Isolation, Culture, Preliminary Identification And Therapeutics Research Of Brain Tumor Stem Cells Within Glicoma Tissue In Vitro

Objective:The expression of ABCG2 and CD133 and their significance in human glioma.Isolation,Incubation and Preliminary Identification of Cancer Stem Cell in Human Gliomas.Experimental study of the killing effect on human brain Glioma stem cells by brachytherapy with¹²⁵I seeds.Effects of BCNU on cell proliferation and cell cycle in human brain tumor stem cells.Methods:The expression of ABCG2 and CD133 were determined by immunohistochemical staining in 59 surgical specimens of human brain glioma and 5 normal brain tissue to observe the positive cells.these specimens were also stained by HE to get the total number of tumor cells and classificate the gliomas.Gliomas samples were got from twenty-one patients.Tumor from patients undergoing tumor resections were acutely dissociated,triturated into single calls in sterile DMEM-F12 medium and then filtered.The tumor cells were seeded at a concentration of 100000 viable cells per mL into serum free DMEM-F12 medium simply supplemented with B27(1:50),human basic fibroblast growth factor(20ug/L),human epidermal growth factor(20ug/L),leukaemia inhibitory factor(10ng/L),insulin(4uL), L-Glutamine,penicillin and streptomycin.After the primary brain tumor spheres(BTs) generated,they were separated again and passaged in fresh medium.Monoclonal culture were progressed simultaneously.Brain tumor spheres were seeded in serum DMEM-F12 medium,then the differentiation capability was detected by immunocytochemistry and immunofluorescence.The tumor from patients undergoing tumor resections were acutely dissociated,triturate into single cell and then filter,the cells were seeded into the serum-free medium,make the gliomas stem cells of gliomas cells proliferating.We perform immunocytochemistry of gliomas stem cell for CD133 express,then calculate the proportion of CD133 positive cells. Use a part of gilomas stem cells serial subcultivation,after 3-5 days there are a lot of cell sphere forming.Make single cell suspension which density is 13×104.Separate the cells into 4 groups and culture in 96 shadow mask.Each group set three time stage(20,40,60hours)according to irradiation dose,each stage have four hole,each hole has 200μl.The number of cells are 2.6×104.Use three of four holes to calculate the dead cells and the one hole for growth inhibiting.Meanwhile take neural stem cells(provided by kunming medical college institute of neuroscience)to culture 3-5 days,after forming cell sphere,make single cell suspension.Setting control group in the same dose as gliomas stem cells.Then use different dose of¹²⁵I seeds in plate and culture together for 20,40,60 hours.Count the CD133 positive cells and observe the change of cells;determine the growth inhibiting of the glioma stem cells by brachytherapy of ¹²⁵I seeds by the measure of cell clone formation.,the same time, compare to neural stem cells,observe the sensibility difference by brachytherapy of ¹²⁵I seeds.Fresh surgical specimen was obtained from one case with ependymocytoma,and was prepared in single cell suspensions firstly,then cultured in serum-free medium and in serum supplemented medium in order to acquire BTSCs and BTCs respectively.7d,cell will be identifid by immunocytochemistry method(CD133 and nestin),Cells were be treated with different concentrations of BCNU(1ug/ml,0.1ug/ml,0.01ug/ml,0.001ug/ml,0.001ug/ml)for 72h,then the viability of each group was measured by MTT method.Cells were also treated with 0.01ug/mL BCNU for a variety of time spans,then cell cycle was examined by PI fluorescence flow cytometry.Results:(1)There were no expression in normal brain tissue.(2)ABCG2 and CD133 positive cells were observed in all glioma specimens.Respectivly,the differences of positive cell rates of ABCG2 or CD133 in different pathological grades were statistically siginificant(p＜0.05),the higher pathological grades have higher positive cell rates.(3)There were siginificant difference between the positive cell rates of ABCG2 group and CD133 group(p＜0.05).The expressions of ABCG2 group were lower than CD133 group.There was a siginificant correlation between ABCG2 group and CD133 group(r=0.765,p＜0.01).