【作者】 江庆萍；

【导师】 姚开泰；

【作者基本信息】 南方医科大学 ， 肿瘤病理学与病理生理学， 2008， 博士

【副题名】表面标志物及分离鉴定的探索

【摘要】 研究背景和目的干细胞分为胚胎干细胞（embryonic stem cells）和成体干细胞（progenitorsomatic stem cells）。胚胎干细胞既全能干细胞能分化成机体的所有细胞。成体干细胞（如血液干细胞、间充质干细胞等）具有定向的分化功能和不对称分裂的特性,一个子代细胞和母代完全一样,另一个显示组织分化的特点。干细胞因此获得了自我更新增殖及分化能力。在正常干细胞的基础上人们逐渐认识到肿瘤干细胞的存在。白血病干细胞最初被发现。急性髓性白血病（AML）细胞根据不同表型被分为两类,CD34⁺CD38⁺和CD34⁺CD38^-。将这两类细胞分别注射入NOD/SCID小鼠,只有CD34⁺CD38^-细胞能成瘤。他们认为具有CD34⁺CD38^-表型的细胞为白血病起始细胞。近些年来,越来越多的实体瘤肿瘤干细胞（cancer stem cells,CSCs）存在被得到证实。AL^-HAJJ等发现乳腺癌CD44⁺CD24^-/low亚型能在小鼠体内长出第一代和第二代同原患者相似的异质性肿瘤,提示肿瘤起始细胞即CSCs能自我更新并且分化成非起始的肿瘤细胞。有学者在脑肿瘤中发现CSCs,能分化成神经元和胶质细胞,并且增生形成“神经球”。其他实体瘤,如肺癌、肠癌、前列腺癌等也都发现CSCs的存在。除致瘤性外,肿瘤干细胞很多方面和正常干细胞相似,如自我更新,增殖分化能力,耐药性,及相似的细胞表型。所以,人们提出肿瘤干细胞可能通过两种途径一是由于正常干细胞基因突变或表观遗传学改变而来,二是体细胞去分化突变而来。一般认为,正常干细胞以非对称分裂为主,在CSCs,则推测既有非对称分裂,也有对称分裂。近些年来,有学者提出一种“neosis”的分裂方式,即在肿瘤细胞中,老化的肿瘤细胞在基因突变等情况下逃脱死亡,形成多倍体核,通过“核出芽”方式一个细胞分裂成多个子细胞,从而满足肿瘤迅速生长的需要,这些子细胞具有短暂干细胞功能。“neosis“是一种新的概念,有待于进一步深入研究。如何分离和鉴定干细胞?目前,国内外主要是应用干细胞表面标志物进行分离与鉴定。如1)利用干细胞多药耐药相关的ABCG2蛋白进行干细胞分离;2)干细胞表面标志物,如CD133,其首先在鼠的神经上皮干细胞中被发现,后来相继被证实为人类血液系统及其他各器官如神经、尿道、肠及前列腺等正常和肿瘤干细胞标志物。另外,在上皮组织及其肿瘤干细胞中,人们发现特殊表面标志物即角蛋白,如皮肤干细胞角蛋白标志物为CK19等。3)成体干细胞的另一个特征是可以被长久标记,因此也被称为标记滞留细胞（label-retaining cells,LRCs）;标记滞留实验是在活体组织内对干细胞进行标记与检测的主要方法,通常使用的标记物为5溴-2脱氧尿苷（Brdu）或氚标胸腺嘧啶核苷（³H^-TdR）等,即在新生小鼠细胞分裂活跃时掺入Brdu或氚标的胸苷或胸苷类似物,在小鼠成年后滞留有标记的细胞即是LRCs。正常干细胞和肿瘤干细胞均是非常稀少的一个亚群,这给相关研究工作带来很大困难,如何分离和富集干细胞,是人们在一直关注的课题。关于鼻咽部干细胞的研究已有一些报告,Zhang等利用Brdu标记LRCs证实鼻咽黏膜基底部有散在干细胞也发现散在LRCs。BrdU标记的鼻咽癌（Nasopharyngeal carcinoma,NPC）细胞株5-8F接种到裸鼠皮下,8周后也可发现散在LRCs;Wang等利用Hoechst 33342在NPC细胞株CNE2中分离出类似干细胞的侧群细胞,且认为CK19是NPC干细胞标志物。CD133作为上皮和间叶正常及肿瘤干细胞的重要标志物,但其是否为NPC肿瘤干细胞的标志物尚不清楚。所以本研究首先采用免疫组化等技术检测NPC组织和细胞株中包括CK19在内的角蛋白系列和CD133以寻找鼻咽干细胞标志物;利用Brdu体外标记鼻咽癌干细胞5-8F,经体外培养和裸鼠体内接种,获得LRCs细胞,比较LRCs和CD133的表达,从另一角度证明CD133可能是鼻咽癌干细胞标志物。在此基础上,利用免疫磁珠分选系统从鼻咽癌细胞株5-8F细胞中分选CD133⁺细胞,接着通过球囊形成、平板克隆、光学显微镜、电镜观察等实验对所获得的CD133⁺干细胞细胞特性进行检测;最后通过TPA和Brdu药物作用,试图寻找富集干细胞方法。方法1.寻找鼻咽癌干细胞表面标志物CK19等角蛋白和CD133在NPC组织和细胞株中检测免疫组化检测58例NPCs、28例鼻咽粘膜慢性炎、NPC细胞株裸鼠移植瘤和鼻咽癌细胞株中CK19、CK18、CK8、CK14和CK5/6等角蛋白表达;39例带有不典型增生和正常粘膜上皮的鼻咽癌组织以及各类细胞株中检测CD133表达。根据免疫组化结果判断CK19等角蛋白和CD133作为鼻咽癌干细胞的可能性。2 CD133⁺细胞分离与纯度分析1)免疫磁珠法分选CD133⁺细胞2)分选后细胞纯度分析:a.流式细胞仪分析分选后的CD133⁺细胞的纯度;b.免疫细胞化学检测分选后的CD133⁺细胞的纯度;c.实时荧光定量PCR检测分选后的CD133⁺细胞和CD133^-细胞中CD133mRNA水平。3鼻咽癌细胞株5^-8F中LRCs细胞检测,并与CD133+细胞率进行比较a.5-8F细胞株体外LRCs实验细胞生长至指数期时,向培养基中加入Brdu（10ng/ml）,连续培养7天后,撤除Brdu,继续培养14天。培养过程中,分别在第2天、第7天和第14天,在洁净的盖玻片上做细胞爬片,然后进行免疫细胞化学及免疫荧光染色。光镜下选取十个高倍视野进行计数。b.裸鼠体内成瘤5-8F细胞处于对数生长期时,向培养基中加入Brdu（10ng/ml）,48小时后,以1×10⁶个细胞分别注入2只裸鼠双侧腋下,观察成瘤情况。8周后,取出瘤块,固定、石蜡包埋并切片,免疫组化检测Brdu,Brdu阳性细胞即为标记滞留细胞（LRCs）。光镜下选取十个高倍视野进行计数。c.比较CD133和Brdu表达情况,从另一角度验证CD133可能为鼻咽癌干细胞标志物。