| Objective: Exploring the effects of nicotine on the biological characteristics of human umbilical cord mesenchymal stem cell(hUC-MSC) and lung cancer cell line A549 to illustrate the potential toxic effects of nicotine on human body, which will add a lot to the understanding of the relationship between nicotine, mesenchymal stem cell and tumor, thus providing experimental basis for the prevention and treatment of smoking related diseases.Methods:(1) We adopted tissue block adhering wall method to isolate and culture hUC-MSC, and identified the biological characteristics according to its morphology, differentiation potential, cell surface antigen expression and related gene level.(2) MTT was used to detect the influence of nicotine on the proliferation of hUC-MSC, and 0.1 mg/ml, 0.2 mg/ml and 0.3 mg/ml were chosed for the following study after concentration gradient screening; the influence of different concentration of nicotine on hUC-MSC migration was tested through Transwell assay; the influence of nicotine on hUC-MSC cell cycle and apoptosis were analyzed by Flow Cytometry; the hUC-MSC related proteins level in different concentrations of nicotine were detected by Western blot and immunofluorescence.(3) A549 was co-cultured with hUC-MSC which was pretreated by 0.3 mg/ml of nicotine for 24 h.(4) The migration ability of lung cancer cell line A549 was detected by Transwell assay and scratch assay.(5) To evaluate the tumorigenic ability of A549 cell after co-cultured with hUC-MSC, we constructed nude mice subcutaneously tumor model.(6) Hematoxylin Eosin(HE) staining observation and immunohistochemical analysis were performed on the tumor.Results:(1) A class of long spindle shaped cells, which was isolated from the umbilical cord tissue, has been successfully isolated. After high passage cultivation, the purified cells can be induced into osteoblasts and adipogenic cells, and the corresponding genes were expressed respectively; Flow cytometry results detected cells did express hUC-MSC surface markers, the results of Flow cytometry analysis were consistent with hUC-MSC surface markers.(2) The results of MTT assay showed that nicotine could inhibit the proliferation of hUC-MSC, which showed a dose and time-dependent manner. When the concentrations of nicotine were between 0.1mg/ml to 0.3mg/ml, nicotine would not inhibit the proliferation of hUC-MSC; with the concentrations of nicotine increasing, the migration ability of hUC-MSC has enhanced; Flow cytometry analysis of cell cycle showed that hUC-MSC was severely blocked in S phase after treated with different concentrations of nicotine and the apoptosis cell number was also increased; western blot and immunofluorescence results showed that the E-cadherin protein expression was decreased and the expression of N-cadherin, Vimentin, β-catenin which were EMT related proteins were increased; the expression of Sox2, Nanog, Sall4, Oct4 related proteins were also increased in varying degrees and Cyclin D1 and Cyclin D3 and other cycle related proteins’ expression were also increased.(3) Transwell experiment and scratch test showed that under the condition of co-culture, the migration ability of experimental lung cancer cell line A549 was higher than the control group; in nude mouse experiments, the tumor growth rate, tumor size, volume and quality of the experimental group were higher than those of the control group; HE and immunohistochemical staining showed that compared with the control group, the cells of experimental group showed disordered arrangement, highly atypia, increased nuclear cytoplasm ratio, pathologic mitosis and increased PCNA expression.Conclusions:(1) Under the culture condition in vitro, nicotine can enhance hUCMSC’s ability of migration and multilineage differentiation.(2) After co-cultured with hUC-MSC which was pretreated by nicotine, lung cancer cell line A549 have stronger ability of migration, malignant degree and tumorigenic ability in vivo. |
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