【作者】 文加斌；

【导师】 张阳；

【作者基本信息】 大连医科大学 ， 中西医结合临床， 2009， 博士

【摘要】 背景随着肿瘤干细胞研究的深入,越来越多的证据表明除白血病外,乳腺癌、肾癌、脑肿瘤也存在肿瘤干细胞,肺癌等实体瘤也可能存在肿瘤干细胞,但肺癌肿瘤干细胞研究相对滞后。动物实验模型提示肿瘤干细胞的存在。犬的正常支气管上皮没有Ⅱ型肺泡上皮,但Ⅱ型肺泡上皮细胞可出现在重度不典型增生和侵蚀性病灶中。Ⅱ型肺泡上皮存在增殖分子标记物Ki267和增殖细胞核抗原（PCNA）,提示其可能是支气管癌的多潜能干细胞。2005年Kim等报道支气管肺泡干细胞很可能就是肺癌的源头。他们在非小细胞肺癌小鼠模型从小鼠肿瘤的最初阶段分离到这种干细胞,并将其命名为支气管肺泡干细胞（brochioalveolar stem cells,BASCs）。他们利用流式细胞仪筛选发现,BASCs表达干细胞抗原Scal（stem cellantigen1）和CD34,但不表达血小板内皮细胞黏附分子PECAM（plateletendothelial cell adhesion molecule,PECAM/CD31）和CD45,即具有Sca1⁺CD45^- PECAM^- CD34⁺特征的肿瘤干细胞。BASCs在正常情况处于静止状态,当体内支气管和肺泡损伤时发生增殖。BASCs表现出明显的自我更新特性,能够分化成其它类型的肺上皮细胞,表现出干细胞的特性。他们认为BASCs是远端肺上皮的干细胞,可能局限于支气管Clara细胞和肺泡Ⅰ型及Ⅱ型细胞。在K—ras基因突变活化的终未支气管和肺泡上皮的不典型增生和腺瘤中,可以观察到BASCs数量明显增加,提示BASCs可能是肺腺癌的起源细胞（亦即肿瘤干细胞）。Maziers等证实了在肺癌细胞信号转道通路中存在与上述干细胞相似的Wnt过渡表达,非经典途径JNK通路激活和Wnt拮抗剂W IF—1的抑制和失活调节作用。黄盛东等研究A549肺癌细胞株中肿瘤干细胞与克隆形成的关系,观察克隆形态是否与肿瘤细胞的不断自我更新相关。他们将A549肺癌细胞株单细胞悬液接种到96孔板,观察克隆形成率和克隆形态及传代情况,测量克隆大小,计数细胞数目,分别观察早期、中间和晚期的黏附细胞数目,检测其细胞增殖活性和细胞免疫表型,结果发现A549肺癌细胞株可形成3种类型的克隆集落,其中Holoclone型占总克隆数的4%,克隆体积大,可连续传代20代以上,并可分化为其他两种类型的集落,增殖活性不随时间延长而降低,并表达干细胞标记,认为A549肺癌细胞株Holoclone型克隆集落存在不断自我更新的肿瘤干细胞。董强刚等研究人肺腺癌干细胞分离及鉴定,采用磁性细胞分选（magnetic activated cellsorting,MACS）技术将肺腺癌细胞分成多个亚群,然后应用流式细胞及RT-PCR技术检测干细胞相关标志表达,通过NOD-SCID小鼠移植评估其致瘤性。结果在A549和SPC-A肺腺癌细胞中发现一个新颖的癌细胞亚群,此类癌细胞的表型特征为CD 24⁺IGF^-.1R⁺,具有高侵袭性和高致瘤性,在NOD-SCID小鼠中仅需移植100个细胞即可形成肿瘤,其致瘤能力是上述标志阴性细胞的1000倍。此外,这些细胞表达胚胎干细胞标志（OCT4和Bmi-1）及肺干细胞标志（CCSP,SP-C,TTF-1和Gremlin）,类似小鼠肺脏细支气管肺泡干细胞（bronchioalveolar stem cells,BASCs）,并具有自主生长特性,能够在无血清条件下长期培养。他们认为人体肺腺癌中CD24⁺IGF^-1R⁺细胞属于BASCs样癌干细胞。当前肿瘤干细胞相关研究中细胞表面标志物是分选肺癌肿瘤干细胞的关键。研究显示白血病肿瘤干细胞、脑肿瘤肿瘤干细胞、毛细血管瘤、前列腺癌肿瘤干细胞均表达CD133分子,肿瘤干细胞可能与正常组织干细胞有相似的表面标志物,Hilbe等在非小细胞肺癌内皮祖细胞发现CD133分子表达,肿瘤中的内皮祖细胞可能来源于肺癌的肿瘤干细胞。由此推论肺腺癌肿瘤干细胞亦可能存在与BASCs相似的表现标志物,如:Sca1⁺、CD45^-、Pecan^-、CD34⁺等。肿瘤干细胞包括肺癌肿瘤干细胞是否具有能够与正常组织干细胞区分开来的特异性细胞表面标志物尚需探索。关于BMI-1与肿瘤干细胞的关系研究中,仅有有关白血病肿瘤干细胞的报道。Lessard等发现,表达BMI-1的小鼠外周血白血病细胞明显高于BMI-1缺失纯合子小鼠,后者的骨髓细胞输注给第2供者小鼠后没有使之发生AMI,再将第2供者小鼠的骨髓细胞经BMI-1转染后再移植小鼠,受者小鼠即可发生AML。对BMI-1-/-白血病细胞进行体外培养发现,培养10 d后,小鼠白血病细胞数较对照组明显下降,细胞阻滞于G1期且凋亡细胞增加,这类细胞的集落形成能力也有所下降。提示BMI-1对白血病干细胞的增殖与致白血病的能力具有决定性作用。本研究拟通过对NCI-H446小细胞肺癌细胞株单细胞悬液进行细胞培养,观察克隆形成率和克隆形态;通过体外实验探索研究多靶点抗肿瘤新药索拉非尼（Sorafenib）是否对不同克隆肿瘤细胞有效及疗效之间存在差别,指导临床化疗失败患者的治疗。进一步比较不同克隆细胞周期分布差别;检测比较表面标记物CD133、ABCG₂、BMI-1基因表达。探讨NCI-H446小细胞肺癌细胞株克隆异质性与肿瘤干细胞的关系,即其细胞生物学行为表明肺癌肿瘤干细胞存在,为下一步肺癌肿瘤干细胞分离鉴定提供实验依据和基础。本研究国内外尚未见同类报道,具备创新性及一定临床指导价值。目的1.研究NCI-H446小细胞肺癌细胞株克隆形态、异质性、细胞周期差别。2.研究多靶点抗肿瘤新药索拉非尼对NCI-H446小细胞肺癌细胞株不同克隆细胞的体外疗效差别。