【作者】 苗霞；

【导师】 郭国祯； 张杰；

【作者基本信息】 第四军医大学 ， 放射医学， 2013， 博士

【摘要】 化疗是临床肿瘤治疗的必要手段之一，而长时间使用化学药物后，肿瘤细胞的复发、转移始终是制约最终疗效的瓶颈问题。目前研究认为，肿瘤中少量存在的肿瘤干细胞是化疗或放疗后肿瘤复发的根源，肿瘤干细胞不仅具有自我更新和增殖能力，同时对化学药物和射线等具有高度抗性。然而，关于肿瘤干细胞的来源始终存在较大争议。有学者认为，肿瘤干细胞可能来源于正常干细胞的恶性转化；但近期研究表明，在肿瘤化疗过程中，DNA损伤可能一方面消除肿瘤细胞，同时也可能加剧基因组的不稳定性，进而诱导普通肿瘤细胞演变成干细胞样肿瘤细胞（stem-likecancer cells, SLCCs），其相关分子机制尚不明确。现行的临床肿瘤治疗策略中，放疗往往是在阶段性化疗后开展，那么，化疗药物诱导SLCCs的产生和累积可能对放疗效果产生巨大影响。因此，阐明化疗药物诱导SLCCs产生的特点，进而围绕DNA损伤修复和基因组稳定性，寻找新的关键靶分子，对于深入探讨化疗过程中肿瘤细胞向SLCCs演变的分子机制，优化临床肿瘤治疗策略，将具有重要参考意义。翻译调控肿瘤蛋白（Translationally controlled tumor protein，TCTP）是一个在多种组织中，如肝癌、肺癌、胰腺癌、胃癌、黑色素瘤、宫颈癌、乳腺癌、骨肉瘤、神经胶质瘤、白血病等，广泛表达的高度保守小分子蛋白质。它不仅参与调控细胞周期、增殖和抗凋亡等多种重要生理过程，还与肿瘤的发生、发展密切相关。我们的前期研究证实，TCTP是维持细胞基因组稳定的重要分子，在DNA损伤修复中发挥着关键作用。另有研究表明，TCTP参与了肿瘤细胞的恶性转化调控，敲除TCTP可以有效促进乳腺癌细胞发生恶性逆转，更可导致动物乳腺癌中“类肿瘤干细胞”的数量显着减少，提示TCTP可能在SLCCs的生成及其生物学特性维持中具有重要作用。值得注意的是，既往研究还提示，TCTP的表达改变可能与肿瘤耐药的产生相关，但其机制和意义尚未见报道。基于TCTP在维系基因组稳定和促进细胞恶性转化中的重要作用，本研究首先利用人胶质瘤及乳腺癌临床组织芯片和临床肿瘤标本，明确TCTP蛋白表达在不同临床分级肿瘤中的分布特点和意义；同时，结合现行的临床肿瘤治疗策略，选用不同组织来源（胶质瘤和乳腺癌）、具有不同转移潜能的五种肿瘤细胞系为对象，建立了九种耐药细胞模型；在此基础上，研究化疗药物诱导SCLLs产生的特点，并通过体外细胞试验和在体裸鼠成瘤实验，观察了TCTP表达改变与耐药细胞的干细胞样生物学特性的相关性；利用siRNA转染等技术，给予耐药细胞复合辐射暴露，进一步探讨了TCTP在维系耐药细胞辐射抵抗中的作用机制。方法：1、利用胶质瘤及乳腺癌临床组织芯片，通过免疫组织化学方法，显微镜下，从每张免疫组织化学染色阳性的结果中随机观察5个不同视野。观察在细胞浆和细胞核的阳性染色时才记分。评分标准如下：阳性细胞数为<10%，0分；10%-30%，1分；30%-50%，2分；50-70%，3分；>70%，4分。观察TCTP在不同等级的临床胶质瘤及乳腺癌标本中的表达变化；同时，采集临床胶质瘤组织，利用Western-blot方法，观察TCTP在不同等级的临床胶质瘤组织中的蛋白表达。2、选用人乳腺癌细胞MCF-7、MDA-MB-231和人胶质瘤细胞系SHG44、U251和U87五种肿瘤细胞为对象，通过MTT实验选择合适的药物剂量，对乳腺癌细胞进行三种方式（5-氟尿嘧啶：5-FU、盐酸阿霉素:ADM及5-氟尿嘧啶+盐酸阿霉素:5-FU+ADM）的长期化疗药物诱导筛选（MDA-MB-231连续给药3次/代，MCF-7连续给药2次/代，持续8个月），对胶质瘤细胞给予TMZ诱导筛选（HG44、U251和U87连续给药2次/代，持续6个月），建立肿瘤细胞耐药模型。通过免疫印迹法实验，观察多药耐药蛋白ABCG2的表达变化。3、利用细胞划痕、Transwell侵袭以及克隆形成等实验，观察筛选出的耐药细胞模型中，其形态学、细胞增殖能力、迁移和侵袭能力的变化规律及特点。4、利用流式细胞技术，观察乳腺癌耐药细胞中的CD44+/CD24-干细胞亚群比例变化；采用免疫印迹法，检测耐药细胞的干细胞标记物Oct4和TCTP的蛋白表达变化。5、对裸鼠进行肿瘤细胞皮下注射实验，在体观察筛选出的耐药细胞的成瘤性；通过免疫组织化学方法，观察TCTP在不同细胞成瘤组织中的表达变化。6、利用TCTPsiRNA转染技术，观察降低TCTP表达后，乳腺癌耐药细胞中OCT4的表达改变。7、耐药细胞株接收0Gy，1Gy，2Gy，4Gy，8Gy X射线辐照后，通过克隆形成实验评价其辐射抗性。8、通过siRNA转染联合辐射暴露，利用CCK-8试剂盒和台盼蓝染色实验，观察降低TCTP表达对乳腺癌耐药细胞增殖能力和存活状况的影响。9、通过siRNA转染联合辐射暴露后，利用凋亡检测试剂盒，观察降低TCTP表达对乳腺癌耐药细胞凋亡的影响；应用western-blot方法，观察TCTP表达改变对辐照后自噬标志物LC3B表达的影响。结果：1、临床组织芯片检测结果显示，在不同临床分级的乳腺癌及胶质瘤组织中，TCTP的表达随着肿瘤恶性程度的增高而升高；Western-blot检测结果显示，恶性程度越高的临床胶质瘤组织中TCTP的蛋白表达量越高。2、肿瘤耐药模型的建立：乳腺癌细胞系给予5-FU1μg/ml，ADM0.08-0.1μg/ml连续给药8个月，胶质瘤细胞系给予TMZ500μM-1000μM连续给药6个月。同时，以ABCG2表达变化为主要耐药标志物，研究显示，同对照组相比，筛选出的五种化疗抵抗细胞系中，ABCG2表达均显着增高，提示本研究中的乳腺癌和胶质瘤耐药细胞模型建立成功。