Identification Of Biological Characteristics Of Xuanwei Lung Adenocarcinoma Stem Cells

[Objective] From Xuanwei lung adenocarcinoma cell line established in new, we selected the SP cells from it by flow cytometry. Let them for cloning culture.We got two cell subsets with different morphology and named SPs, SPcm. Through the Cloning culture Separatly, We got four different forms Cell subsets:SPs-C11, SPs-E8, SPcm-G10, SPcm-B10. We could identify the biological characteristics of stem cells and compare the difference between the cell subsets.[Methods] we selected SP cells from Xuanwei lung adenocarcinoma cell lines by flow cytometry. The SP was cloned culture.We got two different forms which had different biological morphology, malignant biological behavior and stem cell properties. We name them SPs and SPcm. The two cell subpopulations were cloned culture. We selected the long fusiform cells named SPs-C11and the irregular polygon cells named SPs-E8from Sps subgroups. And then, we selected the irregular polygon cells named SPcm-G10and the long fusiform cells named SPcm-B10from SPcm subgroups. The four groups of cell subsets which were new sorting were used for comparative study.(1). The cell morphology:the cell morphology was observed by light microscopy, the ultrastructure was observed by transmission electron microscope.(2). The cell mitotic index:The cells were climbed piece and stained with Giemsa to calculate the cell mitotic index.(3). The cell growth curve:we make the cell into the single cell suspension.The cell number is105/ml. And then we Inoculated the cell in96well plates. Each hole is200μ L. Edge hole filled with sterile PBS.At last, we put them in37℃cell culture box. In the24h,48h,72h,96h,120h,144h,168h, we take a96hole board testing OA value by Mtt method.and drawing cell growth curve.(4). Soft agar colony formation test:Using the double soft agar colony formation to Calculate the rate of cloning efficiency:Colony forming efficiency (%)=(number of clones/inoculation cell number) x1000‰.(5).The distribution of cell cycle and DNA index:Flow cytometry was used to detect the cell cycle and DNA ploidy.(6). Invasion assay in vitro:Testing the Invasive ability of cell used the Transwell assay.[Results](1). Cell morphology:SPs-C11, SPs-E8, SPcm-G10, SPcm-B10cell subsets belonged to the moderate differentiation of lung adenocarcinoma cells. They had the characteristics of morphology of malignant tumor cells. The atypia of cells were large.The size of cells were unequal. The morphology of cells was diversity.The cells were long fusiform or irregular polygon. Cell refraction become weakened. Nuclear volume increased significantly. Nuclear and cytoplasmic ratio was larger than normal cells.There were Dual-core, multi-core, megakaryocyte and bizarre nuclei. Nuclear staining deepened. Chromatin were Coarse and granular. The distribution of Chromatin were Uneven. It Often accumulate in the nuclear membrane. It made The nuclear envelope become thickening. The nucleolus were Hypertrophy. There were asymmetry or multipolar mitosis and other pathological karyokinesis phenomenon. Cells were arranged in adenoid structure. Ultrastructural changes under electron microscope were that the organelles compared with normal cells were decreased, but the number of mitochondria increased slightly, and abnormal shape. Rough endoplasmic reticulum swelled and abnormal shape.(2). The cell mitotic index: SPs-C11and SPs-E8cell divisions index were higher than SPcm-G10, SPcm-B10cell subsets. It illustrated that the cell division and reproduction ability of SPs-C11cell subsets, and SPs-E8cell subsets were larger than SPcm-G10, SPcm-B10cell subsets.(3).The cell growth curve:The cell growth rate of SPs-C11and SPs-E8was higher than that of SPcm-G10, SPcm-B10cell subsets.(4). Soft agar colony formation test:All four groups of cells formed clones.The Clone formation rate: SPs-C11> SPs-E8> SPcm-G10>SPcm-B10. This showed that the four kinds of cell division and transfer capability are extremely strong, and the higher the degree of malignancy, low differentiation. SPs-C11, SPs-E8two groups of clone formation rate is greater than SPcm-G10, SPcm-B10cell subsets. This showed that C11and E8subsets of SPs proliferation and metastasis are slightly higher than that of G10and B10subgroup of SPcm.(5). The distribution of cell cycle and DNA index:The experimental results showed that the four groups of cell subsets were aneuploid tumors, a high degree of malignancy. SPs-C11, SPs-E8two cell subsets of nuclear DNA content greater than SPcm-G10and SPcm-Bl0.(6).Cells Invasive ability: Testing the Invasive ability of cell used the Transwell assay, we can drew a conclusion.The number of the cells which through ECM glue of SPs-Cll and SPs-E8were more than SPcm-G10, SPcm-B10cell subsets.It showed that C11and E8cell invasion ability were slightly higher than that of G10and B10.[Conclusions](1). SPs-C11, SPs-E8, SPcm-G10, SPcm-B10four groups of cells which from Xuanwei lung adenocarcinoma cell line were moderate differentiated lung adenocarcinoma cells.They had morphology and biological characteristics of cancer cells.(2). SPs-C11, SPs-E8two groups of cell subsets in the growth rate, proliferation, metastasis and invasion ability and cell malignant degree is higher than SPcm-G10, SPcm-B10two cell subsets.