【作者】 詹军芳；

【导师】 陈龙华； 袁亚维；

【作者基本信息】 南方医科大学 ， 放射肿瘤学， 2010， 博士

【摘要】 研究背景和目的越来越多的实验证据[1-4]表明肿瘤是一种干细胞疾病,即只有小部分肿瘤干细胞才有致瘤及维持恶性表型的作用。对这一小部分细胞不同的学者有不同的命名,有称为“肿瘤干细胞”(CSCs)、“肿瘤源性癌细胞”、“肿瘤始动细胞”,都是要表明只有这部分细胞移植入免疫缺陷小鼠体内时才能产生新生的肿瘤。经过对肿瘤细胞亚群的分选、特异的表面分子标志及移植动物的功能性实验,已在血液、脑肿瘤、乳腺癌、前列腺癌、肝癌、胰腺癌、鼻咽癌和结肠癌等中分离出各自的肿瘤干细胞[5-12]。2003年,Al Hajj等[13]应用FACS技术从乳腺癌组织中分离出表型为CD44+CD24-ESA+ LIN-的细胞具有干细胞特性,200个即可在小鼠体内成瘤。2005年,Ponti等[14]证实乳腺癌干细胞可以在体外繁殖成非贴壁的球囊细胞,这为以后研究乳腺癌干细胞及分子靶向特异干扰乳腺癌干细胞的自我更新和存活机制提供了合适的模型。肿瘤干细胞数量稀少,它不仅是肿瘤的来源,而且与肿瘤的进展和转移有关,因此,尽管已经治愈了原有疾病,但肿瘤干细胞tumor stem cell,TSC)的存活会最终导致肿瘤的复发,这预示着只有杀死所有肿瘤干细胞,抗肿瘤治疗才能治愈肿瘤。放疗是乳腺癌的重要治疗手段之一,术后放疗可以杀灭残留的肿瘤细胞,术前放疗可以缩小肿块的大小。已有研究发现电离辐射后的肿瘤干细胞的凋亡率明显低于普通肿瘤细胞,提示肿瘤放射抵抗可能与肿瘤干细胞有关[15-18]。随着分子生物学的发展,一些与肿瘤放疗抵抗相关的基因被鉴定出来,使人们能够通过各种手段作用于这些新的靶点以提高肿瘤的放射敏感性。PI3K-AKT信号传导通路在经受电离辐射后被一些外源性刺激因子激活,促进肿瘤细胞存活,已有研究证实其与放射抵抗相关[19,20]。十余年前已被发现的AKT1是PI3K的重要的下游靶因子,目前已发现3个AKT家族成员,AKT1、AKT2和AKT3。AKT/蛋白激酶B信号作用通道调控细胞增殖和生长,促进肿瘤发生[21,22][19,23,24],已有许多证据证实PI3K-AKT是乳腺癌的致癌基因[25-27]。转录信号传导子与激活子(STAT)通路在十多年前作为干扰素诱导的基因表达介质被发现的,包括7个成员,可以被多种细胞因子、生长因子等分子激活,与细胞增殖、分化及凋亡密切相关[28,29],该通路持续激活可导致细胞异常增殖、恶性转化和阻断细胞凋亡。目前已发现STAT1与头颈部癌的放射抵抗相关[30]。AKT-1和STAT-1是否在乳腺癌干细胞中特异性表达将关系到他们是否可以成为乳腺癌放疗和化疗的新靶点。本研究通过对乳腺癌干细胞进行51种相关辐射抵抗基因的qRT-PCR筛选,并针对AKT-1和STAT-1与乳腺癌干细胞的辐射抵抗关系进行探讨。方法1、悬浮培养联合放射线富集具放射抵抗的乳腺癌干细胞MCF-7细胞复苏后加入完全培养基,分别用有无血清的培养基中培养,至对数生长期时制成单细胞悬液,准备流式染色。一部分悬浮培养细胞经varian2100C/D以300-500cGy/min的剂量率照射8Gy后继续悬浮培养,分别检测第二代、第四代、第五代、第六代、第七代的CD44+/CD24-/low的含量。2、对乳腺癌干细胞进行51种与肿瘤增殖及周期调控有关的基因表达微阵列分析采用Primer premier 5软件设计引物,由invitrogen公司合成51种与肿瘤增殖及周期调控有关的基因微阵列组成(具体见正文)。对MCF-7普通培养和悬浮培养的球囊细胞照射前后分别提取细胞RNA,应用荧光定量逆转录聚合酶链式反应(qRT-PCR)进行51种与肿瘤增殖及周期调控有关的基因表达微阵列分析。3、STAT1与AKT1在乳腺癌始动细胞CD44+/CD24-/low的表达水平对MCF-7普通培养和悬浮培养的球囊细胞照射前后应用qRT-PCR重复检测STAT1与AKT1的mRNA表达水平,并应用SPSS 13.0软件进行统计学处理,采用student’s t检验分析STAT1与AKT1在MCF-7与球囊细胞照射前后的表达水平；4、加用STAT1与AKT1抑制剂后对乳腺癌始动细胞CD44+/CD24-/low放射生物学特性的影响分别在MCF-7和球囊细胞中加用STAT1和AKT1抑制剂后,对标本照射前后Annexin V-100 FITC/PI流式细胞仪法检测细胞凋亡、检测克隆形成率和生存曲线,并应用SPSS 13.0软件和3rap hPad Prism(4.0)统计软件处理。5、统计方法：所有数据均以均值x±SD表示,应用student’s t检验分析,P≤0.05为有统计学差异。结果1、流式细胞仪检测结果MCF-7普通培养的含量是0.1%-1.8%,照射后是4.8%-8.3%,悬浮培养后55.5%-69.1%,照射后第二代是75.5%-83.1%,第四代是96.5%-98.8%,第五代是98.3-98.7%,第六代是93.3-95.7%,第七代是90.3%-93.9%。,单纯悬浮培养比普通培养含量高,差异具有显著性(P<0.001)；悬浮联合放射线照射后培养比单纯悬浮培养含量高,差异具有统计学意义(P<0.001)。悬浮联合放射线照射后培养第四代到第五代细胞球CD44+CD24-含量最高,其后逐渐稳定。2、51种与肿瘤增殖及周期调控有关的基因表达微阵列分析应用qRT-PCR高通量筛选相关基因,分组为MCF-7组、球囊空白组和球囊照射8Gy组,分别检测了51种基因mRNA的表达水平,可见悬浮培养的球囊细胞经过放射线照射后ABL1、AKT1、APC、BAX、BAX3、BRAF、BRCA1、BTK、E2F1、MDAC1、IGFIR、BAF1、SIVA1-1、SIVA1-2、STAT1、TGFB1、TIMP3、TRADD、VDAC1、CDK4、CDK5、CDKN1A、CDKN2B、CDKN2D、ING1、JUN、LCK、GNAS-1、ING1、JUN、LCK、GNAS-1表达明显上调,而CDKN1B、FGR、NFKB1变化不明显,ATM、CDKN1C明显下降。综上所述,BCl-2, BRCA-1,Statl,Ing1,E2F1,AKT1,HDAC1,Kit在照射后的乳腺癌干细胞CD44+/CD24-的mRNA表达水平比照射前升高。3、STAT1与AKT1在乳腺癌始动细胞CD44+/CD24-/low照射后的表达水平明显上调。AKT1在乳腺癌MCF-7细胞株照射后和照射前的表达水平无明显差别(P=0.20),但是在球囊细胞CD44+/CD24-/low照射后比照射前明显上调(P<0.01);STAT1在乳腺癌MCF-7细胞株照射后比照射前的表达水平稍有上调(P=0.02),但是在球囊细胞CD44+/CD24-/low照射后比照射前明显上调(P<0.01)。