3.研究NCI-H446小细胞肺癌细胞株不同克隆细胞干细胞表面标记物CD133、ABCG₂、BMI-1表达情况。方法1.细胞培养:实验取对数生长期的细胞。NCI-H446小细胞肺癌细胞株以RPMI1640培养基培养,实验取对数生长期的细胞,细胞数为0.5×10⁶-1×10⁶个/ml。2.有限稀释法克隆培养制备单细胞悬液:对数生长期的细胞贴壁生长铺满培养瓶后,应用有限稀释法将单细胞悬液接种于96孔培养板培养,标记单个细胞生长的培养孔,2～3周后细胞形成克隆,观察不同克隆形态和生长速度。转移到普通培养瓶扩大培养2—3周。3.MTT法检测细胞增殖抑制率:不同克隆细胞传代制备单细胞悬液。将实验药物索拉非尼按终浓度分别为6μmol/L、3μmol/L、1.5μmol/L进行MTT实验,96孔板用酶标仪于570纳米（nm）处测定各孔吸光度（A）值,细胞增殖抑制率（%）=（1—实验组吸光度值/阴性对照组吸光度值）×100%。4.流式细胞术测定各克隆细胞周期:各克隆培养细胞铺满培养瓶壁后按细胞传代常规方法制备细胞悬液,流式细胞仪常规检测细胞周期分布。5.免疫组化染色干细胞标志物CD133、ABCG₂检测:各克隆培养细胞铺满培养瓶壁后按细胞传代常规方法行细胞爬片。免疫组化法检测干细胞标志物CD133、ABCG₂。6.不同克隆核蛋白Western-blotting法免疫印迹分析检测BMI-1蛋白:将不同克隆细胞核蛋白应用Western-blotting法免疫印迹分析检测BMI-1蛋白。7.统计学处理:实验所得CD133和ABCG₂阳性率采用卡方分析检验,细胞周期分布以配对t检验,实验所得细胞增殖抑制率以组间比较检验采用卡方分析检验,数据处理采用SPSS11.5软件统计分析。结果1.15个单细胞克隆生长形成克隆,形态上大致分为两种克隆,索拉非尼对NCI-H446人小细胞肺癌细胞株克隆A细胞增殖抑制作用不随时间及浓度变化,抑制率为4.96%-6.49%;对克隆B抑制率为3.86%-30.39%,呈剂量-时间依赖效应。索拉非尼对两种克隆细胞增殖抑制具有显著性差异。2.15个单细胞克隆生长形成克隆,形态上大致分为两种克隆。克隆A共染色6片次,CD133、ABCG2表达阳性率为（66.67±8.57）%、（33.33±5.17）%;克隆B共染色26片次,CD133、ABCG2表达阳性率为（15.38±2.68）%、（7.69±1.64）%。克隆A与克隆B的CD133、ABCG2表达具有显著性统计学差异（χ²=38.12,40.24,P＜0.05）。克隆A细胞处于G0/G1者多于克隆B,分别是（70.13+3.26）%和（54.61±1.38）%。3.15个单细胞克隆生长形成克隆,形态上大致分为两种克隆,克隆A的BMI-1蛋白阳性表达率为75.00%,克隆B的BMI-1蛋白阳性表达率为12.50%,NCI-H446人小细胞肺癌细胞株BMI-1蛋白阳性表达率为33.33%,克隆A和克隆B的BMI-1蛋白阳性表达率具有显著统计学差异。结论1.NCI-H446人小细胞肺癌细胞株细胞存在异质性肿瘤细胞克隆,可能来源于肿瘤干细胞的分化。2.索拉非尼能抑制NCI-H446人小细胞肺癌细胞株细胞增殖,部分呈剂量-时间依赖效应,索拉非尼对不同克隆细胞增殖抑制率不同,可能与不同克隆细胞生物学特性有关。3.克隆形态观察,肯定2种克隆形态差别明显,BMI-1蛋白在小细胞肺癌不同克隆细胞亚群的表达可能与其发生发展生物学行为和异质性有密切关系,高表达BMI-1蛋白克隆可能存在肺癌肿瘤干细胞。更多还原

【Abstract】 BACKGROUND:There is an increasing evidence supporting the cancer stem cell hypothesis.It has been proven that tumor cells are heterogeneous comprising rare tumor initiating cells and abundant nontumor initiating cells.Tumor initiating cells-cancer stem cells have the ability of selfrenewal and proliferation,are resistant to drugs,and express typical markers of stem cells.It is not clear whether cancer stem cells originate from normal stem cells in consequence of genetic and epigenetic changes and/or by redifferentiation from somatic tumor cells to the stem-like cells. Probably both mechanisms are involved in the origin of cancer stem cells. Dysregulation of stem cell self-renewal is a likely requirement for the development of cancer.Isolation and identification of cancer stem cells in human tumors and in tumor cell lines has been successful.To date,the existence of cancer stem cells