| Background: Cancer stem cells (cancer stem cells, CSCs) is the researchhotspot in recent years, Cancer stem cell theory which is put forward for theclinical treatment of tumor targeting pointed out the direction, also broughtopportunity to cure tumor. Cancer stem cell theory is that cancer stem cells arethe few cells that present in the tumor cells, has unlimited proliferation abilityand differentiation potential, has the strong ability of self-renewal,enlightenment, drug resistance, and have a close relationship with metastasisand recurrence of tumor. The proportion of cancer stem cells in tumor cells isextremely low, that brought difficulties to cancer stem cells research, under thedilemma, it is very important how to purification and enrichment of cancer stemcells. At present, enrichment and sorting cancer stem cells mainly throughself-renewal, proliferation, resistant properties and related biomarkers of cancerstem cells. The commonly used method is Drug-resistant cell sorting，(magneticActivated cell sorting, MACS),(fluoresc-owing-Activated cell sorting,FACS)and (side population, SP) separation method，Conditions of cultivation sorting，and so on, but these methods have their defects. Drug-resistant cell sorting is theuse of resistant properties of cancer stem cells, with low dose chemotherapy drug intervention so as to achieve the purpose of enrichment of cancer stemcells, but in the long process of resistance to chemotherapy, cancer stem cellsurface markers and cancer stem characteristics may be changed, which is notuseful toresearcht the cancer stem cell biology.IMACS and FACS needs to becombined with the specific markers of cancer stem cells, but the problem ofsorting cancer stem cells is the determination of surface markers, due to the lackof specific surface markers of cancer stem cells, which limits the developmentof the two methods. SP is to use Hoecsht33342nucleic acid fuel cells to do withtumor cells, Hoecsht33342can emit blue fluorescence under uv excitation, dueto the cancer stem cell surface ABCG2gene, the nucleic acid Hoecsht33342fuel pump out of cells and shows low dye or don’t dye, using flow cytometryinstrument according to the strength of the tumor cell staining sorting out thesecells that don’t dye or low dye cells, but the purity of tumor stem cells by thatmethod was more and more questioned.Conditions of cultivation sorting is thesuspension culture separation method, it is though adding suitable amount ofepidermal growth factor and basic fibroblast growth factor instead of fetalbovine serum in serum-free medium, the two factors was confirmed to havethe effect of promoting the proliferation inhibition differentiation for stem cell,serum-free stem-cell cultures keep the undifferentiated state of stem cells,cancer stem cells in serum free medium conditions can survive but thenon-cancer stem cells can not, to enrichment cancer stem cells, but thesuspension culture separation method completely changed the growth conditionof cancer stem cells, may go against the cancer stem cells research. In recentyears,researchers have proposed using unicellular culture to enrich cancer stemcells, according to the single cell cloning can be divided into (holoclone, H), (meroclone, M) and (paraclone, P), in which H is proved to contain CSCs. Inthis study, this research is based on the resistance characteristics of cancer stemcells, using nude mice in vivo adriamycin intervention preliminary enrichmentof human hepatocellular carcinoma cell, establishing the drug-resistant cell lineBEL-7404-ADM-P4, but the resulting proportion of cancer stem cells is stillvery low, did not achieve the expected results.In order to explore a moreeffective method, in subsequent studies, this research selects the parent cellBEL-7404and drug-resistant cell line BEL-7404-ADM-P4cells limited dilutionunicellular culture constructe the three forms clones and detected stemcharacteristics, hoping to find out the clone enrichment mode which containshigh proportion of liver cancer stem cells，to build solid foundation for thesubsequent study on cancer stem cells.Objectives:Taking BEL-7404as the research object, nude mice in vivoadriamycin intervention based on tumor drug resistance characteristicsestablished resistant cell line; Selecting the parent cell BEL-7404anddrug-resistant cell line BEL-7404-ADM-P4unicellular culture form three typesof clones and detecting its cancer stem characteristics, in order to extractcontaining high proportion of liver cancer stem cell cloning enrichment method.Methods: Select HCC BEL-7404cell line as the research object, throughthe nude mice in vivo adriamycin intervention extend the preliminary filterringof liver cancer stem cells in the sample cell, then joint unicellular cultureconstruction of enrichment model of liver cancer stem cell in vitro. Theexperiment method is as follows:(1) nude mice in vivo adriamycin interventionextend the preliminary screening of liver cancer stem cells characteristics: toestablish nude mice subcutaneous transplantation tumor animal models by human liver cell line BEL-7404, with adriamycin (8mg/kg) tail intravenousinjection, once a week，give original generation of culture until the tumordiameter was1.5cm, and four generations consecutive batches in the body, andname the4th generation tumor cell BEL-7404-ADM-P4.