3、耐药肿瘤细胞的生物学特性研究：①与对照组相比，六种乳腺癌耐药细胞在形态上呈现出间质细胞样外观，而三种胶质瘤耐药细胞形态上无明显改变；②与对照组相比，耐药细胞的划痕伤口愈合能力均显着增加，同时，Transwell小室穿膜细胞数量增多，提示耐药细胞的迁移和侵袭能力明显提高；③克隆形成实验结果表明，与对照组相比，耐药细胞的增殖能力显着增强。4、干细胞样肿瘤细胞（SLCCs）筛选分析表明，五种耐药细胞株中的干细胞标记物OCT4表达均明显升高，同时伴随TCTP的表达升高；流式细胞术检测显示，与对照组相比，乳腺癌耐药细胞群中的CD44+/CD24-细胞亚群比例显着提升，提示干细胞样乳腺癌细胞比例增加。5、裸鼠在体植瘤实验结果表明，同对照组肿瘤细胞相比，耐药细胞的皮下种植成瘤率明显增高，并且肿瘤生长速度快；免疫组化染色发现，在耐药肿瘤细胞的成瘤组织中，TCTP阳性颗粒的表达量明显高于对照组。上述结果表明，TCTP的异常高表达与耐药细胞的高增殖能力密切相关。6、利用siRNA质粒转染技术降低TCTP表达后可见，乳腺癌抗药细胞的Oct4表达随TCTP降低而降低，两者变化呈正相关性。综合4-6实验结果分析表明，TCTP可能通过调控Oct4，在维持耐药细胞的干细胞样生物学特性中发挥关键作用。7、耐药细胞的辐射抗性研究表明，不同剂量X射线照射后，与对照组相比，耐药细胞的克隆形成率显着增加，提示耐药细胞辐射抗性明显提高。8、乳腺癌耐药细胞在siRNA转染联合辐射暴露后的实验结果显示，降低TCTP表达，乳腺癌耐药细胞的增殖能力和存活能力均显着降低。9、乳腺癌耐药细胞在siRNA转染联合辐射暴露后的实验结果显示，降低TCTP表达，受照细胞的凋亡显着增加，自噬小体形成增多，同时，自噬标志物LC3B的表达明显升高。结论：1、TCTP在胶质瘤和乳腺癌组织的蛋白表达主要位于细胞质和细胞核，并伴随着肿瘤恶性程度的增加而增加，提示TCTP可能成为未来判断肿瘤临床预后或分级的新的标志物。2、肿瘤细胞持续使用化疗药物后，耐药肿瘤细胞群中干细胞样细胞亚群的比率显着增加，筛选出的耐药肿瘤细胞出现明显的干细胞样肿瘤细胞（SLCCs）生物学特性，包括OCT4表达增加、细胞增殖能力、迁移能力和克隆形成能力均显着增加，TCTP主要通过维持上述SLCCs生物学特性，在耐药细胞中发挥了重要作用。3、耐药肿瘤细胞同时具有显着的放疗抗性，TCTP可能通过抗凋亡和抗自噬两种方式参与化疗耐药细胞辐射抗性的调控。更多还原

【Abstract】 Chemotherapy is one of the necessary methods of clinical cancer treatment. Althoughmost malignancies initially respond to chemotherapeutic treatments, after an unpredictableperiod, developed chemoresistance and recurrence of tumor cells could always lead to thefailure of treatment. Multiple mechanisms which cancer cells may use to developresistance to the cancer treatment have been proposed. Notably, Cancer Stem Cells (CSCs),also named tumor-initiating cells and stem-like cancer cells (SLCCs) have been postulated recently as responsible for recurrent tumours after chemotherapy. CSCs not only have theability of self-renewal and proliferation, but also have developed highly resistant toionizing radiation and chemical drugs. Current studies showed that, during the process ofchemo-or radio-therapy, while DNA damage induced by ionizing radiation or chemicalsmay eliminate tumor cells, it may aggravate the instability of genome and promote thetumor cells to stem-like cancer cells（SLCCs）. The molecular mechanism is still unclear.Translationally controlled tumor protein（TCTP）is a small molecular, which is highlyconservative in evolution and widely expressed in many tissues. It has been shown thatTCTP is involved in regulating a variety of important physiological processes, includingthe cell cycle, proliferation, anti-apoptosis and development of cancer. Our previous studyconfirmed that TCTP is one of the key molecules in the maintenance of genome stabilityand plays a critical role in DNA damage repair. Other