4.CD133⁺细胞生物学特性鉴定1) Sphere形成实验培养基配方:无血清培养基DMEM^-F12+EGF+B27;CD133⁺细胞、CD133^-细胞和未分选5-8F细胞以1000/ml的密度进行培养,适时观察Sphere形成时间、数量和大小。2)克隆形成实验把三种细胞（即CD133⁺细胞、CD133^-细胞和未分选5-8F细胞）分别200个于12孔培养板中培养,一种细胞3个复孔,当见肉眼可见的克隆形成,终止培养,显微镜下计数大于50个细胞的克隆,并进行统计学分析3)光学显微镜、扫描及透射电镜观察4)TPA和Brdu对5-8F细胞中CD133、ABCG2mRNA水平、蛋白水平以及CD133+细胞数量和生物学行为的影响1)荧光定量PCR检测mRNA水平变化2)流式细胞仪分析四种处理组细胞中CD133⁺的百分率3)体外Boyden小室侵袭实验验验证四种处理细胞的侵袭行为结果:1.寻找鼻咽癌干细胞标志物1)免疫组化检测CK19等角蛋白标志物在鼻咽癌和鼻咽癌细胞株裸鼠移植中表达结果发现虽然CK19在三个NPC细胞株5-8F、6-10B和CNE2中几乎没有见到阳性细胞表达,但三个细胞株移植瘤中CK19呈灶性或散在表达,NPC标本中67.2%（39/58）的癌组织更呈弥漫强阳性表达,粘膜慢性炎和NPC癌旁腺上皮均阳性,部分化生鳞状上皮（8例）也阳性。CK18和CK8为配对角蛋白,NPC中它们除了在化生的鳞状上皮中表达低或不表达外,在腺上皮和癌中的表达同CK19类似,均呈泛表达状态。癌细胞株裸鼠移植瘤中,除CK18在CNE2裸鼠移植瘤中为阴性外,其余均有表达。CK14和CK5/6为另一对配对角蛋白,一般认为它们表达于鳞状上皮和柱状上皮基底层细胞,在本实验中,CK14表达于化生鳞状上皮全层及纤毛柱状上皮基底层储备细胞,只有少数癌组织的散在区域癌细胞阳性（6/58,10.3%）。三种癌细胞株裸鼠移植瘤中均表达。CK14部分表达于NPC细胞株5-8F中。CK5/6虽然和CK14为配对角蛋白,但它的表达情况并不和CK14一致,在鼻咽粘膜中它主要强表达于化生的成熟鳞状上皮和假复层纤毛柱状上皮胞浆内,而基底层细胞呈阴性,在部分癌组织中呈散在弱阳性表达（22/58,37.9%）。三种癌细胞株裸鼠移植瘤中均阴性。根据干细胞数量稀少,组织中只位于上皮基底部和癌巢边缘特点,角蛋白免疫组化结果提示,CK19以及CK18、CK8和CK5/6不能作为合适的NPC干细胞标志物,关于CK14,因为它在组织中表达于鳞状上皮和腺上皮基底细胞,移植瘤癌巢中散在表达,细胞株5-8F中部分表达,所以,我们认为需要寻找其他干细胞标志物,观察CK14与之共表达情况,确定其是否为鼻咽干细胞角蛋白标志物。2)NPC组织和细胞株中CD133检测a.免疫组化检测NPC组织中CD133表达39例NPC组织中仅见8例个别癌巢的边缘有散在CD133⁺细胞,2例粘膜上皮基底部细胞散在呈阳性。11例鼻咽粘膜组织和39例NPC中不典型增生区域均未见阳性细胞。b.免疫细胞化学、PCR检测CD133在鼻咽癌细胞株、永生化上皮细胞和正常上皮细胞中的表达鼻咽正常上皮细胞及永生化细胞株NP69中均见有散在的CD133⁺细胞,其中NP69细胞在十个高倍视野下阳性细胞平均率为1.5%。但三种癌细胞株5^-8F、6-10B和CNE2细胞爬片免疫细胞化学均未查到阳性细胞。分析原因,可能是因为三种癌细胞株中CD133⁺细胞含量太少而难以检测出。PCR扩增凝胶电泳结果显示5-8F、6-10B和CNE2均见见明显条带。结果提示,CD133⁺细胞在癌巢和粘膜上皮中分布的部位和数量（癌巢边缘和上皮基底层）以及细胞株中的表达数量均提示CD133可能是鼻咽癌干细胞的指标之一。2.分离CD133⁺细胞并进行纯度检测1)流式细胞仪检测CD133⁺细胞纯度免疫磁珠分选前后细胞计数,结果显示CD133+细胞约为总数的0.5%,因为每次分选阳性细胞数量太低,不能达到检测要求,所以未成功2)免疫荧光观察CD133⁺几乎所有细胞胞质和胞膜发出红色的荧光,而CD133^-细胞只见个别细胞微弱荧光信号,说明分选纯度较高。3)实时荧光定量PCR5-8F细胞经CD133磁珠分选后,CD133⁺细胞中CD133mRNA含量是CD133^-细胞的7.656倍（P=0.000）。3.鼻咽癌细胞株5^-8F中LRCs细胞检测,并与CD133+细胞率进行比较a.鼻咽癌细胞株5^-8F体外LRCs实验加入Brdu两天和7天几乎所有细胞都阳性,撤除Brdu14天后,只有个别细胞呈阳性。撤除Brdu14天的阳性细胞即LRCs,十个高倍视野下LRCs细胞百分率约为0.67±0.32%。b.裸鼠体内成瘤Brdu免疫组化检测结果显示只有散在的阳性细胞即LRCs,一般在肿瘤组织中位于癌巢周边部,十个高倍视野下LRCs细胞百分率约为0.55±0.36%。c.LRCs含量与第四章流式细胞仪检测NPC5-8F细胞株CD133⁺细胞含量0.5%相接近,从另一个侧面证明CD133可能为NPC干细胞标志物。4.CD133⁺细胞特性鉴定1)Sphere形成实验从第四天开始CD133⁺细胞长出大小不一的圆形球囊,悬浮于培养基中;而CD133^-细胞和未分选细胞未见球囊形成,最终死亡沉积于瓶底。2)克隆形成率测定统计结果发现不同细胞之间平板克隆集落形成率具有显著性差异。CD133⁺细胞组的平均集落形成数明显高于未处理的58F细胞组（P=0.000）,而5-8F细胞组的平均集落形成数高于CD133^-细胞组（P=0.019）。比较3组细胞克隆形成能力,CD133⁺细胞组的克隆形成能力最强;58F细胞组次之;CD133^-细胞组最差。3)形态观察a.光学显微镜观察CD133⁺细胞:小细胞背景中见有多个大细胞,个别大细胞核空泡变。部分大细胞中见有多个核。CD133^-细胞:大小基本一致,偶尔见个别大细胞。b.扫描电镜观察CD133+细胞大部分细胞胞体小,圆,透亮,微绒毛短而小。而CD133^-细胞大部分细胞体积相对较大,呈多角形,颜色较暗,与玻片贴附紧密,微绒毛明显,相互间形成连接。只有散在小圆形细胞存在。另外,我们在CD133⁺细胞中见有多个细胞从母体生出芽苞的现象。一些芽胞已部分脱离母体,伸出绒毛贴附于玻片上。c.透射电镜观察CD133⁺细胞大部分核形态规整,呈圆形。CD133^-细胞核异型性大,可见核沟形成。5.TPA和Brdu对5-8F细胞中CD133、ABCG2表达及CD133+细胞生物学行为的影响1)荧光定量PCR与未处理组相比,TPA作用下,CD133mRNA水平降低,与文献相符。