| Objective: To study the sorting of CD133+liver cancer cells have thefeatures of cancer stem cells,131I-CD133mAb prepared by chloramine Tmethod.using its radiation immune function, analyze131I-CD133mAbbiological effect on CD133+human liver cancer stem cells in vivo and invitro, to discuss the feasibility of its radiation immune therapy for livercancer.Part1Sorting of CD133+liver cancer cells and confirmation oftheir stemness propertiesMethods：Using flow cytometry (FCM), detected the high expressionof CD133cells from liver cancer cell line Huh-7and HepG2cells. Usingmagnetic-activated cell sorting (MACs), sorted out CD133+cells fromHuh-7cells, and the expression of CD133after sorting were detected byflow cytometry. The stemness properties of sorted CD133+cells werevalidated by spheres-forming assay, colony formation assay in vitro andxenograft experiment in vivo.Results：After sorting by flow cytometry, CD133expression rate ofHuh-7and HepG2cells is18.80%and5.2%, respectively, so we choseHuh-7cells to next experiment.CD133+cells were successfully obtainedfrom Huh-7cells by MACs, and the sorted CD133+cells showed a highpurity of99.28%. Tumor-sphere formation assay showed that CD133+cells could form spheres and grow suspended in serum-free culture mediacontaining grow factors. As time passed, the cell spheres increased in size,whereas the CD133-cells failed to stay alive in the serum-free culture media.The results of colony formation assay showed that CD133+Huh-7cellsshowed higher ability of clone formation than CD133-Huh-7cells (P<0.01).Xenograft tumorigenicity assay showed that as few as1×103CD133+cellswere sufficient to form subcutaneous xenografts in BLAB/c mice4weeksafter inoculation. However, no tumors were observed in inoculation of1×103CD133-cells.Conclusion：These results reveal that CD133+Huh-7cells demonstrathigher tumor-spheres formation capability，colony formation capabilityand tumorigenesis capacity than CD133-cells, which could be considered asCSC-like subsets of liver cancer cells.Part2The preparation of131I-CD133mAb and identificationMethods：By chloramine T method,CD133mAb marked131I anddetermine the markup rate by the paper chromatography.131I-CD133mAbwould be purified by Sephadex G50chromatography column, anddetermination of specific activity and radiochemical purity. Under thecondition of37℃, the purification of131I-CD133mAb mixed with humanserum, after24,48,72h, respectively, detection its radiochemical purity ofchange.131I-CD133mAb and CD133+Huh-7cells in37℃incubationbox on30,60,90,90min, detection the cellular immunity in combinationwith rate.Results：The markup rate of131I-CD133mAb is87.92%by paperchromatography, the radiochemical purity of131I-CD133mAb is97.54%after Sephadex G50chromatography column purification and the radioactivity specific activity is0.91MBq/ug. After purification of131I-CD133mAb mixing with fresh people serum, after incubation for24,48,72h in37℃, the radioactive chemical purity is95.43%,93.06%and90.57%, respectively.on each time the radiochemical purity were higher than90%, there was no significant difference (P <0.05).131I-CD133mAbcombined with CD133+Huh-7cells after30min, the immune rate was (38.49+0.91)%, after60min,the immune rate was (56.36+0.45)%, after90min,the immune rate was (69.10+0.55)%, after120min,the immune rate was(64.80+0.35)%, they were higher than the one of131I-CD133mAbcombined with CD133-Huh-7cells.Conclusion：131I-CD133mAb prepared by chloramine T have thehigher mark rate,the better stability and the higher cellular immuneactivity.Part3131I-CD133mAb effect on CD133+human liver cancer stemcells in vitroMethods：The first time,131I-CD133mAb,131I, CD133mAb werediluted into five levels,9.6ug/100ul,4.8ug/100ul,2.4ug/100ul,1.2ug/100ul,0.6ug/100ul, respectively, they join in the sorting