studies have also shown that TCTPis involved in the regulation of tumor cell malignant transformation; knocking-down ofTCTP can effectively promote the malignant reversion of breast cancer cells and thenreduce the amount of the SLCCs. Interestingly, the change of TCTP expression in tumorcells has been suggested to associate with the chemoresistance, but its mechanisms hasnot yet been reported. The above studies implicates that TCTP may be involved in theprocess of SLCCs generation during chemotherapy.To learn the distribution characteristics and significance of TCTP in different clinicalclassification of tumor, we initiated our study by detecting the tissue and tissue chips ofglioma and breast cancer. Using5tumor cell lines, with different potential metastasis fromglioma and breast cancer, we set up9kinds of chemoresistant models through continualselection in6generation under different drug pressure. Then, we observed thecharacteristics of the generation of SCLLs which may be induced durng chemotherapy andfurther studied the correlation of the TCTP expression change and SCLLs biologicalcharacteristics.Results1. Clinical tissue chips detection shows that, the expression of TCTP increased withtumor malignant degree in different clinical classification of breast cancer and glioma tissues; Also, Western blot confirms that the amount of protein expression of TCTPincreased with tumor malignant degree.2. Through MTT analysis, we optimized the selection concentration of drugs,5-FU：1μg/ml，ADM：0.08-0.1μg/ml and TMZ:500μM-1000μM, respectively. As ABCG2expression changes the main resistance markers, compared with the control group,After continual selection through6generation, the expression of ABCG2, one of themarkers of chemoresistance, increased significantly in all9selected cell lines,which manifests that the chemoresistant cell models of breast cancer and glioma areestablished successfully.3. The study of biological characteristics of chemoresistant tumor cell lines shows that,①Compared with the control group, while interstitial cells emerge in two breastcancer resistant cells in morphology appearance, no obvious changes in threeTMZ-resistant glioma cells;②Compared with the control group, scratch woundhealing ability of five kinds of drug-resistant cells were significantly increased,meanwhile, Transwell Chambers in membrane the populations of cells through theTranswell Chambers membrane increased, which implicated that the migration andinvasion abilities of drug-resistant cell