has been proven in acute and chronic myeloid leukemia,in breast cancer,in brain tumors,in lung cancer and gastrointestinal tumors.Cancer stem cell model is also consistent with some clinical observations.Although standard cheroot-herapy kills most cells in a tumor, cancer stem cells remain viable.Despite the small number of such cells, they might be the cause of tumor reccurrence,sometimes many years after the "successful" treatment of primary tumor.Growth of metastases in distinct areas of body and their cellular heterogeneity might be consequence of cancer stem cell different-tiation and/or dedifferentiation and asymmetric division of cancer stem cells.Further characterization of cancer stem cells is needed in order to find ways to destroy them,which might contribute significantly to the therapeutic management of malignant tumors. Kim and his colleages had isolated such a regional pulmonary stem cell population in 2005,termed bronchioalveolar stem cells(BASCs).Identified at the bronchioalveolar duct junction,BASCs were resistant to bronchiolar and alveolar damage and proliferated during epithelial cell renewal in vivo. BASCs exhibited self-renewal and were multipotent in clonal assays, highlighting their stem cell properties.Furthermore,BASCs expanded in response to oncogenic K-ras in culture and in precursors of lung tumors in vivo.These data support the hypothesis that BASCs are a stem cell population that maintains the bronchiolar Clara cells and alveolar cells of the distal lung and that their transformed counterparts give rise to adenocarcinoma.Although bronchiolar cells and alveolar cells are proposed to be the precursor cells of adenocarcinoma,this work points to BASCs as the putative cells of origin for this subtype of lung cancer.Dong Qiang-gang and his assistants had isolated and characterized the highly tumorigenic cancer stem cells with stem cell feathers from the established human lung adenocarcinoma cell lines in 2008.The cell subtypes were separated by MACS and the stem cell-related markers on these cells were detected.They concluted that the CD 24~+ IGF-1R~+ cells in human lung adenocarcinoma belongs to the BASC-like cancer stem cells.Renewing cancer stem cells exist in Holoclone in A549 lung cancer stem.CD133、ABCG2 maybe the signs on the surface of lung neoplasm cancer stem cells.BMI-1 gene overpressing maybe related with the growth of lung neoplasm cancer