CD133+Huh-7cells and after24h, detect each group of CD133+Huh-7cell growthinhibition of the optimum dose by MTT method; Then the CD133+Huh-7,Huh-7cells and HepG2cells be took experiments, each cell can be dividedinto four groups (131I-CD133mAb group,131I group, CD133mAb group,131I+CD133mAb group), with131I concentration is about4.8MBq/mL,CD133mAb concentration is4.8ug/100ul. After24h,48h,72h, detect thecell inhibition rate of the group by MTT method. Using flow cytometry totest131I-CD133mAb role respectively CD133+Huh-7, Huh-7cells andHepG2cells after72h, the change of the three kinds of cell apoptosis and cell cycle;Results：In concentration screening experiment,131I-CD133mAb,131Iand CD133mAb on CD133+Huh-7cell growth all have different degrees ofinhibitory effect. when the concentration of131I radioactive4.8MBq/100ml, CD133mAb concentration of4.8ug/100ul, there have the greatestdifference between the131I-CD133mAb group,131I and CD133mAb group (p<0.05). so we selected experiment that131I concentration is about4.8MBq/mL, CD133mAb is4.8ug/100ul.We determined to detect in the131I-CD133mAb of CD133+Huh-7cell inhibition rate was significantlyhigher in131I, CD133mAb group,131I+CD133mAb group by MTT, Theinhibition rate was (30.69+0.11)%,(46.54+0.70)%and (63.47+0.96)%on24h,48h and72h,respectively. The cell apoptosis rate of131I-CD133mAb effect on CD133+Huh-7cells after72h by flow cytometry,CD133+Huh-7group cell apoptosis rate (the sum of the early apoptosis andlate apoptosis) was31.21%, significantly higher than the Huh-7cell groupwere12.09%and6.66%of HepG2cells group (p <0.05). G0/G1phase ofCD133+Huh-7group cells was from81.02%to26.25%, the most cells intoG2phase and S phase, and gradually apoptosis. G0/G1phase ofCD133+Huh-7cell group decreased by54.77%, significantly higher thanHuh-7cells19.04%and HepG2cells10.32%(p <0.05).Conclusion：All the experimental results showed that the strongestinhibitory effect on131I-CD133mAb of CD133+Huh-7cells group,significantly higher inhibitory effect than the131I and CD133mAb onCD133+Huh-7cells. the CD133mAb inhibition was the weakest. And131I-CD133mAb have a specific target inhibition on high CD133expression ofCD133+Huh-7cells. Part4131I-CD133mAb effect on CD133+human liver cancer stemcells in vivoMethods：The cell concentration of5×104/200ul CD133+Huh-7cellsand Huh-7cells grown in BLAB/c nude mice subcutaneous, being xenografformation, the differences of CD133expression in xenograf tissue undermicroscope after immunohistochemical staining. The xenograf formation ofCD133+Huh-7cells as5×104/200ul, the12mice were divided into normalsaline control group,131I-CD133mAb group,131I group and CD133mAbgroup.Through tail vein we injected into physiological saline100ul, or131Iradioactive intensity4.8MBq/100ul and CD133mAb concentration4.8ug/100ul related fluid100ul, respectively. After28days, measuring xenografsize,weight, and the tumor inhibition rate. Observe the histological changesunder microscope after H&E staining.Results：Immunohistochemical study shows the CD133expression ofCD133+Huh-7cells in xenograf tissue was a little higher than Huh-7cellsxenograf tissue expression.131I-CD133mAb groups xenograf volume wereaccumulated (0.19+0.01) cm3, it had significantly less than the other groups.The xenograf weight were (1.66+0.07),it was significantly lighter than theother groups, YiLiuLv (85.21+2.53)%is significantly higher than the restof the two groups, it was statistically significant difference (p <0.05). H&Estaining showed that in131I-CD133mAb group xenograf the most tumor cellswere necrosis, lysis and the glass sample.Conclusion：131I-CD133mAb can specificity restrain the growth of theCD133+Huh-7cells in BALB/c nude mice body, and play a role of specifictargeting. |
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