Isolation And Identification Of Lung Cancer Stem Cells From NCI-H1650 Cell

BackgroundLung cancer is the leading cause of cancer-related deaths worldwide,which is characterized with high incidence, high malignancy, poor prognosis, high mortality.Cancer stem cells are a population of heterogeneous cells, they are capable of self-renewal, immortalization and multi- directional differentiation potential. Lung cancer stem cells play a critical role in occurrence and development of lung cancer, and also are involved in maintenance, metastasis, recurrence and chemotherapy-resistant of lung cancer. Therefore,the research of lung cancer stem cell may provide a novel direction for treatment. Howerver, the number of lung cancer stem cell is extremely low, the important basis for study on lung cancer stem cell is effectively method of isolated it. We will isolated lung cancer stem cells from human lung cancer cell line NCI-H1650 in two different ways.Objectives:Isolation and identification of lung cancer stem cells from human lung cancer cell line NCI-H1650.Methods:1. The NC I-H1650 cells were cultured in serum- free medium combination with paclitaxel treatment to form tumor spheres.2. Flow cytometry were used to compare the expression levels of CD133 and CD326 between NCI-H1650 cells and induced NCI-H1650 spheres. The immunofluorescent staining used to verification the expression levels of C D133 and C D326 in induced NCI-H1650 spheres. The expression of Oct4, Nanog and Sox-2 were evaluated by RT-PCR and Western blot.3. CD133+ and C D 133-subpopulations were sorted from NC I-H1650 by magnetic cell separation(MACS).4. The immunofluorescent staining used to verification the expression levels of CD133 and C D326 in cells sorted by MACS. The expression of Oct4, Nanog and Sox-2 were evaluated by RT-PCR and Western blot.Results:1. Induced of NCI- H1650 stem cells were spherical growth in SFM.2. Induced of NCI-H1650 stem cells express C D133 and CD326 which are tumorassociated surface markers.3. Induced of NCI-H1650 stem cells express Oct-4, Nanog, Sox-2 which are tumorassociated gene.4. NCI-H1650 cells sorted by MACS were spherical growth in SFM.5. NCI-H1650 cells sorted by MACS express CD133 and C D326 wich are tumor-associated surface markers.6. NCI-H1650 cells sorted by MACS express Oct-4, Nanog, Sox-2 which are tumorassociated gene.Conclusions:1. The serum- free condition culture with paclitaxel treatment enrich cancer stem like cells from lung carcinoma cell line NHI-H1650, which expression high levels of CD133, CD326, Oct4, Nanog and Sox-2.2. Cancer stem cells were sorted from NCI-H1650 cells by magnetic cell separation(MACS), which expression high levels of CD133, C D326, Oct4, Nanog and Sox-2.