stem cells.SP(side population) cells maybe useful for isolation of cancer stem cells in surficant-unkown cells.OBJECTIVE:This study is to investigate the character of heterogeneous clones derived from a single cell,and to explore the mechanism of their heterogeneity;to investigate the expression of BMI-1 in human lung cancer H446(small cell lung cancer,SCLC) line heterogenous subpopulation cells;relationship between them and cancer stem cells in human lung cancer H446(small cell lung cancer,SCLC) line cells;to investigate the Inhibitory effects of sorafenib on NCI-H446 human lung cancer line cells.METHODS:NCI-H446 lung cancer line cells were cultured in vitro, heterogeneous clones were isolated by limiting dilution.CD133 and ABCG2 were detected by immunohistochemistry(IHC) in each clone cells.Cell cycle was examined by flow cytometry(FCM).BMI-1 prote(?) were detected by Western-blotting.The inhibitory effects of sorafenib(?) NCI-H446 human lung cancer line cells were determined by MTT ass(?) after at 24h,48h and 72h after the addition of sorafenib to the NCI-H446 culture. The concentration of sorafenib were 6umol/l、3umol/l、1.5umol/l.RESULTS:15 clones were derived from H446 line cultured cells,they were divided into two sorts termed clone A and clone B morphologyly.The positive rate of CD133 and ABCG2 expression in clone A were(66.67±8.57)%、(33.33±5.17)%;while(15.38±2.68)%、(7.69±1.64)%in clone B(P＜0.05).Furthermore,the ratio of cells in G0/G1 phase were(70.13±3.26) %70.13%and(54.61±1.38)%.The positive rate of BMI-1 protein expression in clone A was 75%while 12.5%in clone B(P＜0.05).Totally,the positive rate of BMI-1 protein expression in H446 line cells was 33.33%. Inhibitory ratio of sorafenib on clone A was 4.96%-6.49%,in contrast, it was about 3.86%-30.39%on clone B.The inhibitory effect of sorafenib on clone B cells could be developed as the time and concentration increased.CONCLUSIONS:Charecters of morphology and tumorigenesis are different in clones,cancer stem cells maybe exist in clone A which over expresses CD133 and ABCG2.BMI-1 is related to tumor progress in different clones,cancer stem cells maybe exist in clone A which over expresses BMI-1.NCI-H446 lung cancer cells may be inhibited by sorafenib, and the effects were increased partly with the time and concentration developed.The different effects on the two clone cells were related to their biological mechanism.更多还原