4、加用STAT1与AKT1抑制剂后对乳腺癌始动细胞CD44+/CD24-/low放射生物学特性的影响在MCF-7和球囊细胞中分别加用AKT1抑制剂后,照射前后的凋亡率、生存分数改变无明显差异(P>0.05),球囊细胞加用抑制剂后未照射组的凋亡率、生存分数改变无明显差异(P>0.05),加用抑制剂后照射组的凋亡率上升,生存分数减少,差异具有统计学意义(P<0.05)；同样,在MCF-7和球囊细胞中分别加用STAT1抑制剂后,照射前后的凋亡率、生存分数改变无明显差异(P>0.05),球囊细胞加用抑制剂后未照射组的凋亡率、生存分数改变无明显差异(P>0.05),加用抑制剂后照射组的凋亡率上升,生存分数减少,差异具有统计学意义(P<0.05)；结论1.悬浮培养联合放射线照射得到的干细胞比例高达98%,稳定于90%左右。2.BCl-2, BRCA-1,Sta1,Ing1,E2F1,AKT1,HDAC1,Kit与乳腺癌干细胞的放疗抵抗可能有关.3.AKT1和STAT1与乳腺癌干细胞的放射抵抗密切相关,阻断各自的通路有可能加强乳腺癌的放疗效果。本研究的创新之处1、利用悬浮培养联合放射线照射成功富集了乳腺癌干细胞,比之前所有的分离方法得率都高。2、第一次应用qRTPCR的方法定量检测乳腺癌干细胞的相关放疗抵抗基因,发现BCl-2, BRCA-1, Stat1,Ing1,E2F1,AKT1,HDAC1,Kit与有关；3、分别应用AKT1和STAT1特异性抑制剂来抑制PI3K/Akt、JAK/STAT通路的活化,进一步验证了AKT1、STAT1与乳腺癌干细胞CD44+/CD24-/low的放射抵抗相关。4、乳腺癌干细胞的放射抵抗基因研究目前尚未有报道,本研究填补了空白。更多还原

【Abstract】 Background and Objection:In recent years, a growing body of evidence has been reported supporting the hypothesis that tumors are driven and maintained by a minority subpopulation of cells that have the capacity to self-renew (i.e.,give rise to progeny with similar properties as themselves) and to generate the more differentiated progeny which make up the bulk of a tumor.The minority population has been termed cancer stem cells (CSCs), tumorigenic cancer cells, or tumor-initiating cells, by various investigators, to indicate that only they can give rise to new tumors when transplanted into immunodeficient animals. Tumor-initiating cells have been identified in blood, brain, breast cancers,prostate, liver, pancreas, and colon cancers (4-6) through an experimental strategy that combines sorting of tumor cell subpopulations, identified on the basis of the different expression of surface markers, with functional transplantation into appropriate animal models. The subpopulation with the CD24-/low/CD44+ phenotype was shown to identify breast cancer-initiating cells in primary patient specimens which 200 cells were transplanted into NOD/SCID mice and formed tumor by Al Hajj using fluorescence-activated cell sorting (FACS) etc in 2003.Then in 2005,Ponti etc showed that breast tumorigenic cells with stem/progenitor cell properties can be propagated in vitro as nonadherent mammospheres and represent a suitable in vitro model to study breast cancer-initiating cells and to challenge them with molecularly targeted agents specifically interfering with breast cancer-initiating cells self-renewal and survival.According to the hypothesis, CSCs limited number within the bulk of the tumor are not only the source of the tumor but also may be responsible for tumor progression,metastasis,thus leading to subsequent tumor relapse although the primary lesion is eradicated and resistance to therapy; Radiotherapy, as an integral part of the current comprehensive breast cancer treatment regimen, may be used to eradicate remaining cancer cells in the breast, chest wall, or axilla after surgery or to reduce the size of an advanced tumor before surgery.hence, it is imperative to understand the molecular mechanism underlying this radioresistance. A number of candidate genes have been implicated in the response of eukaryotic cells to ionizing radiation, including cell cycle, checkpoint, and