(2)single cell culturein vitro enrichment of liver cancer stem cells: take parental cell line BEL-7404and the four generations of nude mice in vivo adriamycin intervention tumorcell line BEL-7404-ADM-P4, single cell clone culture with limited dilutionmethod, and observed the clone morphological classification under invertedmicroscope on the12th day, clones can be divided into three types, that is(holoclone, H),(meroclone, M) and (paraclone, P).The three type clones cancer stem characteristics detection:(1) theproliferation ability detection:①single cell clone growth curve;②theagain-clone forming experiment: to compare the clone formation rate ofdifferent types of cloning and different types of cloning than;③suspension ballformation experiment: to compare the different types of cloning floating ballformation rate;(2) resistance experiment:Hoechst33342dying condition;(3)real time fluorescence quantitative PCR to detect cancer stem markers;(4) nudemice tumor experiment.Results:(1) Low doses of doxorubicin in the body nude mice intervention,in the process of straight into a tumor in the body, nude mice subcutaneoustransplantation tumor tumor rate were100%, and time from the first generationto the fourth generation into tumor have been shortened, what is more，thefourth generation of resistant index is higher22times than the first generation,toachieve the preliminary enrichment of the liver cancer stem cells;(2) Proliferation ability:①single cell clone growth curve:the H proliferation was the fastest, cell volume is the largest,the M was second,and theP proliferation was the slowest, and on the10th day started to shrivel to death;②Clone formation rate: the BEL-7404, BEL-7404-H, BEL-7404-M,BEL-7404-ADM-P4, BEL-7404-ADM-P4-H, BEL-7404-ADM-P4-M cloneformation rate is respectively (72.03+1.54)%,(80.67+1.80)%,(68.90+1.28)%,(74.13+7.64)%,(84.77+1.46)%,(69.60+3.32)%, the BEL-7404-ADM-P4-H is higher than the BEL-7404, BEL-7404-H,BEL-7404-M,andBEL-7404-ADM-P4-M, P <0.05, the difference was statistically significant；And its again clone formation experiment in BEL-7404-H, BEL-7404-M,BEL-7404-ADM-P4and BEL-7404-ADM-P4-M, BEL-7404-ADM-P4-Hcomponent ratio is higher than BEL-7404, P <0.05, the difference wasstatistically significant.③Suspension ball formation rate: only the BEL-7404-H and BEL-7404-ADM-P4-H can form floating ball, the rest of the celllines do not form a floating ball,BEL-7404-H and BEL-7404-ADM-P4-Hfloating ball formation rate is respectively (8.79+5.62)%,(13.78+10.49)%,and BEL-7404-H and BEL-7404-ADM-P4-H floating ball formation ratecomparison, P>0.05, no statistical significance;(3)resistance experiment:dyeing conditions by hoechst33342: there were afew cells don’t been dyed and low dye cells exist in the BEL-7404-H andBEL-7404-ADM-H,but M and P were whole dyed, and all BEL-7404monoclonal clones hoechst33342fluorescence intensity is stronger than theBEL-7404-ADM-P4monoclonal clones.(4) Real-time fluorescent quantitative PCR detection of cancer stemmarkers: detected five cancer stem surface markers from the BEL-7404,BEL-7404-H,BEL-7404-ADM-P4and BEL-7404-ADM-P4-H,ABCG2, ALDH1A3, CD44, CXCR4and OCT4； ABCG2, CD44, CXCR4inBEL-7404-ADM-P4-H is higher than the BEL-7404, BEL-7404-H andBEL-7404-ADM-P4, P <0.05, the difference was statistically significant. Andeach cell line ALDH1A3, OCT4expression has no obvious difference.(5) Nude mice tumor experiment:in BEL-7404,BEL-7404-H, BEL-7404-M,BEL-7404-ADM-P4, BEL-7404-ADM-P4-H and BEL-7404-ADM-P4-M tumorexperiment, under the condition of cell amount1.0×104，only the BEL-7404-Hand BEL-7404-ADM-P4-H into tumor with nude mice,BEL-7404-H andBEL-7404-ADM-P4-H nude mice tumor rate is respectively1/8，7/8，BEL-7404-ADM-P4-H nude mice tumor rate higher than that of BEL-7404-H,BEL-7404-ADM-P4-H tumors had the expression of Ki67is stronger than theBEL-7404-H, P <0.05, the difference was statistically significant.Conclusions:①Nude mice in vivo adriamycin intervention based ontumor drug resistance characteristics build drug-resistant cell line successfully;②Drug resistance cell line BEL-7404-ADM-P4-H showed stronger abilityof self-renewal and proliferation, drug resistance and tumorigenicity;③Basedon resistance characteristics of cancer stem cells joint unicellular culture method,the holoclone that was intervened by ADM contains a higher proportion of livercancer stem cells. |
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