(4)The differences of positive cell rates in different age groups,not sex and location groups,were statistically siginificant(p＜0.05).The elder groups have higher positive cell rates.(5)In ten samples,only a few tumor cells survived in serum-free medium and suspendedly proliferated into the cell sphere.The cell sphere still kept the capability of self-renewing and proliferation after subculture presenting CD133 and ABCG2 positive products.In serum medium,cells adhered and specific protein of neurons and glial cells could detected after they differentatiated.In tissue slice,the CD133+ and ABCG2+ cells diffused distribution,and a few distributed with nest form. Furthermore,in the hypso- hierarchy pathology tissue,tumor stem cell sphere were more and proliferated prosperity in the primary culture,the percentage of CD133+ and ABCG2+ cells was high also.(6)The dead cells in radiation group are obviously higher than in the control group. So does growth inhibition.The proportion of gliomas stem cell(CD133 positive)is 0.25,0.14,0.10,0.04 in different irradiation dose.The radiation group compare to the control group,P＜0.05,which is statistics significance.In the radiation group 20 hours compare to 40hours,which isn't statistics significance;20 hours compare to 60 hours, P＜0.05.which is statistics significance.The growth curve of clone inhibition in radiation group is obviously lower than in the control group,the growth fraction in different time stage is 1,0.75,0.52,0.25.the survival curve of gliomas stem cells is a straight line,linear regression equation is Y=40.979-38.205X,equations matching grade is F=51.713,P＜0.001,which indicate that the survival rate of gliomas stem cells have regression relationship with the radiation activity of ¹²⁵I seeds,furthermore,there are line negative correlation between the two ones(r=-0.981,P＜0.05);compare to neural stem cells,brain tumor stem cells have more sensible and are statistics significance.(7)BCNU could inhibit both the growth of BTSCs and BTCs,while BTCs(IC50=0.157μg/ml)was more sensitive than BTSCs(IC50=0.072μg/ml)(P＜0.05) With the prolonged treatment of BCNU,flow cytometry analysis showed that not only cells at S phase but also apoptosis cells increased gradually,but these changes of BTSCs occurred(24h)later than that of BTCs(6h).Conclusions:(1)ABCG2 and CD133 did not expressed in normal brain tissue.(2)The differences of positive cell rates in different pathological grades were statistically siginificant,it revealed the positive expression of ABCG2 and CD133 may promote the development of glioma.(3)The ABCG2 and CD133 positive cells may represent different cells.(4)Detecting the expression of ABCG2 and CD133 is helpful to evaluate the biological behavior of glioma,and also provides the evidence for clinic treatment.(5)A few of tumor stem cells are present in human gliomas,which can be isolated,incubate and induced to differentiate in vitro.(6)we prove that CD133,which is specificness marker of nerve stem cell,can mark gliomas stem cell.Use ¹²⁵I seeds to irradiate gliomas stem cell in a short distance.The result indicate that the dead rate of gliomas stem cells is very obviously difference with different radiation dose and time.(7)Use ¹²⁵I seeds to irradiate nerve stem cells and gliomas stem cells.The result indicate that in the same radiation dose,gilomas stem cell is more sensitive than nerve stem cell.The difference of dead cell is obviously significance,which can provid some evidence for the identification of the two kinds of cell.(8)the result indicate in 30Gy the dead rate of gliomas stem cell is the highest in all radiation dose,the cytocidal effect of ¹²⁵I seeds brachytherapy is significance, however,in vitro and in vivo there are a lot of influencing factor,thus a lot of things still are to do.In a shot,brachytherapy of ¹²⁵I seeds can kill the glioma stem cell and inhibit the proliferation in two groups,it should play an important role in the therapy of gliomas and have great perspective.(9)BCNU could inhibit the growth of BTSC in vitro,The mechanism was probably through blocking the cell cycle on S phase to induce BTSC apoptosis(10)Compared with BTC,BTSC was resistant to BCNU by MTT method and flow cytometry.