人脑胶质瘤干细胞的分离、培养、初步鉴定以及脑胶质瘤治疗的新探讨
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摘要
目的:通过检测ABCG2和CD133在人脑胶质瘤中的表达证明人脑胶质瘤中存在BTSC;再进行人脑胶质瘤干细胞的分离、培养、鉴定研究其生物学特性,然后进一步观察研究~(125)Ⅰ及BCNU对BTSC的影响。
方法:1、利用免疫组化方法测定5例正常脑组织及59例胶质瘤中ABCG2和CD133的表达并记录阳性细胞数,同时HE染色计数肿瘤细胞总数并分级,统计分析ABCG2和CD133阳性细胞率与各因素之间关系。
2、术中取21例患者胶质瘤组织,分离成单细胞悬液,接种于添加生长因子的无血清培养基中,使其中的脑肿瘤干细胞克隆增殖;对增殖形成的肿瘤干细胞球连续传代培养,部分克隆球细胞做单克隆培养;将脑肿瘤干细胞球接种于含血清培养基,观察其分化现象。
3、术中取髓母细胞瘤细胞,接种于含生长因子的无血清培养基中行原代培养,使其中的脑肿瘤干细胞增殖,形成悬浮生长的细胞球。将细胞球吹打成单细胞后,取其中部分细胞,利用细胞免疫荧光检测脑肿瘤干细胞在细胞培养CD133的表达,并计算CD133阳性细胞的比例。另一部分细胞继续培养,3-5天后有大量细胞球生成,取细胞对数生长期离心、吹打成单细胞悬液。并将~(125)Ⅰ籽粒置入,培养皿中共同培养。细胞悬液密度为13×10~4,按照射吸收剂量分4组,每组为一96孔板培养。每组按照射剂量(空白对照、10Gy、20Gy、30Gy)设四个时间段0、20、40、60个小时,每个时间段设四孔,每孔液体量200μl,细胞数为2.6×10~4,其中三孔记细胞死亡数取平均值,另一孔做生长抑制实验。同时取神经干细胞,按神经干细胞培养方式传代培养3-5天后,形成细胞球,吹打成单细胞悬液,取相同剂量设定为对照组,与脑肿瘤干细胞进行照射比较。对脑肿瘤干细胞给予不同剂量、不同时间的近距离照射,检测CD133阳性细胞死亡率,增殖抑制的改变;通过细胞克隆形成法,测定~(125)Ⅰ籽粒近距离照射对脑肿瘤干细胞的生长抑制作用,同时与神经干细胞进行照射对照,检测两组细胞对~(125)Ⅰ籽粒射线的敏感性差异。
4、术中取新鲜人脑室管膜瘤标本一例,制备为单细胞,接种于无血清培养基中获得脑肿瘤干细胞,接种于含血清培养基中获得肿瘤细胞,7d时,利用免疫细胞化学染色的方法鉴定两组细胞中干细胞标志物CD133和nestin的表达,并用培养7d的细胞进行以下实验:(1)、MTT法检测药物敏感性:将两组细胞接种于96孔,将BCNU稀释为1ug/ml、0.1ug/ml、0.01ug/ml、0.001ug/ml、0.001ug/ml五个不同的浓度,作用于细胞72h后,MTT法测OD值,分别计算抑制率(IR)及半数抑制率(IC50);(2)、细胞周期检测:将细胞接种于6孔板中,以0.01ug/ml浓度的BCNU作用细胞6h、12h和24h,设不加药组为空白对照组,记为0h,流式细胞仪检测细胞周期变化
结果:1、ABCG2与CD133在成人正常脑组织中无表达,在不同类型胶质瘤中有表达。
2、ABCG2与CD133阳性细胞率之间的差异有统计学意义(p＜0.05),CD133阳性细胞率大于ABCG2。二者呈正相关(r=0.765,p＜0.01)。
3、ABCG2及CD133的阳性细胞率在多个病理级别之间有差异(p＜0.05),两者在Ⅳ级胶质瘤中的表达最高。
4、ABCG2及CD133的阳性细胞率与患者性别、肿瘤生长部位无关(p＞0.05)。多个年龄组的阳性细胞率之间有差异(p<0.05),大年龄组具有更高的阳性率。
5、本组中有10例胶质瘤中均仅有少数肿瘤细胞能够在无血清培养基中存活并悬浮增殖、形成克隆性脑肿瘤干细胞球,传代后脑肿瘤干细胞仍保持很强的自我更新和增殖能力,表达特异性标志物CD133和ABCG2;在含血清培养基中贴壁分化,产生具有多种形态且表达NSE、GFAP或O4的分化细胞;组织切片中CD133和ABCG2阳性细胞呈散在分布,部分呈巢状分布;且肿瘤病理级别越高,原代肿瘤干细胞球越多,增殖越旺盛,组织切片中CD133或ABCG2阳性细胞越多。
