【作者】 邓永文；

【导师】 方加胜；

【作者基本信息】 中南大学 ， 外科学， 2008， 博士

【摘要】 1.目的第一部分实验旨在运用磁珠分选技术(magnetic cell sorting,MACS)分选人多形性胶质母细胞瘤U251细胞系中的CD133+和CD133-亚群细胞;比较两个亚群细胞在增殖、分化及体内致瘤性等方面的差异,重点探讨CD133+细胞生物学特性;评价CD133作为脑肿瘤干细胞表面标志物的特异性与磁珠分选系统分选脑肿瘤干细胞的效率及特异性。第二部分实验的主要目的是研究和比较CD133+细胞亚群与CD133-细胞亚群耐药性的差异,并检测两个亚群细胞在ABC转运体、DNA修复及抗凋亡基因等表达的差异,初步分析CD133+细胞亚群的耐药机制。2.方法第一部分实验通过磁珠分选人多形性胶质母细胞瘤U251细胞系中的CD133+和CD133-细胞亚群,通过流式细胞仪检测分选的纯度和两个亚群细胞的细胞周期;利用MTT试验和单克隆形成率实验检测两个亚群细胞的增殖能力;运用免疫荧光检测CD133+细胞亚群的多向分化能力以及CD133+细胞增殖形成的BTS的分化情况;最后通过裸鼠移植实验,检测两个亚群细胞在裸鼠体内致瘤性的差异。第二部分实验在第一部分磁珠分选细胞的基础上,观察CD133+细胞与CD133-细胞在抗肿瘤药物作用下形态学的变化;利用MTT试验和流式细胞仪检测CD133+细胞亚群与CD133-细胞亚群对抗肿瘤药物替尼泊苷、卡莫司汀和顺铂的敏感性;通过RT-PCR方法检测两个亚群细胞在ABC转运子(MDR1、BCRP、MRP)、DNA修复基因(MGMT)及抗凋亡(Bcl-2)基因的表达差异。3.结果第一部分实验结果表明,在U251细胞系中只有大约4.5%的CD133+细胞,经过磁珠分选后,CD133+亚群细胞的细胞纯度可以提高到92.4%;分选后的CD133+细胞接种到含EGF和bFGF的无血清培养基中,具有强大的增殖能力,能增殖形成典型的BTS,而CD133-细胞不具有形成BTS的能力,生长曲线亦显示CD133+细胞增殖能力明显高于CD133-细胞;流式细胞仪实验显示CD133+细胞的细胞增殖率为40.1%,CD133-细胞的细胞增殖率为30.6%;单克隆形成率实验显示CD133+细胞能形成BTS的细胞比率达到78.5%-92.4%,而CD133-细胞仅有0.8-2.4%;分化实验免疫荧光实验显示CD133+细胞能分化为具有GFAP和NeuN成熟表型的肿瘤细胞,且CD133+细胞增殖形成的BTS亦能分化为具有GFAP和NeuN成熟表型的肿瘤细胞;裸鼠致瘤实验显示CD133+的致瘤率为71.42%,而CD133-细胞没有致瘤性。第二部分实验结果表明,CD133-细胞在抗肿瘤药物作用下凋亡形态学改变明显,CD133+细胞仍能维持生长;MTT实验显示,CD133-细胞对抗肿瘤药物替尼泊苷、卡莫司汀和顺铂的敏感性明显高于CD133+细胞;流式细胞仪试验显示在中等浓度的替尼泊苷作用下,CD133-细胞的凋亡明显,并出现明显的“亚G1峰”,而CD133+细胞并未受到明显抑制和杀灭;流式细胞仪检测显示CD133+细胞内的药物蓄积明显低于CD133-细胞;RT-PCR实验显示,MDR1、BCRP、MRP、MGMT和Bcl-2表达在CD133+明显高于CD133-细胞。4.结论U251细胞系中存在少量具有增殖、多向分化与体内致瘤能力的CD133+细胞,CD133+细胞是符合肿瘤干细胞定义的细胞亚群;磁珠分选是理想的脑肿瘤细胞分选系统;CD133+是U251细胞系中的耐药细胞亚群,高表达ABC转运体,DNA修复和抗凋亡基因可能是CD133+细胞耐药的重要机制。更多还原