但Brdu处理组CD133含量却明显升高,高达13.23倍,有显著性差异（P=0.037）,Brdu+TPA组CD133含量升高,但升高的倍数小于Brdu组。这可能与TPA抑制CD133mRNA水平有关。ABCG2在三个处理组均增高,但在TPA组,升高不明显,1.72倍,无显著性差异（P=0.173）,Brdu组和Brdu+TPA组均明显增高,具显著差异（P值分别为0.000和0.006）,其中Brdu组增高的倍数8.86,Brdu+TPA组为2.9倍。在四组细胞中,CD133和ABCG2mRNA的变化基本一致,证明5-8F细胞中,Brdu对CD133和ABCG2mRNA表达有促进作用,而TPA抑制mRNA表达。2)流式细胞仪分析四种处理组中CD133⁺细胞的含量结果显示,5-8F-Brdu和5-8F-Brdu+TPA组与5-8F组相比,CD133⁺细胞数量的增加均具有统计学差异（P值均为0.000）,而5-8F-TPA组虽然CD133⁺细胞数量有增加,但无显著性意义（P=0.086）。5-8F-Brdu+TPA组与5-8F-Brdu组及5-8F-TPA组相比,CD133⁺细胞数量的增加也具有统计学差异（P=0.000）。结果说明Brdu和TPA能促进5-8F细胞中CD133⁺细胞数量增加,并且Brdu作用比TPA明显,两者共同培养细胞时,有协同增强作用。掘文献报道,TPA抑制CD133mRNA水平表达,而增强蛋白水平表达,存在转录后调节作用,所以我们的实验中CD133+细胞数量在TPA组增加,我们考虑是与CD133蛋白水平增高有关,尚有待实验证实。3)体外Boyden小室侵袭实验验证四种处理组细胞的侵袭行为结果显示,四种细胞的侵袭能力不同,具有显著统计学差异（F=127.360,P=0.000）。并且四组之间LSD检验两两比较,均有显著性差异（P＜0.005）,提示加入Brdu或TPA后细胞侵袭能力增强,并且Brdu和TPA具有协同作用。结论及创新之处:1.本实验证实了鼻咽癌中CSCs的存在;通过免疫组化等方法,并和LRCs做比较,初步证明CD133⁺癌细胞可能是鼻咽癌干细胞;并对前人提出的CK19作为鼻咽癌干细胞标志物提出疑问。2.免疫磁珠分选法是分离CD133+细胞的一个行之有效的方法。3.Sphere实验和体外克隆实验显示CD133+细胞和CD133-细胞生物学行为不同;在光学显微镜和电镜下观察,CD133+和CD133-细胞形态上有差别。4.Brdu除作为标记物的功能外,在mRNA水平促进细胞中CD133、ABCG2的表达,同TPA作用相反,后者抑制CD133和ABCG2的表达。在细胞水平上,它促使CD133⁺细胞数量增多,侵袭行为增强,同TPA有协同作用。更多还原

【Abstract】 BACKGROUND & OBJECTIVEStem cells were classified as embryonic stem cells（ESCs） and adult stem cells（ASCs）.ES Cs are totipotent,precursors of all cells of the organism.Tissuespecific ASCs reside in adult tissues,which are capable of self-renewal and multipotent differentiation.The ASCs divide asymmetrically,whereby one daughter cell retains as the stem cell,whereas the other daughter cell begins the process of differentiation.On the basis of nomal stem cells（NSCs）,cancer stem cells（CSCs） were recognized.The hematopoetic cancer stem cells were described first.Acute myelogenous leukemia（AML） were categorized into two subtypes with phenotypes CD34+,CD38+ and CD34+,CD38-.These two types of cells were injected to NOD/SCID mouse.Only a fraction of CD34+,CD38-cells could transfer human AML to mouse.These cells were named as leukemic initiating cells which are necessary and sufficient to maintain the leukemia.AL-Hajj et al discovered that CD44⁺CD24^-/low subtype in breast cancers could generate the same first and second generation of heterogenous tumors as the original tumor.These cells featured as the stem cells,therefore were named as cancer stem cells.CSCs in brain tumors could differentiate into neuron and neuroglia and clonally proliferated into "neurosphere". CSCs were also discovered in other solid tumors,such as lung,colon and prostate cancers.Except of high tumorigenity,there are a lot of similarities between NSCs and