In Vitro Study Of The Rat Lung Homogenate Supernatant On Differentiation Of Bone Marrow Derived Mesenchymal Stem Cells

Object:To investigate the influence of rat lung homogenate supernatant on differentiation of bone marrow-derived mesenchymal stem cells into alveolar epithelial cells.Method:Mesenchymal stem cell were harvested and purified from rats bone marrow by adherent culture. Cellular morphological was observed, growth curve was drawn by a CCK-8 kit. Cells were also identified by FACS for the antigen expression on cell surface. The staining method was chosen to detect the potential differentiation of bone marrow-derived mesenchymal stem cells. The rat normal and bleomycin damaged lung homogenate supernatant were used to induced the third passage of bone marrow-derived mesenchymal stem cells for ten days. The experiments were carried out in three groups, the control group:BMSCs, the intervention group 1:BMSCs+the rat normal lung homogenate supernatant, the intervention group 2:BMSCs+the rat bleomycin damaged lung homogenate supernatant. The expression of SP-C protein were identified by immunofluorescence method, the differentiation rate was calculated among the three groups, the expression of SP-C mRNA were detected by RT-PCR.Results:The third passage adherent cells were arranged in whorls and fusiform shape in a relatively homogeneous morphology. FACS showed that cells were positive for CD29 and CD90, and negative for CD49d, CD34 and CD45. Cells which were treated by differential inducers, which showed red to alizarin red staining, orange red to oil red O and blue to toluidi-ne blue. With differentiation in vitro, the expression of SP-C mRNA and protein were negative in control group, while positive in intervention group. Statistical analysis indicated that the positive rate of SP-C protein detected by immunofluorescence method in rat normal and bleomycin damaged lung homogenate supernatant were 13.29% and 25.27%; RT-PCR tests showed the rat bleomycin damaged lung homogenate supernatant has a higher rate of inducing bone marrow-derived mesenchymal stem cells into alveolar epithelial cells than that of the normal lung homogenate supernatant.Conclusions:The separation method of adherence in this study was identified to be practical and efficient, and resulted in sufficient quantities of mesenchymal stem cells with potential differentiation. The rat lung homogenate supernatant can induce the bone marrow-derived mesenchymal stem cells into alveolar epithelial cells. The rat bleomycin damaged lung homogenate supernatant has a higher rate of inducing bone marrow-derived mesenchymal stem cells into alveolar epithelial cells than that of the normal lung homogenate supernatant.