6、与对照组比较,照射组细胞死亡率,增殖抑制率明显升高。脑肿瘤干细胞(CD133阳性)在不同的照射时间段内(即0、20、40、60小时)的细胞比例分别为0.25、0.14、0.10、0.04。实验组与对照组比较,P＜0.05.有显著性意义;实验组内20小时与40小时比较,无显著性差异;20小时与60小时比较,P＜0.05。有显著性意义。~(125)Ⅰ籽粒各实验组生长曲线抑制克隆形成,较空白组明显降低。不同照射时间段内的生长分数分别为1、0.75、0.52、0.25。~(125)Ⅰ籽粒对脑肿瘤干细胞的存活曲线为一条直线,直线回归方程为Y=40.979-38.205X,方程配合度检示:F=51.713,P＜0.001,表明细胞存活率和~(125)Ⅰ籽粒放射活度线性有回归关系,且两者相关性检验呈直线负相关(r=-0.981,P＜0.05);与神经干细胞比较,脑肿瘤干细胞对于~(125)Ⅰ籽粒的敏感性具有显著性差异。
7、BCNU对人脑肿瘤干细胞及脑肿瘤细胞均有明显的抑制作用(IC50＜10μg/mL),呈浓度依赖性,其对肿瘤干细胞(IC50=0.157μg/mL)的抑制低于肿瘤细胞(IC50=0.072μg/mL),有统计学差异(P＜0.05)。与阴性对照比较,两类细胞均表现为随药物作用时间的延长,S期细胞比例增高,并伴有凋亡细胞的增多,但肿瘤干细胞的改变(24h)出现晚于肿瘤细胞(6h)。
结论:1、ABCG2与CD133在成人正常脑组织中无表达,在不同类型胶质瘤中有表达。
2、ABCG2与CD133的表达可能促进了胶质瘤的发生发展。
3、ABCG2与CD133标记不完全相同的胶质瘤细胞群。
4、ABCG2与CD133的表达在不同胶质瘤病理级别之间有差异,病理级别越高,阳性率越高。
5、人脑胶质瘤中存在少量的具有自我更新和增殖能力、表达CD133和ABCG2的肿瘤干细胞,并能在体外将其分离、培养和诱导分化。
6、利用放射性物质~(125)Ⅰ籽粒近距离照射脑肿瘤干细胞,结果表明,不同剂量、不同照射时间细胞的死亡比例不同,随着照射剂量的增加,细胞死亡比例增加,同时脑肿瘤干细胞的生长抑制也随着放射剂量的增加而增加。这点也与实验预期的设想相吻合。
7、对脑肿瘤干细胞与神经干细胞分别进行了近距离照射,结果表明,相同的放射剂量,脑肿瘤干细胞对~(125)Ⅰ籽粒的射线更敏感,两者的细胞死亡数具有显著性差异。这也说明神经干细胞与肿瘤干细胞在细胞特性上存在一定的差异。但是,对于两者对于射线敏感性的差异的机制,还有待于进一步研究。
8、本实验为体外照射实验,结果表明,~(125)Ⅰ照射剂量达到30Gy时,对脑肿瘤干细胞的杀伤作用最明显,与其他剂量组有统计学意义。但是,体内与体外的影响因素很多,所以对于~(125)Ⅰ籽粒的临床照射剂量的研究还有很多工作要做。在体外实验中,BCNU对人脑肿瘤干细胞有明显的生长抑制作用,其抑制作用与药物浓度成正比例关系。
9、流式细胞仪检测显示,药物作用于脑肿瘤干细胞后细胞周期的改变表现为S期细胞比例增多,并伴有凋亡细胞的增多,提示BCNU对脑肿瘤干细胞的作用可能为作用于细胞的S期,干扰DNA的合成起抑制细胞生长的作用。
10、通过脑肿瘤干细胞与脑肿瘤细胞进行比较,发现脑肿瘤干细胞的药物敏感性低于脑肿瘤细胞,并且药物作用后脑肿瘤干细胞在细胞周期上的改变也晚于脑肿瘤细胞,两者均提示较脑肿瘤细胞而言,脑肿瘤干细胞对BCNU具有一定的耐药性。
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 with~(125)I 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 of~(125)I 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 ~(125)I seeds by the measure of cell clone formation.,the same time, compare to neural stem cells,observe the sensibility difference by brachytherapy of ~(125)I 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 ~(125)I 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 ~(125)I 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 ~(125)I 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 ~(125)I 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 ~(125)I 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.