DNA repair genes, as well as mediators of apoptosis, such as p53,Bax,Bcl-2, and so forth.PI3K-AKT signaling is an important cascade to promote cell survival in response to exogenous stress factors such as exposure to ionizing radiation. It has been shown that PI-3K pathway plays a critical role in mediating radioresistance.One of the best characterized downstream targets for the PI-3K lipid products is Aktl, which was discovered about a decade ago and which is now known to be a member of a family of closely related, highly conserved cellular homologues that consists of at least two additional members (Akt2 and Akt3). Akt has been shown to be a critical player in oncogenesis. There is increasing evidence indicating that PI-3K/Akt plays an important role in breast cancer tumorigenesis. An elevated level of Akt activity was associated with increased cellular resistance to the treatment with doxorubicin or tamoxifen in breast cancer cell lines.STAT transcription factors were discovered 10 years ago as mediators of interferon-induced gene expression. They now form an important group, comprising seven members, that are activated by virtually every cytokine and growth factor. Cell culture work has further delineated their importance in cellular proliferation, transformation, apoptosis, differentiation and growth control. STAT1,STAT3 and STAT5 may play an important role in breast carcinogenesis. Recently, signal transducer and activator of transcription 1(STAT1)has been implicated in the presence of radioresistance,first demonstrated in a head-and-neck cancer cell line (4).To date, AKT1 and STAT1 involvement in CD24-/low/CD44+ breast cancer-initiating cells in general and their specific relationship with radioresistance of CD24-/low/CD44+ breast cancer-initiating cells have never been explored.This study intend to discover the genes which may play a key role in radioresistence of CD24-/low/CD44+ breast cancer-initiating cells by qRT-PCR and investigate the effects of AKT1 and STAT1 in radiosensitivity by the additional of specific inhibitors.METHODS AND MATERIALS1.Suspension culture combined with radiation to enrich CD24-/low/CD44+breast cancer-initiating cells that have more radioresistance.MCF-7 cultured in SSM and SFM respectively. Then some mammaspheres were cultured with radiation at a dose rate of 300-500 cGy/min by varian2100 C/D. The cells cultured to achieve 80-90% confluence on the day of experiments were stained with CD24-PE and CD44-FITC and analyzed the percentage of CD44+CD24- by flow cytometry.2. The 51 genes chip was designed by Primer 5 software and syntheses by Invitrogen Co.Real-time quantitative reverse transcription polymerase chain reactions performed to detect the mRNA level of 51 genes in MCF-7 and mammaspheres before and after radiation.3.AKT1 and STAT1 mRNA levels were examined d by qRT-PCR repeatedly.4. Evaluated apoptosis by flow cytometrywith Annexin-V100 FITC/PI double staining method and calculated the plating efficiency by the number of