| Objectives:We used flow cytometry and Hoechst 33342 dye efflux assay to isolateSide population cells and Main population cells from MHCC97; Then we usedsoft agar cloning methods and tumor cell implantation experiments in test fortumorigenicity of two subpopulations; The two Subpopulations were inducedby 10-hydroxycamptothecin; RT-PCR showed their differentiation potential.METHODS:1. Culture MHCC97 cells as usual and prepare the liver cancer cellsuspension .2. Divide the cancer cells suspension into two labeled centrifuge tube. Alltwo tubes of cancer cells stain with the Hoechst dye, but one of tube addinhibitor of Ca2+ channel in additionally. Both of two tubes incubate at 37oC onwater bath. Then place Hoechst-stained cancer cells on the cytometer, excite theHoechst dye at 355 nm and use dichroic mirror to separate the emissionwavelengths, collect Hoechst blue fluorescence with a 450nm band-pass filter and Hoechst red fluorescence with a 675nm edge filter long-pass. Display thehistogram of Hoechst red (x-axis) versus Hoechst blue (y-axis). Side populationcan be found on the histogram by establish gate. Compare the Hoechstfluorescence profile of two groups of cancer cells suspension.3. Then culture SP cancer cells and MP cancer cells respectively into softagar.Observe the clone formation of two types of liver cancer cells in vitro.4. Subcutaneously inject the 1×105,1×104,1×103 SP phenotype and MPphenotype liver cancer cells into nude mouse respectively. Observe the growthof xenograft tumors.5. The two Subpopulations are induced by 10-hydroxycamptothecin .AT thesame time we culture SP cells and MP cells as usual for contrast. Then weobserve the proliferation and shape of SP cells and MP cells by microscope.6. After induction, we extract the total RNA from the SP phenotype cellsand MP phenotype cells respectively. RT-PCR test the changes of AFP,ALB,GGT and G6P of SP cells and MP cells.ResultsResults:1. MHCC97 cells grow well. After digestion we can get liver cancer cellsuspension at 1×109/L.2. The side population can be observed on the fluorescence profile whenthe emission of Hoechst analyzed by dual-wavelength. The percentage of SPcells is about 3.9%. When the inhibitor of Ca2+ channel is used, the sidepopulation eliminate because of inhibition of efflux mediated by the ABCtransporter. The SP cells enter into the main population on the fluorescenceprofile because not have low fluorescence characteristics.3. The sorted SP cancer cells grow well and have very highrate of clone formation in the soft agar; In contrast, the MP cancer cells shows growth retardation and have very low rate of clone formation under the samecondition. It demonstrates that the SP cancer cells have very large capacity ofproliferating.4. Tumor cell implantation experiments showed that only 1×103 SP cellswere sufficient for tumor formation, whereas an injection of 1×105 MP cellscould initiate tumors. It demonstrates that the SP cancer cells have very largecapacity of tumorigenicity.5. After 3 days,induction, the morphology changes greatly. The shape ofSP cells become large and stretch, binuclear cells can observed about 15% ,andabout 87% of nucleoluses have 2~4 nucleoluses.But MP cells only have 2% ofbinuclear cells, and about 90% of nucleoluses have only one nucleolus.6. The expression of alpha fetoprotein andγ-glutamyltransferase werelower in induced SP cells, while albumin was higher after induction. Theglucose-6-phosphate was only been detected in induced SP cells.ConclusionsConclusions:1. We can use flow cytometry and Hoechst 33342 dye efflux assay toisolate SP cells and MP cells from MHCC97.2. SP cells that show higher proliferative potential,tumorigenicity anddifferentiation potential compared with MP cells., indicat that they arehepatocarcinoma stem cells.
|
PDF Full Text Request