方法:1、利用免疫组化方法测定5例正常脑组织及59例胶质瘤中ABCG2和CD133的表达并记录阳性细胞数,同时HE染色计数肿瘤细胞总数并分级,统计分析ABCG2和CD133阳性细胞率与各因素之间关系。
2、术中取21例患者胶质瘤组织,分离成单细胞悬液,接种于添加生长因子的无血清培养基中,使其中的脑肿瘤干细胞克隆增殖;对增殖形成的肿瘤干细胞球连续传代培养,部分克隆球细胞做单克隆培养;将脑肿瘤干细胞球接种于含血清培养基,观察其分化现象。
3、术中取髓母细胞瘤细胞,接种于含生长因子的无血清培养基中行原代培养,使其中的脑肿瘤干细胞增殖,形成悬浮生长的细胞球。将细胞球吹打成单细胞后,取其中部分细胞,利用细胞免疫荧光检测脑肿瘤干细胞在细胞培养CD133的表达,并计算CD133阳性细胞的比例。另一部分细胞继续培养,3-5天后有大量细胞球生成,取细胞对数生长期离心、吹打成单细胞悬液。并将~(125)Ⅰ籽粒置入,培养皿中共同培养。细胞悬液密度为13×10~4,按照射吸收剂量分4组,每组为一96孔板培养。每组按照射剂量(空白对照、10Gy、20Gy、30Gy)设四个时间段0、20、40、60个小时,每个时间段设四孔,每孔液体量200μl,细胞数为2.6×10~4,其中三孔记细胞死亡数取平均值,另一孔做生长抑制实验。同时取神经干细胞,按神经干细胞培养方式传代培养3-5天后,形成细胞球,吹打成单细胞悬液,取相同剂量设定为对照组,与脑肿瘤干细胞进行照射比较。对脑肿瘤干细胞给予不同剂量、不同时间的近距离照射,检测CD133阳性细胞死亡率,增殖抑制的改变;通过细胞克隆形成法,测定~(125)Ⅰ籽粒近距离照射对脑肿瘤干细胞的生长抑制作用,同时与神经干细胞进行照射对照,检测两组细胞对~(125)Ⅰ籽粒射线的敏感性差异。
4、术中取新鲜人脑室管膜瘤标本一例,制备为单细胞,接种于无血清培养基中获得脑肿瘤干细胞,接种于含血清培养基中获得肿瘤细胞,7d时,利用免疫细胞化学染色的方法鉴定两组细胞中干细胞标志物CD133和nestin的表达,并用培养7d的细胞进行以下实验:(1)、MTT法检测药物敏感性:将两组细胞接种于96孔,将BCNU稀释为1ug/ml、0.1ug/ml、0.01ug/ml、0.001ug/ml、0.001ug/ml五个不同的浓度,作用于细胞72h后,MTT法测OD值,分别计算抑制率(IR)及半数抑制率(IC50);(2)、细胞周期检测:将细胞接种于6孔板中,以0.01ug/ml浓度的BCNU作用细胞6h、12h和24h,设不加药组为空白对照组,记为0h,流式细胞仪检测细胞周期变化
结果:1、ABCG2与CD133在成人正常脑组织中无表达,在不同类型胶质瘤中有表达。
2、ABCG2与CD133阳性细胞率之间的差异有统计学意义(p＜0.05),CD133阳性细胞率大于ABCG2。二者呈正相关(r=0.765,p＜0.01)。
3、ABCG2及CD133的阳性细胞率在多个病理级别之间有差异(p＜0.05),两者在Ⅳ级胶质瘤中的表达最高。
4、ABCG2及CD133的阳性细胞率与患者性别、肿瘤生长部位无关(p＞0.05)。多个年龄组的阳性细胞率之间有差异(p<0.05),大年龄组具有更高的阳性率。
5、本组中有10例胶质瘤中均仅有少数肿瘤细胞能够在无血清培养基中存活并悬浮增殖、形成克隆性脑肿瘤干细胞球,传代后脑肿瘤干细胞仍保持很强的自我更新和增殖能力,表达特异性标志物CD133和ABCG2;在含血清培养基中贴壁分化,产生具有多种形态且表达NSE、GFAP或O4的分化细胞;组织切片中CD133和ABCG2阳性细胞呈散在分布,部分呈巢状分布;且肿瘤病理级别越高,原代肿瘤干细胞球越多,增殖越旺盛,组织切片中CD133或ABCG2阳性细胞越多。
6、与对照组比较,照射组细胞死亡率,增殖抑制率明显升高。脑肿瘤干细胞(CD133阳性)在不同的照射时间段内(即0、20、40、60小时)的细胞比例分别为0.25、0.14、0.10、0.04。实验组与对照组比较,P＜0.05.有显著性意义;实验组内20小时与40小时比较,无显著性差异;20小时与60小时比较,P＜0.05。有显著性意义。~(125)Ⅰ籽粒各实验组生长曲线抑制克隆形成,较空白组明显降低。不同照射时间段内的生长分数分别为1、0.75、0.52、0.25。~(125)Ⅰ籽粒对脑肿瘤干细胞的存活曲线为一条直线,直线回归方程为Y=40.979-38.205X,方程配合度检示:F=51.713,P＜0.001,表明细胞存活率和~(125)Ⅰ籽粒放射活度线性有回归关系,且两者相关性检验呈直线负相关(r=-0.981,P＜0.05);与神经干细胞比较,脑肿瘤干细胞对于~(125)Ⅰ籽粒的敏感性具有显著性差异。
