| Summary: | 碩士 === 國立陽明大學 === 生命科學暨基因體科學研究所 === 96 === Cancer stem cell (CSC) hypothesis is one of the tumor formation models. The concept of this hypothesis is that only a few fractions of cancer cells can form tumor, and these cells have the same characteristics with normal stem cell such as self-renew, long lifespan, resistant to apoptosis, etc. Until now, cancer stem cells have been found in acute myeloid leukemia (AML), breast cancer, brain tumor, lung cancer, liver and gastrointestinal cancer by selection of specific cell surface markers or side population (SP). However, these kinds of cells have not been found in esophageal cancers.
We used specific cell surface markers and side population assay to select candidate of cancer stem cell in 8 esophageal cancer cell lines (TE1 large, TE1 small, TE2, TE6, TE9, CE48T/VGH, CE81T/VGH, and CE146T/VGH). These markers included CD133, CD44 and CD24: the common cancer stem cell markers, CD71, CD49f: the normal mouse esophageal stem cell markers. The candidate of cancer stem cells were then tested with cell proliferation rate assay, colony formation assay, and evaluated the expression of stemness genes to prove them have identical characteristics with normal stem cells. In the cell surface marker analysis, CD133+, CD44+ CD24- cells percentage were almost 0% in all cell lines. Side population (SP) was only 0~2.5% in all cell lines. CD71- CD49f+ cells (suspected candidate of esophageal cancer stem cell) percentage were 0~0.5% in TE1 large and small cell, 5~10% in TE2 cell, 15% in TE6 cell, 2~6% in TE9 cell, 48~60% in CE48T/VGH, 30~70% in CE81T/VGH cell, 30~40% in CE146T/VGH cell. After flow sorting only the TE2 CD71- CD49f, CD71+ CD49f+, SP and non-SP cells could survive. Therefore we selected these cells to do further experiments. In the experiment of cell proliferation rate, TE2 CD71- CD49f+ cell grew faster than CD71+CD49f+ cell; SP cell also grew faster than non-SP cell. But between them, TE2 parental cell had the fastest proliferation rate. In colony formation assay, all CD71- CD49f, CD71+ CD49f+, SP cells could not form colony on soft agar. Instead, non-SP cell could form tiny colony and TE2 parental cell could form vast colonies after 3 weeks culture. About stemness genes expression, the Q-PCR results revealed that TE2 parental and CD71+CD49f+ cells were much higher than TE2 CD71- CD49f+ cell. And RT-PCR results showed that TE2 CD71- CD49f+ and SP cell didn’t have higher stemness genes expression. Taking these data, it seems that CD71, CD49f may not the cancer stem cell markers of esophageal cancer cell line and SP cells of esophageal cancer cell line did not enrich with cancer stem cells.
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