【Abstract】 OBJECTIVE The aim of the first part was to sort subpopulation of CD133+ and CD133- cells in the human Glioblastoma Multiforme(GBM)U251 cell line.To explore the difference of proliferation、differentiation and tumorigenic potential in vivo between the two subpopulation.The cytobiologic character of CD133+ subpopulation was the emphasis of this study;To investigate the specificity of CD133 to be specific surface marker(SSM)of brain tumor stem cells(BTSC)and the specificity and efficiency of the system of FACS sorting BTSC.The purpose of the second part was to compare the difference of drug sensitivity between the two subpolulation,and analysis the mechanism of chemoresistance by comparing the different expression of gene of ABC transporter、DNA repair and apoptosis.METHODS In the first part,CD133+ and CD133- subpopulation cells in the human GBM U251 cell line were sorted by MACS.Single isolated CD133+ and CD133- cells was cultured in serum-free DMEM/F12 medium,containing B27(1∶50),basic fibroblast growth factor(bFGF,20μg/L),epidermal growth factor(EGF,20μg /L)or medium containing 10%fetal bovine serum.Cell cycle of the two subpopulation was analyzed by flow cytometry(FCM).MTT and monoclone forming rate(MFR)assay was performed to investigate the capacity of self-rewal and clonogenic potential of the subpopulation cells. Immunofluorescence staining was performed to investigate the multidiferentiation of CD133+ cells and brain tumor sphere(BTS), formed by CD133+ cells cultured in serum-free medium.At last,in order to investigate the tumorigenic potential of the two subpopulation in vivo, xenograft in BALB/c nude mice brain was performed.On the foundation of cell-sorting,the second part was aimed to analyze drug sensitivity of the two subpopulation,the cell morphology change was examined by phase microscopy after treated with anti-tumor drug.MTT and flow cytometry was performed to analyze the drug sensitivity of the two subpopulation to anti-tumor drug,Teniposide,Carmustine and Cisplatin. Finaly,ABC transporter,DNA repair and apoptosis gene expression of the two subpopulation was analyzed by RT-PCR.RESULTS The first part show that,only4.5%cells was CD133+ in the total popul- ation of U251 cell line,and the purity of CD133+ cells in the CD133+ population can reach 92.4%after cell-sorting.The single CD133+ and CD133- cell was cultured in serum-free DMEM/F12 medium,containing B27(1∶50),basic fibroblast growth factor(bFGF, 20μg/L),epidermal growth factor(EGF,20μg /L)and serum-contain medium respectively.The CD 133+ cells exhibited powerful proliferation capacity.The single CD133+ cells could proliferate and form typical brain tumor sphere(BTS),while CD133- cells couldn’t.And the cell growth curve showed that CD133+ cells proliferated remarkably faster than CD133- cells while cultured in serum-free medium.The cell proliferation index of CD133+ and CD133- cells was 40.1%and 30.6% respectively,showed by FCM.And the MFR of CD133+ and CD133-cells was 78.5-92.4%and 0.8-2.4%respectively.Immunofluorescence staining showed that,CD133+ cells and the BTS,forming by single CD133+ cells,could be induced to differentiate into mature neurocyte cells,which express NeuN and GFAP in the serum-containing medium. Xenograft assay showed that,CD133+ cells had tumorigenic potential in vivo,while CD133- hadn’t.In the second part of this study,apoptotic morphologic change of CD133- cells was obvious in present of anti-tumor drug,while CD133+ cells could maintain survive.Anti-tumor drug assay showed that,CD133- cells were more sensitivite to Teniposide, Carmustine and Cisplatin than CD133+ cells.Treated with Teniposide, apoptotic percentage of CD133- cells were remarkerly higher than CD133+ cells,and "sub-Glpeak" appeared in CD133- subpopulation while CD133+ did not,showed by FCM.And FCM also showed that, accumulation of anti-tumor drug in CD133+ cells remarkably lower than CD133- cells.RT-PCR showed that,CD133+ cells expressed remarkably higher than CD133- cells in MDR1,BCRP,MRP1,MGMT and Bcl-2.CONCLUSION The U251 cell line contained a small proportion of CD133+cells,which had the capacity of proliferation,multidifferentiation and tumorigenic potential in vivo.The subpopulation of CD133+ cells was the brain tumor stem cells subpopulation in U251. MACS was an optimizing cell sorting system in BTSC sorting.The subpopulation of CD133+ cells was the chemoresistant subpopulation in U251 cell line.High expression of ABC transporter,DNA repair and apoptosis maybe the important chemoresistance mechanism of CD133+ cells.更多还原