7、BCNU对人脑肿瘤干细胞及脑肿瘤细胞均有明显的抑制作用(IC50＜10μg/mL),呈浓度依赖性,其对肿瘤干细胞(IC50=0.157μg/mL)的抑制低于肿瘤细胞(IC50=0.072μg/mL),有统计学差异(P＜0.05)。与阴性对照比较,两类细胞均表现为随药物作用时间的延长,S期细胞比例增高,并伴有凋亡细胞的增多,但肿瘤干细胞的改变(24h)出现晚于肿瘤细胞(6h)。
结论:1、ABCG2与CD133在成人正常脑组织中无表达,在不同类型胶质瘤中有表达。
2、ABCG2与CD133的表达可能促进了胶质瘤的发生发展。
3、ABCG2与CD133标记不完全相同的胶质瘤细胞群。
4、ABCG2与CD133的表达在不同胶质瘤病理级别之间有差异,病理级别越高,阳性率越高。
5、人脑胶质瘤中存在少量的具有自我更新和增殖能力、表达CD133和ABCG2的肿瘤干细胞,并能在体外将其分离、培养和诱导分化。
6、利用放射性物质~(125)Ⅰ籽粒近距离照射脑肿瘤干细胞,结果表明,不同剂量、不同照射时间细胞的死亡比例不同,随着照射剂量的增加,细胞死亡比例增加,同时脑肿瘤干细胞的生长抑制也随着放射剂量的增加而增加。这点也与实验预期的设想相吻合。
7、对脑肿瘤干细胞与神经干细胞分别进行了近距离照射,结果表明,相同的放射剂量,脑肿瘤干细胞对~(125)Ⅰ籽粒的射线更敏感,两者的细胞死亡数具有显著性差异。这也说明神经干细胞与肿瘤干细胞在细胞特性上存在一定的差异。但是,对于两者对于射线敏感性的差异的机制,还有待于进一步研究。
8、本实验为体外照射实验,结果表明,~(125)Ⅰ照射剂量达到30Gy时,对脑肿瘤干细胞的杀伤作用最明显,与其他剂量组有统计学意义。但是,体内与体外的影响因素很多,所以对于~(125)Ⅰ籽粒的临床照射剂量的研究还有很多工作要做。在体外实验中,BCNU对人脑肿瘤干细胞有明显的生长抑制作用,其抑制作用与药物浓度成正比例关系。
9、流式细胞仪检测显示,药物作用于脑肿瘤干细胞后细胞周期的改变表现为S期细胞比例增多,并伴有凋亡细胞的增多,提示BCNU对脑肿瘤干细胞的作用可能为作用于细胞的S期,干扰DNA的合成起抑制细胞生长的作用。
10、通过脑肿瘤干细胞与脑肿瘤细胞进行比较,发现脑肿瘤干细胞的药物敏感性低于脑肿瘤细胞,并且药物作用后脑肿瘤干细胞在细胞周期上的改变也晚于脑肿瘤细胞,两者均提示较脑肿瘤细胞而言,脑肿瘤干细胞对BCNU具有一定的耐药性。
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 with~(125)I 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 of~(125)I 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 ~(125)I seeds by the measure of cell clone formation.,the same time, compare to neural stem cells,observe the sensibility difference by brachytherapy of ~(125)I 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 ~(125)I 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 ~(125)I 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 ~(125)I 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 ~(125)I 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 ~(125)I 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.
引文
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