mount of alveolus grew downwards, alveolar septum increased thickness, more mesenchymal cells proliferation. After 12-hours of scarification, we found all of them only few cells enter into scarification. At 24-hours, migrating cells increased all of three group, but it was very obviously in experimental group A. At 48-hours, migrating cells in experimental group A are obviously more than in experimental group B and control group. Injuries lung tissues co-culture with bone marrow mesenchymal stem cells could increase the immigration ability of MSCs. Experiment of infestation:After culture 48-hours, We count cells of superficies inferia of PET, there had significant difference compare experimental group A with experimental group B and control group. Injuries lung tissues co-culture with bone marrow mesenchymal stem cells could increase infestation rate of MSCs. The result of immunofluorescence:We could find blue fluorescence tagged by hoechst33342, red fluorescence tagged by AQP5, green fluorescence tagged by SP-C in experimental group A. Blue fluorescence tagged by hoechst33342, red fluorescence tagged by AQP5 could be seen in experimental group B and control group, but green fluorescence tagged by SP-C not been fould. Real-time PCR: SP-C mRNA was expressed in experimental group A, and was not expressed in experimental group B and control group. AQP5 mRNA and TGF-β1 mRNA were expressed in three group, their expression levels were significantly higher in experimental group A than in experimental group B and control group (P＜0.01), but no statistical difference was found between the experimental group B and control group (P＞0.05).conclusion; We had successfully established the BPD model, and established co-culture model in vitro to induce the marrow mesenchymal stem cells to differentiate into lung epithelial cells. We confirmed Immigration ability of MSCs can be increased by co-culture with lung tissues injuried by hyperxia and the injury lung tissue could promote the mouse MSCs to differentiate into alveolar epithelial cells. |
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