Heterogeneous Expression Of AFP And CK19 In Different Subpopulations Of Hepatocellular Carcinoma Cells

There is no unified opinion of tumorgenesis in academic circles.Recently,researchers proposed the theory of cancer stem cells because the sameimmature cell phenotype was included in the tumor cell and stem cell.Researchers have isolated leukaemia cells that have the markers of HSCs fromacute myeloid leukaemia (AML) It reveals that the cancer stem cells might existin many tumors, which have both capacities of stem cells and tumorigenicity.Now the cancer stem cells have been found in many solid tumors, for examplebreast cancer, human train tumor etc. With the development of research,manyapproaches have been used for the disassociation and purification of cancer stemcells. Recently, more and more studies showed that there is increased evidence offinding a small side population (SP) both from established cancer cell lines andprimary tumors.SP cells were identified of having stem like characteristics, thecapacity for self-renewal to allow the maintenance of an undifferentiated stemcell population and the ability to undergo differentiation.HCC was common inour country.It is possible for the research of the carcinogenesis, palindromia, metastasis and drug fast through the disassociation of SP.AFP belongs to embryo antigen and plays important role in the body.CK19 iszighly expressed in the liver stem cell and regarded as the specific marker of bileduct.The expression of CK19 has negative correlation with the differentiation ofliver cancer cell.Aim:To investigate the significance of AFP and CK19 different expression indifferent subpopulations of MHCC97.Methods:1.To analyze the side population cells from human hepatocellular carcinomacell lines MHCC97, and to sort them in MHCC97. To assay the differentiationability of side population cells1)Culture cells as usual;2)0.25% trypsin digest cells, centrifuge them as usual and wash them by HBSS;3)Detect and sort SP cell and Non-SP cell.Aliquot the cells suspension into two labeled centrifuge tube. All two tubes ofcancer cells stain with the Hoechst dye, but one of tube add inhibitor of Ca2+channel to inhibit the exocytosis of ABCG2 in additionally. Both of two tubesincubate at 37℃on water bath. Then place Hoechst-stained cancer cells on thecytometer, excite the Hoechst dye at 355 nm and use dichroic mirror to separatethe emission wavelengths, collect Hoechst blue fluorescence with a 450nm bandpassfilter and Hoechst red fluorescence with a 675nm edge filter long-pass.Display the histogram of Hoechst red (x-axis) versus Hoechst blue (y-axis). Sidepopulation can be found on the histogram by establish gate. Compare the Hoechstfluorescence profile of two groups of cancer cells suspension4) According to the sequences of AFP and CK19 from Genebank, we design the primers of two genes by Primer5 soft, and send them to biological company forcomposting. Then, using the cDNA from side population and non-side populationcells as template, we do the semi-quantity assay.5) After being cultured on coverslips, we carry the immunofluorescence stainingassay of AFP, CK19 in two cells.2. Tissue chip of liver cancer was purchased for the immunohistochemicalstain of AFP and CK19.ResultResults:1. The side population can be observed on the fluorescence profile when theemission of Hoechst analyzed by dual-wavelength. The percentage of SP cells inMHCC97 is about 0.25%.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.2. Reverse transcription–polymerase chain reaction (RT-PCR) for AFP andCK19 gene.The results of AGE showed that the transcriptional level of AFP and CK19 insp cell group was significantly higher than that in the Non-SP cell group.3. ImmunocytochemistrySP cells and Non-SP cells were grown on coverslips respectively for 4 h, fixedwith 4% paraformaldehyde , then cells were exposed to 0.3% H2O2 in absolutemethanol at room temperature for 15 minutes to inhibit endogenous peroxidaseactivity. Immunocytochemical staining was performed with commerciallyavailable primary antibody and other reagents completed according to themanufacturer's protocol.Under confocal microscopy,AFP-positive grains andCK19-positive grains could be detected both in SP cell and Non-SP cell. Lg IOD(integrated optical density)was used for measurement of the expression ofAFP and CK19.These results showed that there were obvious difference of AFPand CK19 between SP cell and Non-SP cell.4. Immunocytochemistry of AFP and CK19 were performed in the Tissue chipof liver cancer.Under fluorescence microscope,buffy grains were partly expressed in thecytoplasm.The results showed that AFP was expressed in most liver cancer,CK19 was expressed in partial liver cancer and only a few liver cancer expressedboth AFP and CK19.ConclusiConclusion:1. The side population cells existed in human hepatocelluar carcinoma cell linesMHCC97.2. The side population cells from MHCC97 co-expressed AFP and CK19, it wasinferred that the side population cells had bilateral differentiation ability tosome extent. It suggested that side population cells had some primitivecharacteristics.3. The liver cancer with the expression of CK19 has the high relapserate.Because of the absence of ck19 in normal liver,it suggested that ck19positive HCC origined from the progenitor cell.