CSCs,including self-renewal,the ability of unlimited proliferation,differentiation, resistance against drug,and similar surface biomarkers.Therefore,it is generally accepted that CSCs might be derived from NSCs due to mutation or epimutation,or from progenitor cells and differentiated cells which have acquired the ability of dedifferentiation and self-renewal as the results of oncogenic mutations.Generally,division of NSCs are considered as asymmetrical.However,Division ways of CSCs are conjectured not only asymmetrical but also symmetrical division. In recent years,some researchers have raised another type of division—neosis. Neosis still is a new concept and needs advanced research.At present,ASCs were isolated and identified mainly according to the following characteristics.1.tumors contain a subpopulation of cells that excludes the DNA binding dye,Hoechst 33342,out of the cell membrane.These cells,with stem cell characteristics are named as side population（SP） cells.2.Because of the asymmetrical division feature,ASCs can be labeled by 5-bromo-2-deoxyuridine （BrdU） and ³H-TdR for a long time,so the ASCs are also called as label-retaining cells（LRCs）.3.Compared with other cells,stem cells have distinctive surface biomarkers.For example,CD133,a mouse prominin-1 homologous analog,has been verified as a biomarker for normal and tumor stem cells of many tissues and organs such as hematological system,renal,colon and prostate et al.A dilemma for stem cell research is that though important,NSCs or CSCs are very low in number.That fact hinders the study of stem cells.Therefore,it is very important to enrich stem cells in vitro. Up to now,there are only two articles which are involved in the investigation of nasopharyngeal normal and cancer stem cells.Zhang found long-term BrdU-labeled LRCs in nasopharyngeal epithelia of adult mice and subcutaneous xenografts of human NPC cell lines in nude mice.Wang isolated SP cells in nasopharyngeal carcinoma cell line CNE2 and considered that CK19 is a biomarker of nasopharyngeal stem cells.CD133,an important biomarker of normal and tumor stem cells of many epithelial and mesenchymal tissues,is still not applied to stem cell researches of nasopharyngeal carcinoma.Our purpose of this study is to clarified whether cytokeratin 19 and CD133 are possible surface biomarkers of NPC stem cells and then try to look for a way to enrich CSCs of NPC.Methods1.Exploration of the possibility of several cytokeratins and CD133 as biomarkers of nasopharyngeal cancer stem cells.Cytokeratins comprised of CK19,CK18,CK8,CK14 and CK5/6 were detected by immunohistochemistry（ISH） in specimens of NPC and chronic