The Expressed Significance Of ABCG2 In Human Tongue Cancer Cell Line TCA8113 And The Dissociation Of Side Population Cells

At present, tumor is a great disease of threatening mankind's health. Tumor stem cells theory presumes that tumor is a stem cell disease and it is abnormal tissue of tumor cell which has the capacity of forming tumor proliferating. There is few cell having the capacity of forming tunor in tumor cell . Nowadays, the research of tumor stem cell has became a hot spot.In 1996,Goodell at first proposed that there were a few colored fairly shallow cells in Hoechst33342 colored bone marrow cells. He named them side population cells which contained many stem cell components. Not only their phenotype was the same as stem cell ,but they had very fortis capacity of reestablishing hematopoietic tissue. ABCG2 is a transport protein . It plays important effect in keeping cell's homeostasis and organism normal physiological functions .With the development of stem cell research, it is though that it participates the formation SP phenotype which is stem cell character.Side population cells expressed highly ABCG2 protein. They could pump Hoechst33342 out from cells. Compared with main population cells, side population cells showed fairly lepto Hoechst33342 stimulating fluorescence character.We sorted SP(Side Population) cells and MP(Non-SP) cells from Human Tongue Cancer Cell Line Tca8113 through fluorescence-activated cell sorting(FACS)and investigated the expression rate and significance of ABCG2 in SP cells ,MP cells and T(Tca8113) cells. The research may be a useful method to purity alike Tumor Stem Cell(TSC) and isolate TSC in following research.Research method and result1. the expressed significance of ABCG2 in human tongue cancerMethod: cultivate the human tongue cancer cell line Tca8113 in the RPMI1640 (15%CS), put the culture bottle in the CO2(5%) ,37℃incubator. When the cells proliferated to 70%~80% of the culture bottle's bottom, trypsinase(0.125%) digested and passaged. After creeped piece on the cover glass. cultivate Tca8113 in the six shadow mask.When seeing the cells growing well, we fixed cell creeping piece on the glass slide and used formaldehyde(10%) to fix it about 60~90 minutes. Then added ABCG2 antibody and FITC antibody, and detected ABCG2 expression of Tca8113 under immunofluorescence . In addition, collected the cells growing well , used 200 ul PBS to suspend the cells and added ABCG2 antibody and FITC antibody. Then detected ABCG2 expression of Tca8113 through FACS. Results: the cells which FITC colored emited virent fluorescent light and ABCG2 protein which FITC colored emited few virent light spot. The expression rate of ABCG2 in Tca8113 is about 5℅ by flow cytometry measuring. Experimental results indicated that human tongue cancer cell line Tca8113 contained SP cells like some cancer cells.2. side population cells dissociated from human tongue cancer cell line Tca8113Method: Collected 25 bottles of cultivated Tca8113 cells , Prepared about 108 cells for staining. Then used PBS(0.01%) to washout the cells and trypsinase(0.125%) digested them. After broke off diagesting, we used HBSS to washout them , suspended the cells and added Hoechest33342. At the same time, added Verapalmil in the control group.Used PI to mark dead cells. Detected and sorted SP cells which illuminated slightly through FACS. After removed PI+ dead cells, collected sorted SP, MP cells. Results: After colored by Hoechest33342, there were expressed only in 0.8% of the cells in human tongue cancer cell line Tca8113. Experimental results indicated that SP cells exited in human tongue cancer cell line Tca8113.3. detection and analysis of ABCG2 in human tongue cancer cell line Tca8113,MP cells and SP cellsMethod: Prepared about (1~3)×105 SP,MP,T cells for detecting.Used 200 ul PBS to suspend the cells and added ABCG2 antibody and FITC antibody. Then detected ABCG2 expression of Tca8113 through FACS. Results: The expression rate of ABCG2 in SP cells was about 13.16℅, and in T cells the expression rate was about 3.29℅, on the contrary ,there was nearly no expression in MP cells(1.07℅). Experimental results indicated that SP cells in human tongue cancer cell line Tca8113 overexpressed ABCG2.Research results indicated that SP cells exited in human tongue cancer cell line Tca8113 and we sorted them. So isolation of ABCG2+ cell from SP cells may lay experimental basis for puriting alike Tumor Stem Cell(TSC) . At the same time, it could provide new method and thinking for targeting to Tumor Stem Cell therapy.