Role Of Chemokine CCL19in The Transformation Of Alveolar Cells Type â…¡ Induced By Yunnan Tin Mineral Dust

[Objective] To explore the role of Chemokine CCL19in the transformation of alveolar cells type Ⅱ induced by Yunnan tin mineral dust.[Methods](1) Immortalized rat lung fibroblasts were treated with200μg/ml Yunnan tin mineral dust for72h on every other generation until the9th generation. Since the10th generation to the40th generation were cultured as convention. The9th generation of the normal fibroblasts and dust-exposed fibroblasts were routinely cultured for72hours, then clear away the medium, continually cultured in serum-free medium, harvested the cell supernatants after24hours. The CCL19level in the supernatant of fibroblasts was examined by enzymelinked immunosorbent assay (ELISA).(2) Immortalized rat lung alveolar cell type Ⅱ have been treated with200μg/ml Yunnan tin mineral dust for72h on every other generation until the9th generation. Since the11th generation epithelial cells were treated with chemokine CCL19and CCR7mAb. The groups were devided into E(Control), E200, E200(L), and E200(L-RmAb) groups.(3) E200cells were treated with CCL19and the cellular proliferation was evaluated by MTT method and the optimum concentration was selected.(4) The morphology of cells was observed with inverted phase contrast microscope.(5) The expressions of a-SMA in fibroblasts, CCR7, TGF-β and Ki-67in epithelia were investigated by the method of immunocytochemistry.(6) The transformation of epithelial cells was identified by ConA agglutination test and anchorage independent growth in soft agar.(7) The levels of CCR7, Akt, p-Akt, Bcl-2and Bax were detected through Western blot.(8) SPSS17.0statistical software was used for data analysis.[Results](1) The Yunnan tin mine dust could induce morphology change of immortalized rat lung fibroblasts (RLF-6), and promote it to produce chemokine CCL19, and the level was higher than nonmal fibroblasts.(2) MTT method shows that the higher concentration of CCL19, the more obvious cell proliferation between0-400ng/ml; When the concentration of CCL19is200ng/ml, it has significant proliferation effect, however, there was no statistical significance between the200ng/ml group and the higher concentration group (p>0.05). The difference of the same concentration for I2h and24h was not statistically significant(p>0.05), while the differences were statistically significant in the same concentration for12h and48h,24h and48h,(p<0.05). When the epithelial cells was dealed with CCL19for48hours, there was no statistical significance between each group (p>0.05).(3) Treated with200μg/ml Yunnan tin mine dust, the morphology of RLE-6TN changes gradually from polygon to irregular polygon, and then transfomes into short spindle since35th generation.(4) The expression of a-SMA in RLF-6was negative in the normal fibroblasts. The expression of a-SMA became positive in RLF-6cells treated with the200μg/ml Yunnan tin mine dust in the25th, especially in the35th. The tin mine dust could induce the expression of CCR7in RLE-6TN cells. The level of Ki-67and TGF-β expression was higher in the40th generation of epithelial cells treated with Yunnan Tin mine dust; the expression level was E200(L)group> E200(L-RMAB)group>E200Pgroup in the same generation.(5)The epithelial cells of E200group have malignant transformation characteristics in the40th generations. Its colony forming efficiency was (2.3333±0.5773)%o and ConA agglutination test was positive. E200cells treated with CCL19、mAbCCR7from the11th generation, the malignant transformation of E200(L) group was from35th generation; The colony forming efficiency was up to (5.3333±1.1547)%o in the40th generation and ConA agglutination test time was only8min. The colony forming efficiency of E200(L-RmAb) group was (1.500±0.7071)%o and ConA agglutination test time was also positive. The Control group has no malignant transformation characteristics untill to the40th generation.(6) After treated by tin mine dust, epithelial cells show the expression of CCR7, and its level was higher during culture progression. CCL19could increase the level of p-Akt and Bcl-2. however, the expression of Bax was decreased.[Conclusions](1) Yunnan tin mine dust can induce activation of fibroblasts, and promote the secreting of chemokine CCL19;(2) Yunnan tin mine dust can induce the transformation and the expression of CCR7in alveolar cells type Ⅱ;(3) Chemokine CCL19can promote transformation of epithelial cells induced by Yunnan tin mine dust, which may be through PI3K/Akt signaling pathway.