The Study Of Bone Marrow Mesenchymal Stem Cells’ Differentiation Induced By Alveolar Epithelial Cells Of Rats Exposed To Silica Dust

Objective: To observe whether bone marrow mesenchymal cells(BMMSCs) can differentiated into alveolar epithelial cells(AEC) after induced by alveolar epithelial cells of rats exposed to silica dust. Methods: 1、Rankly selected 6 healthy male SD rats without specific pathogen, cultured BMMSC with the method of whole bone marrow adherent. 2、selected 14 rats randomly from the same batch of rats, seven of them were injected 1.0 ml silica dust suspension at the mass concentration of 40 g/L through trachea to make the model of rats exposed to silica dust, cultured AEC of rats exposed to silica dust by the method of immune adhesion purification, another seven rats injected 1.0 m L 0.9% sodium chloride solution to be the normal control rats, cultured AEC of normal rats by the method of immune adhesion purification. 3、set experimental group:the co-cultured of BMMSCs and AEC from rats exposed to silica dust as experimental group, control group A: the co-cultured BMMSC and AEC from normal rats as control group A, control group B: the co-cultured of BMMSCs alone as control group B. 4、observed the change of morphology in BMMSCs from each group by inverted microscope when co-cultured 4days and 8 days. 5、stained aquaporin 5(AQP5) and surfactant protein C(SPC) in BMMSCs by double immunofluorescence staining when co-cultured 4 days and 8days, and then, observed the expression of the two fluorescence in BMMSCs with inverted fluorescence microscope(IFM) and laser scanning confocal microscope(LSCM), calculated integral optical density(IOD) of the two proteins’ fluorescence observed by the two microscopes with Image-pro plus 6.0 graphic analysis software. 6、detected the expression level of m RNA in AQP5 and SP-C with real time fluorescence quantitative PCR in each group when co-cultured 4days and 8 days. 7、collected the cell culture fluid when co-cultured 4days and 8 days, detected the concentration transforming growth factor-β1(TGF-β1) and epidermal growth factor(EGF) in the cell culture fluid by enzyme-linked immunosorbent assay(ELISA). Results: 1、the cell morphology in each group: BMSC in experimental group and control group A changed from long spindle to cubic and polygonal gradually after co-cultured, the variation of morphology in 8 days was more obvious than 4 days, and the chang in control group A were less obvious than experimental group, the morphology of BMMSCs in control group B did not change obviosly. 2、the results observed by IFM and LSCM:when co-cultured 4 days and 8 days, BMMSC from experimental group and control group A expressed AQP5 which was red and SP-C which was green, In the control group B, a small amount of AQP5 green fluorescent expression was observed in BMMSC but no SP-C red fluorescent expression, compared to IFM, LSCM can clearly observe the expression of different proteins and their distribution in the cell. 3、the results of IOD: under the two kinds of microscope, when co-cultured 4 days and 8 days, the IOD of the two proteins’ fluorescence in BMMSCs from experimental group were strengthen than control group A and control group B in the same time(P<0.01), the IOD of the two proteins’ fluorescence in BMMSC from control group A were strengthen than control group B in the same time(P<0.01). compared the IOD of different time, the IOD of the two proteins’ fluorescence when co-cultured eight days from experimental group and control group A were both higher than co-cultured four days in the sanme group(P<0.01). the comparison of IOD from two kinds of microscope: besides the IOD of SPC’s fluorescence from control group B, the IOD of the two proteins’ fluorescence in BMMSC from three group under IFM were both higher than under LSCM in the same group(P<0.01). 4 、 the results of real time fluorescence quantitative PCR: the expression level of m RNA in AQP5 and SP-C from experimental group were higher than control group A and control group B in the same time(P<0.01), and control group A were higher than control group B in the same time(P<0.01), the expression level of m RNA in AQP5 and SP-C when co-cultured eight days from experimental group and control group A were both higher than co-cultured four days in the same group(P<0.01). 5、the he concentration of TGF-β1 and EGF: the concentration of TGF-β1 from experimental group were lower than control group A and control group B in the same time(P<0.01), the concentration of EGF from experimental group were higher than control group A and control group B in the same time(P<0.01),and TGF-β1 from control group A were lower than control group B in the same time(P<0.01), EGF from control group A were higher than control group B in the same time(P<0.01), comparison between the two time points in the same group, the concentration of TGF-β1 was all no significant difference in the three group(P>0.01), the concentration of EGF from experimental group and control group A both have significant difference(P<0.01). Conclusion: AEC from rats exposed to silica dust can induce BMMSCs differenciate into AEC effectively, the induction of differentiation may be related to the cytokines secreted by BMMSCs.