colonies/cells seeded and fitted the surviving fraction by GraphPad Prism(4.0) in MCF-7 and mammaspheres before and after AKT1 or STAT1 inhibitor before and after radiation..5.All data are represented as means and differences of the means with 95% confidence intervals (CIs). P values of.05 or less, calculated using a paired two-sided Student’s t test or chi-square test, were considered to indicate statistically significant differences.RESULTS1.Flow-cytometric analysis.The percentage of cells from suspension culture combined with radiation higher than those in single suspension culture without radiation and the difference was determined to be statistically significant(P<0.001)2.It was revealed that-mRNA levels of BC1-2, BRCA-1,Statl,Ing1, E2F1, AKT1,HDAC1 and Kit were higher after radiation than those before radiation in mammaspheres.3.The mRNA levels of STAT1 and AKT1 were higher after radiation than those before radiation in mammaspheres and the difference was determined to be statistically significant(P<0.001),whereas,there was no difference between MCF-7 before and after radiation.4. Apoptosis and the surviving fraction had no difference in MCF-7 treated with AKT1 inhibitor or STAT1 inhibitor respectively before and after radiation and mammaspheres with AKT1 inhibitor or STAT1 inhibitor respectively before radiation(P>0.05).However, Apoptosis was enhanced and the surviving fraction decreased in mammaspheres with AKT1 inhibitor or STAT1 inhibitor respectively after radiation.The difference was determined to be statistically significant(P<0.001).Conclusion1.The percentage of CD44+CD24-through suspension culture combined with radiation is up to 98%,stable at about 90%。2.Our data suggest that BC1-2, BRCA-1,Stat1,Ing1,E2F1,AKT1,HDAC1 and Kit may be associated with the radioresistance in breast cancer-initiating cells CD44+/CD24-/low.3.AKT1 and STAT1 may play a key role in radioresistance in breast cancer-initiating cells CD44+/CD24-/low and manipulation of this pathway may enhance the efficacy of radiotherapy.Innovations of our study:1.The enrichment of CD44+CD24-through suspension culture combined with radiation is successful than all the methods previous。2.This is the first study reporting the mRNA level of genes which may be associated with the radioresistance in breast cancer-initiating cells CD44+/CD24-/low by qRT-PCR and suggesting that BC1-2, BRCA-1,Statl,Ing1,E2F1,AKT1,HDAC1 and Kit may be associated with the radioresistance.3.The blockage of PI3K/AKT and JAK/STAT by the addition of a STAT1-specific inhibitor, fludarabine, and AKT1-specific inhibitor, significantly increased the fraction of apoptotic cells.These results confimed that AKT1 and STAT1 may play a key role in radioresistance in breast cancer-initiating cells CD44+/CD24-/low and the targeting of STAT1 and AKT1 could prove to be a promising new therapeutic strategy against breast cancer.4.Genetic studies of radioresistence in breast cancer stem cell has been repoted yet and our study provide a valuable result.更多还原