| Breast cancer,the incidence rate of lower than the most common malignant tumors of the lung in the worldwide,has become the most important factor of death in women 20 to 59-year-old.It’s a serious threat to women’s physical and mental health and even life all over the world.Early breast cancer surgery is easy to relapse after treatment,breast cancer patients with advanced concurrent transfer treatment effect is poorer,and the survival time is short,high mortality rate.The requirements in the clinical work,we are going to further study the molecular mechanism of breast cancer occurrence and development,to seek more safe and effective chemotherapy drugs for breast cancer treatment,prolong the median survival time of patients,and improve the quality of life.In 1994,the separation of cancer stem cells in leukemia,part of scholars believe that tumor tissues with self-renewal and differentiation potential cells plays an important role in the onset of tumor,driven by tumor cells lose control to enhanced resistance to radiation and chemotherapy.Cancer stem cell hypothesis for us the clinical treatment of malignant tumor provides a new research direction.Part of a tumor have self-renewal ability and can produce heterogeneity of tumor cells,with unlimited proliferation and differentiation potential of cells,called Cancer stem cells,(Cancer stem cells,CSCs).There are also such in Breast Cancer cells,called Breast Cancer stem cells(Breast Cancer stem cells,BCSCs).Further study on the biological characteristics of BCSCs,understand and find the distinguish method of BCSCs the clinical treatment of breast cancer has important significance.CD44+CD24-/low,is now the most commonly used sorting BCSCs markers.Mainly by adding bFGF and EGF in laboratory of these two kinds of growth factors DMEM/F12 serum free medium suspension into a ball,set up the biology of breast cancer stem cell model in vitro.Suspension culture techniques for breast cancer stem cells into a ball further research provides a feasible technical support.Arsenic trioxide(As2O3)is the most important in traditional Chinese medicine in malignant cancer therapy.Recent research suggests that arsenic trioxide on acute early young leukemia plays an important role,mainly through the acute early fine grain leukemia protein(PML(RAR)regulation,make its degradation,thus eliminating leukemic cells,to effect a radical cure.PML(RAR)also has high expression in breast cancer stem cells,we explore and research the role of arsenic trioxide on breast cancer stem cells and the related mechanisms and clinical treatment for breast cancer provides a potential theoretical basis.Part 1 Arsenic trioxide after treatment of breast cancer stem cells of biological characteristics and mechanism of PML geneMaterials and methods1.Cell adherent culture and suspension culture MCF-7 cell adherent culture,with RPMI-1640 medium inactivated(containing 10%fetal bovine serum),37℃in5%CO2,cell incubator in conventional culture,1~2 days in liquid,extend the 3~4days.The sidewall of MCF-7 cell suspension in DMEM/F12 medium(including 20ng/mLhEGF,5 ug/mL insulin B27 and 0.4%bovine albumin),37℃in 5%CO2,cell suspension culture in the incubator,3-4 days and an half amount in liquid culture,subculture 7 to 10 days.2.Balloon CD44+CD24-/low proportion of cancer detection Collect serum-free culture of micro balloon cancer cells,pancreatic enzyme digestion after mechanical beat for single cell suspension,with PBS liquid suspension cells,each tube is about 1 x 106/100 ul.Set up the experimental group and type with the control group respectively.In the experimental group with CD24-PE and CD44-FITC monoclonal antibody,the control group,belong to the same type of corresponding control antibody at room temperature away from light incubation for 30 min,PBS with 500 ulpbs heavy suspension after washing twice,upflow cell instrument detection of breast cancer within 1 h MCF-7cell micro balloon CD44+CD24-/low cell proportion,repeat test three times.3.Determine the viability of mammosphere cells Collect serum-free culture of micro balloon cancer cells,pancreatic enzyme digestion after mechanical beat for single cell suspension,concentration of cells in 5 x 105/mL vaccination in 96-well plates,getting 5 ul,every hole set in the control group(without any reagent)and experimental group,each group of six complex hole,37℃in 5%CO2,cell suspension culture in the incubator for 24 h,48 and 72 h,application reduction method to detect different concentrations WST-1 set of MCF-7cell micro balloon breast cancer cell proliferation inhibition,growth inhibition curve drawing.The experiment repeated three times.4.cell cycle examination Collect serum-free culture of micro balloon cancer cells,pancreatic enzyme digestion after mechanical beat for single cell suspension,with 5 x 106/mL cell concentration vaccination in 96-well plates,getting 10 every hole ul,set in the control group,2.0 umol/L,and 8.0 umol/L group,37℃in 5%CO2,cell suspension culture in the incubator of 48 h,collect groups of cells,pancreatic enzyme digestion after the mechanical blowing in single cell suspension,to go on after low-speed centrifugal clear liquid,add 1 mL ice bath precooling 70%ethanol,4℃for 12 h,0.5 mL iodide c organism within 24 h after dyeing liquid flow detection.5.RT-PCR MCF-7cell group analysis of breast cancer,breast cancer MCF-7balloon cells group,the concentration of 2 umol MCF-7suspension balloon As2O3 treatment breast cancer cells after 48 