nasopharyngitis, human NPC cancer cell lines and xenograft tumors of those cell lines.CD133 was also detected by ISH and other methods in 39 cases of NPCs with atypical hyperplasia and normal epithelia and cancer cell lines.2.Isolation of CD133+ cells and purity examination1) MACS isolation of CD 133+ cells2) purity examination of CD133+cellsa.Flow cytometry detected pecentage of CD133+cellsb.CD133 immunofluorescence c.The Quantitive Real-time PCR3.detection of LRCs and numerical comparison between LRCs and CD133+ cells in 5-8F1) LRCs in vitroThe 5-8F cells were grown in standard medium,and pulsed with 10ng/ml BrdU for 7 days,and then washed out Brdu for 14 days.Brdu immunhistochemistry and immunofluorescence were proceeded on the second,the senventh day and the fourteenth day after Brdu washed out,respectively.LRCs were numbered under the light microscope.2) Tumorigentiy in nude mice After being cultivated with Brdu for 48 hours,5-8F cells were inoculated into nude mice subcutaneously.After chasing 8 weeks,xenograft tumors were cut off and LRCs were displayed by ISH and numbered under the light microscope.3) Percentages between LRCs and CD133 were compared,to confirm that CD133 is an important surface marker ofNPC CSCs.4.Identification of CD133+ cells features1) sphere formation assayCD133+cells and CD133- Cells（1000 cells/ml） were cultured in serum-free DMEM-F12 supplemented with some growth factors,and observed everyday under light microscope.2) Test of colony formating efficiency（CFE）3) CD133+ and CD133-cells were observed under light microscope,SEM and TEM. 5.The effect of TPA and Brdu on expression of CD133We classified 5-8F cells as four groups:5-8F,5-8F-Brdu,5-8F-TPA,5-8F-Brdu+TPA to culture 48 days,and then took the following tests:1) Quantitive Real-time PCR2) Western blotting3) flow cytometry4) Boyden chamber testsResults:1.Exploration of cytorkeratins and CD133 as biomarkers of nasopharyngeal stem cells.1) Expression of CK19 and other cytokeratinsCK19 was positively expressed in cancer cell foci of 39 NPC specimans and in pseudostratified ciliated columnal epithelia of all NPCs and chronic nasopharyngitis.Some metaplastic squamous epithelia were also positive for CK19. CK19 expression almost could not be found in cell lines 5-8F,6-10B and CNE2 while xenograft tumors of those cancer cell lines were positively stained with ahti-CK 19.CK18 and CK8 are a pair of partner.The expression ways of them were similar with CK19 in human tissues and xenograft tumors of cancer cell lines.CK14 and CK5/6,another pair of partner,are generally expressed in basal cells of squmaous and columnar epithelial layers and squamous