The in Vitro Study of Serum-free Culture Combined DDP Enriching Human Lung Cancer Stem Cells
|School||Dalian Medical University|
|Keywords||Lung Cancer Cancer stem cells SP side population cells Resistance|
Lung cancer is present to human health and life of one of the most harmful cancer. So far, the existing cancer treatments only prolong the survival of patients and improve their quality of life, almost all patients eventually died of the primary disease. In recent years, some scholars have proposed that in the presence of a very small part of the tumor tissue with indefinite proliferative potential and self-renewal capacity of stem cell population, the incidence of tumor development plays a vital role, for this part of the cell targeted therapy will far-reaching implications for cancer therapy. However, due to cancer stem cells (cancer stem cells, CSCs) in tumor tissue content is extremely low, which restricted the development of the main obstacles to study CSCs. Therefore, the establishment of an efficient method of enrichment CSCs, experimental study on the follow-up is necessary for the smooth conduct, CSCs is currently an important issue for research in the field. Objective: ① To investigate the human lung cancer cell line A549 with the existence of cancer stem cells. ② explore the possibility of using CSCs relatively resistant to conventional chemotherapy drugs characteristics of CSCs in serum-enriched method based on the use and cisplatin (DDP) short-term treatment methods, more convenient and effective from a human lung cancer cell line A549 in Isolation and identification of cancer stem cells. Methods: Grouping: parental A549 cells group; simple serum-free culture of CSCs group; serum-free culture selected cells cisplatin group (Drug-selected cells, DSCs). ① the cells to 5000 cells / seeded to add a serum-free medium (serum free medium, SFM) in 6-well plates, the observed changes in cell morphology. ② limited dilution method using cells 10 cells / well were seeded to 96 well plates, test conditions, a single cell in the SFM capability into a ball. ③ By Cell Counting Kit-8 (CCK-8) assay detects cells in each group of self-renewal and differentiation capabilities and 24h after cisplatin tolerance. ④ through fluorescent dye Hoechst 33342 staining using FASC BD Aria II flow cytometer to establish Hoechst blue-Red analysis diagram, the detection side population (side population, SP) phenotype cell levels. Will add Hoechst 33342 active transporter blockers - reduce the area after reserpine action is set to the SP \Results: ① A549 cells after drug screening morphological changes: parental A549 cells after three cycles a final concentration of 7μg/ml cisplatin treatment, originally arranged in dense polygon morphological changes, cells become large and flat surface particles increased. ② cell self-renewal and differentiation comparison: By using an inverted microscope SFM continuous observation of two weeks in the case of a single cell into a ball, we found DSCs Dir 3 days in SFM conditions to form lung tumors ball (CSCs start in the first five days form), and the resulting suspension ball volume larger quantity more; CCK-8 Experimental results show that, DSCs group have self-renewal and differentiation of cells in the group than the 2-fold increase CSCs. ③ cells to cisplatin resistance capability comparison: CCK-8 assay through the cells in each group at 24h after cisplatin apoptosis, using the SPSS 17.0 software analysis showed that, DSCs drug cisplatin compared with CSCs exhibit higher resistance (IC50 value of 66.52 ± 2.49μg/ml vs. 37.82 ± 0.39μg/ml), both compared with the control group, parental A549 cells (IC50 value of 10.81 ± 0.39μg/ml) resistance were increased by 6 fold and 3.5-fold (P lt; 0.01). ④ cells in each group content of SP cells in comparison: Hoechst blue-Red analysis chart showed that the three groups have significant differences in the content of SP cells, DSCs than CSCs content of SP cells was significantly increased (12.32 ± 1.51% vs.7.93 ± 1.44% ), compared with the control group, both parental A549 cells (1.29 ± 0.48%) of SP cells were increased by 12-fold and 8-fold (P lt; 0.01). Conclusion: ① human lung cancer cell line A549 in the presence of self-renewal and differentiation capacity and highly resistant tumor stem cells. ② with conventional serum-free culture method compared to cisplatin in serum-free culture screening method can be more efficiently from human lung cancer cell lines isolated and cultured lung cancer stem cells.