ABSTRACT
BACKGROUND:Studies have shown that lung cancer stem cels can be isolated from the lung cancer cel lines, But there are few reports on in vitro isolation, culture and identification of lung cancer stem cels in patients with lung squamous carcinoma. OBJECTIVE:To establish the feasible methods of harvesting lung cancer stem cels from fresh lung cancer tissues in patients with lung squamous carcinoma, and to investigate the alterations in cel number and function during primary culture. METHODS: Side population cels were isolated by colagenase digestion, Ficol density gradient centrifugation and Hoechst 33342 efflux properties. The isolated cels were isolated and cultured in conditioned medium. Flow cytometry method was used to detect lung cancer stem cels based on the cel surface markers CD133 and CD44, and the positive rates of CD133+, CD44+ and CD133+/CD44+ were recorded. The single cel clones assay, flat colony formation assay and the cel sphere formation assay were used to identify the stem-like characteristics of lung cancer stem cels between the first and fourth generations. RESULTS AND CONCLUSION:The positive rates of CD133+, CD44+ and CD133+/CD44+ cels at the fourth generation were increased significantly, and the positive rates of CD133+ and CD133+/CD44+ cels at passage 4 were significantly higher than those at the first generation. The abilities of single cel clone formation, the flat colony formation and the cel sphere formation in the fourth-generation cels were greatly enhanced compared with the first-generation cels. Experimental findings showed that stem cel-like lung cancer cels were obtained from fresh lung cancer tissue in patients with lung squamous carcinoma, which stably and rapidly amplified in vitro, laying the foundation for the further study of the heterogeneity and drug resistance of lung cancer stem cels.
ABSTRACT
BACKGROUND:Studies have shown that lung cancer stem cel s can be isolated from lung cancer cel lines. But there are few reports about in vitro isolation, culture and identification of lung cancer stem cel s in patients with lung squamous carcinoma. OBJECTIVE:To explore the feasible methods of harvesting lung cancer stem cel s from fresh lung cancer tissue in patients with lung squamous carcinoma. METHODS:Side population cel s were isolated by col agenase digestion, Ficol density gradient centrifugation and Hoechst 33342 solution. The isolated cel s were suspended in conditioned medium for isolated culture. Flow cytometry method was used to detect lung cancer stem cel s based on the cel surface markers CD133 and CD44, and the positive rates of CD133+, CD44+and CD133+/CD44+cel s were recorded. RESULTS AND CONCLUSION:Cel s adhered at 0.5 hour after incubation;typical cel colony was formed at 4 days of culture;cel s showed paving stone-shape at 7 days in a total number of 10 8. The positive rates of CD133+, CD44+and CD133+/CD44+cel s at passage 4 were increased significantly. These findings indicate that stem cel-like lung cancer cel s were obtained from fresh lung cancer tissue in patients with lung squamous carcinoma, which were stably and rapidly amplified in vitro, laying the foundation for the further study on the heterogeneity and resistance of lung cancer stem cel s in the future.