cell carcinomas.In this study,the whole squamous epithelial layer and basal cells of columnar epithelia were stained with anti-CK14 in all cases.However,in NPCs,only 6 cases were sporadically stained with anti-CK14.Though they are a pair of partner,expression of CK5/6 were not coincided with CK14 in nasopharyngeal tissues.The positive signals of CK5/6 were mainly presented in mature squamous cells and ciliated columnar cells while basal cells were negative for CK5/6.In 58 NPC cases,CK5/6 were weakly and sporadically expressed in 22 cases.The results suggest that CK19 and the other cytokeratins studied are not proper biomarkers for nasopharyngeal stem cells.2) CD133 expression in NPCs biopsies and cell linesa.CD133 detection in NPCs biopsies.In 39 cases of NPC specimens,only 8 cases showed CD133+ signals in some cells just in margin areas of a few cancer nests,and 2 cases were CD133 positive in basal cells of epithelial layer.b.CD133 expression in normal epithelial cells,immortalized cell line NP69 and cancer cell linesSeveral CD133 positive cells could be found in normal epithelial cells and NP69.But we could not find positive cells in 5-8F,6-10B and CNE2.Gel electrophoresis of PCR amplification with CD133 showed that 5-8F and 6-10B and CNE2 had distinct positive bands.The expression model of CD133 in NPC tissues and cell lines suggested that CD133 maybe a potential surface marker of NPC cancer stem cells.2.Isolation of CD33+ cells and detection of puritya) CD133+cell percentage detected by Flow cytometry:Due to very low quantity of CD133 positive cells,the detection was not successful with several times.b) Immunofluorescence:Observed under fluorescence microscope,almost all CD133+cells showed fluorescence,while fluorescence were difficult to be seen in CD133-cells.c) Quantitive real-time PCR The quantity of mRNA of CD133+ cells is 7.656 times higher than that of CD 133-cells.2.Detection of LRCs and numerical comparison between LRCs and CD133+ cells in 5-8Fa) LRCs in vitroBrdu immunohistochemistry discovered that almost all cells were positive on the second and the seventh day after Brdu added.Only sporadic cells were positive on the fourteenth day after Brdu washouted,and these cells were thought of LRCs.The average percentage of LRCs is 0.67±0.32%.b) 5-8F xenograft tumors in nude miceOnly a small quantity of cells in margin area were Brdu positive.These positive cells were considered as LRCs.The average percentage of LRCs under ten high magnified powers is 0.55±0.36%.c) the percentage of CD133+cells（0.5%） were similar to LRCs.This result confirms once again that CD133 is an important marker for NPC CSCs.4.Identification