Osthole Improves Bone Marrow-derived Mesenchymal Stem Cell Differentiation Into Alveolar Epithelial Cells Type ?

Objective:To isolate and culture rat BMSCs in vitro,and to identify the biological characteristics of the rat BMSCs.And to investigate the effects of osthole on differentiation of BMSCs.Methods: The rat primary BMSCs were isolated and extracted by whole bone marrow adherent method and cultured in vitro,purification of BMSCs by cell passage.Cell surface antigen CD29,CD34,CD71,CD90 and CD106 were identified by flow cytometry.Induced the differentiation of rat BMSCs into type II alveolar epithelial cells by the condition media extracted from rat lung tissue.And add the osthole to explore its effect on differentiation of BMSCs to differentiate into type II alveolar epithelial cells.The expression of the surface markers of type II alveolar epithelial cells,SPB and SPC,were identified by immunofluorescence staining and quantitative analysis.The m RNA expression of SPB and SPC was detected by real time PCR,further verified the differentiation.Results: The results of flow cytometry for surface antigen detection and analysis showed that CD29,CD90 expression were 85% and 97.5% respectively,the expression of CD106 was 47.2%,CD34 and CD71 expression were little,0.18% and4% respectively,is in accordance with the characteristics of BMSCs.Immunofluorescence staining results showed that,after differentiation BMSCs expressed cell surface markers of type II alveolar epithelial cell,SPB and SPC,the quantitative analysis showed among 3 groups,the positive expression cells rates respectively were : SPB: control group 24.18%±1.60%,lithium chloride group44.57%±1.71%,osthole group 82.99%±3.74%;SPC: control group 41.64%±7.29%,lithium chloride group 69.18%±7.14%,osthole group 82.42%±0.79%.Compared with the control group,the positive expression rates of Lithium chloride group and osthole group were significantly higher,the difference was statistically significant(P<0.05).Quantitative real-time PCR results showed that in SPB m RNA expression,expression of lithium chloride group and osthole group respectively was 5.05 times(0.027±0.009,P < 0.05)and 7.93 times(0.017±0.003,P < 0.05)higher than that of control group,the difference is statistically significant;in the SPC m RNA expression,expression of lithium chloride group and osthole group respectively was 4.28 times(1.024±0.330,P < 0.05)and 4.18 times(0.980±0.190,P < 0.05)higher than that of control group,the difference Have statistical significance.Conclusion: Bone marrow mesenchymal stem cells were successfully extracted and cultured in vitro.BMSCs could differentiate into the type II alveolar epithelial cells after induction,osthole could facilitate this process,which indicated osthole could be used as an agonist for promoting differentiation of BMSCs,provided a new train of thought in cell engineering in the treatment of acute lung injury,but the mechanism was still not clear,further study was needed.