Study On Biological Characteristics Of CD133~+Cells Separated From A Gallbladder Cancer Cell Line GBC-SD

Purpose To establish the method of isolation and purification of CD133+gallbladdercarcinoma cells and investigate the different biological characteristics of CD133+cellsand CD133-cells in GBC-SD cell lines.Methods Magnetic activated cell sorting (MACS) method was used to isolateCD133+gallbladder carcinoma cells, Flow cytometry was used to determine theproportion of CD133+in gallbladder carcinoma cell lines, CD133mRNA expressionwas evaluated by half quantitative polymerase chain reaction, CD133protein wasevaluated by western blot and immunofluorescence. The growth of single CD133+subpopulation cells in suspension culture was observed by colony formation assay.Transplanted tumor on nude mouse were used to evaluate tumorigenesis abilities. Thedifferences of cell proliferation and drug resistance to5-fluoropyrimidinedione(5-FU)and gemcitabine between CD133+group and CD133-group were evaluated by CellCounting Kit8-assay(CCK8).The invasion ability of CD133+group and CD133-groupwere evaluated by Transwell. The expression of stem cell marks were determined usinghalf quantitative polymerase chain reaction.Results Two gallbladder carcinoma cell lines including GBC-SD, SGC996were foundto contain (68.5±2.9)%,(0.3±0.2)%of CD133+subsets cells respectively. The relativemRNA expression levels of CD133in GBC-SD (0.6466±0.0259) was higher thanSGC996(0.2181±0.0108, P=0.002). CD133mRNA expression in CD133+group(0.7734±0.0217) was significantly stronger than that in CD133-group(0.2146±0.0174,P=0.001). CD133protein expression in CD133+group (0.3689±0.0375) wassignificantly stronger than that in CD133-group(0.0345±0.0040, P=0.003).Thepercentage of CD133+cells in CD133+groups was (90.2±2)%. The colony formationshowed single CD133+cells can form the new big sphere, and the formation rate ofcolony sphere in CD133+group [(36.25±2.99)%] was higher than that in CD133-group[(4.5±1.29)%, P=0.006)]. The nude mouse transplantation tumor experimentshowed a remarkable difference of tumor formation rate between CD133+group(100%),non-sorted group(60%), CD133-group(0). The invasion cells of CD133+group(23.78±8.74) was stronger than that of CD133-group(6.56±3.09, P=0.0007). TheRT-PCR showed the relative mRNA expression levels of expression of ABCG2(0.8774±0.0191) and CD44(1.3681±0.0879) in CD133+group was significantlystronger than that in CD133-group[0.3276±0.0272, P=0.001]，[0.7891±0.0385,P=0.005].Conclusion The high purity of CD133+gallbladder carcinoma cells can be successfullyisolated by MACS. Furthermore, the CD133+subsets of gallbladder cancer possesssome properties including self-renewal, tumorigentic ability, proliferation,chemoresistance and invasion ability in vitro and vivo. Also, they can express somestem cells marks.