of CD133+cells features1)Sphere assay:From the fourth day CD133+ cancer cells formed spheres,the case in CD133-tumor cells was different.CD133-tumor cells failed to form spheres and most of cells died within several days.2) colony forming efficiencyA significant difference was found in clony formation efficiency among different cells（F=68.045,P=0.000）.The CFE of CD133+ cells is higher than that of 5-8F （P=0.000） and CD133-cells（P=0.000）.3) Observation of CD133+and CD133- cells under light microscope,SEM and TEM a.Observation under light microscopeThe most distinguished characteristics of CD133+ cells is that a lot of big cells are dispersed among small cells.Under high magnification,polykaryon could be found in some big cells.On the other hand,most of CD133- cells are similar in size.b.Observation under SEMUnder SEM,appearances of CD133+ and CD133- cells are different.In CD133+ cells,we can see some little spherical bodies on a big mother cell,like budding phenomenon.The other cells are generally small,round and with short villi. With long villi,CD133-cells are bigger,polygonal,and tightly sticking to slides.In CD133- cells,we almost can not find budding phenomenon.b.Observation under TEMMost of CD133+ cells appeared regular and round nuclei,whereas nuclei were beteromorphic and nuclear grooves could be seen in CD133- cells4) The effects of TPA and Brdu on CD133 expression and CD133+cell quantity1) The quantitive real-time PCRCD133mRNA and ABCG2mRNA were significantly higher in the groups of 5-8F-Brdu and 5-8F-Brdu+TPA than 5-8F（CD133:P=0.037 and P=0.003,respectively; ABCG2:P=0.000 and P=0.006 respectively）.CD133 mRNA was decreased in 5-8F-TPA.It proved that Brdu can promote CD133mRNA expression.2) The percentage of CD 133+cells by Flow cytometryThe percentage of CD133+cells in 5-8F-Brdu and 5-8F-brdu+TPA were significantly higher than that of 5-8F（P=0.000）.CD133+ cells in 5-SF-TPA was also higher than 5-8F.However,no statistics difference was found.2) Boyden chamber invasion test in vitroThe invasion ability of four groups is significantly different（F=127.360,P=0.000）. Among them,the ability of 5-8F-Brdu+TPA was the strongest,the combined effects of Brdu and TPA promote the invasion.Conclusions:1.The presence of CSCs in NPC was confirmed once again;CD133+ but not CK19 maybe an important surface marker of NPC CSCs.2.MACS isolation of CD133+ cells is an effective method.3.Biological and morphological characteristics are different between CD133+ and CD133-cells.4.Brdu,besides as a marker of LRCs,can increase the mRNA expression of CD133 and ABCG2,and promote CD 133+ cell proliferation and invasion ability.更多还原