h group PMLmRNA transcription;6.Western bolt MCF-7cell group analysis of breast cancer,breast cancer MCF-7balloon cells group,the concentration of 2 umol As2O3 processing MCF-7suspension balloon breast cancer cells after 48 h after the expression of PML protein group.7.statistic analysis All data were analyzed by variance(ANOVA)method using SPSS(17.0).measurement data using mean±standard deviation(x±s)to show.The comparison between multiple sets of measurement data using single factor analysis of variance,two comparison using LSD test comparison differences between groups.It’s statistically significant for P<0.05.Results1.MCF-7 cell adherent culture and cell suspension culture form2.CD44+CD24-/low proportion of breast cancer stem cells Adherent cultivation and suspension balloon MCF-7in breast cancer cells of CD44+CD24-/low proportion of breast cancer stem cells(1.83+0.25)%,(62.39+2.77)%,statistical analysis showed that the difference was statistically significant(F=7.4878,P=0.000<0.05).3.Determine the viability of mammosphere cells In arsenic trioxide concentration is 0.5~2.0 umol/L,within the limits of a lower overall survival time trend of balloon cells was increased(F=31.778,P=0.000<0.05),the low concentration As2O3 may induce breast cancer stem cells and promote its value,when the concentration is 2.0 umol/L,when handling 48 h balloon cell survival rate for the highest;When the concentration of arsenic trioxide is 2.0~8.0 umol/L range,higher total survival time trend of balloon cells was lower(F=18.781,P=0.000<0.05),indicating high levels of As2O3 is likely to inhibit breast cancer stem cells4.cell cycle examination Set in the control group and experimental group,without the MCF-7balloon As2O3 treatment of breast cancer cells(control group)in G0/G1,G2,and proportion of S phase cells were 91.70±1.16,4.87±0.37 and 0.37±1.09;Concentration is 2.0 umol/MCF-7balloon LAs2O3 processing cells in G0/G1,G2(experimental group)and the proportion of S phase cells were 54.55±1.44,10.89±0.28 and 0.28±1.22;Concentration is 8.0 umol/MCF-7balloon LAs2O3processing cells in G0/G1,G2(experimental group)and the proportion of S phase cells were 23.51±0.61,55.44±0.96 and 0.96±0.41,the apoptosis rate was(6.23+0.61)%.Through the statistical analysis showed that no medicine group from one period to the cell proportion and different drug concentrations in each phase of the cell proportion compared distribution is different,the difference was statistically significant(P<0.05).5.RT-PCR Breast cancer MCF-7cell PMLmRNA transcription level is lower than the MCF-7balloon breast cancer cells;MCF-7suspension balloon and breast cancer cells treated with 2 umol concentration of As2O3 after 48 h,its PMLmRNA MCF-7balloon transcription level and breast cancer cells have no obvious difference.6.Western bolt Without As2O3 MCF-7suspension balloon cells and through the concentration of 2 umol As2O3 after 48 h of MCF-7cell suspension balloon,PML protein expression significantly decreased(P=0.000).Breast cancer MCF-7cell less application of As2O3 MCF-7suspension balloon cell group of PML gene protein expression decreased(P=0.000<0.05).Part 2 The construction of carrier and nude mice into tumorMaterials and methods1.For known PML protein gene cDNA sequence,sequence design and synthesis of four specific interference(PML(shRNA)-1,2,PML(shRNA PMLshRNA--3,the PML(shRNA)-(4)and 1 the nonspecific sequence(PML(shNC).2.Using liposome mediated transfection technology will shRNA MCF-7 cell transfection to breast cancer,through a semi-quantitative reverse transcription-polymerase chain reaction(rt-pcr)and protein imprinting technology(Western blot)detection SNCG gene is interference in the expression of mRNA and protein level after the change.3.Respectively with the turn and then MCF-7 suspension balloon treated cells,arsenic trioxide MCF-7 suspended balloon cells and MCF-7 suspended balloon planting transplanted tumor cells subcutaneously in nude mice.Results1.Liposome mediated transfection technique of PML(shRNA)can be successfully MCF-7 cell transfection to breast cancer.2.Rt-pcr and Western blot results show that the PML(shRNA MCF-7 cell transfection to breast cancer,PML protein gene in the expression of mRNA and protein levels were significantly lower(P<0.05).3.MCF-7 cell suspension balloon in 10~3/mL concentration can subcutaneous tumor,and treated by arsenic trioxide MCF-7 cell suspension balloon and the PML(shRNA transfection to breast cancer has not been MCF-7 cell into a tumor.Conclusion1.With cytokines in serum free medium under the condition of low adhesion can effective enrichment of breast cancer stem cells.2.Low concentration As2O3 MCF-7 balloon cells can be induced differentiation into split proliferation and promoting the proliferation;High concentrations of As2O3can inhibit the differentiated MCF-7 balloon cell proliferation and induce its apoptosis.3.Arsenic trioxide has inhibitory effect on breast cancer stem cells,may cut PML protein way through implementation.4.The PML(shRNA MCF-7 cell transfection to breast cancer,PML protein genes were obviously down-regulated in the expression of mRNA and protein levels.5.Breast cancer stem cells in 10~3/mL concentration level can make the nude mouse skin grow into the tumor,and arsenic trioxide treatment of breast cancer stem cells under the same concentration can’t into the tumor,breast cancer stem cell characteristics were identified. |
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