lines increased significantly;③Cloneformation assay shows that the proliferation ability of drug-resistant cells enhanced inthe culture medium containing drugs and3%FBS.4.Western blot analysis of SLCCs showed that, the expression of the stem cell markersOCT4in five chemoresistant cell lines were significantly increased, along with theup-regulation of TCTP; flow cytometry results show that,compared with the controlgroup, the percentage of CD44+/CD24-cells significantly increased inchemoresistant breast cancer cells. In vivo experiment in nude mice demonstrated that,compared with the control5. group, the growth rate of chemoresistant tumor cells which were subcutaneouslyinjected is much faster; immunohistochemical staining of tumor tissues show that thepositive staining particles of TCTP in groups of chemoresistant tumor cells weresignificantly higher than respective control groups. The above results suggested that TCTP expression is closely related to the proliferation ability of chemoresistant tumorcells.6. In chemoresistant breast cancer cells, down-regulation of TCTP by siRNA plasmidtransfection technology could also induce the reduction of Oct4expression. Thechange of TCTP and Oct4shows a positive correlation. Collectively, the results ofexperiments of four to six implicated that TCTP may play an important role inmaintaining the “stem” biological characteristics of SLCCs.7. We detected the radio-resistance of chemoresistant tumor cells by exposing the cellsto the different dose of X-rays. The clone formation analysis showed that thechemoresistant cells also have the stronger radio-resistant potentials. Interfering theexpression of TCTP by siRNA transfection would significantly reduced theproliferation and survival abilities of chemoresistant tumor cells when exposed to4GyX-rays.8. To understand the mechanisms of TCTP involved in maintaining radio-resistance ofchemoresistant tumor cells, we detect the occurrence of apoptosis and LC3Bexpression, marker of autophagy, in cells exposed to X-rays with down-regulation ofTCTP. The expression of caspase-3and caspase-8are increase in somechemoresistant. Our results showed that TCTP participates in radio-resistance ofchemoresistant tumor cells through both anti-apoptosis and anti-autophagy functions.Conclusion1. The expression of TCTP in glioma and breast cancer tissues is mainly located incytoplasm and nucleus. The expression of TCTP increase significantly with the tumormalignant degree in both glioma and breast cancer tissue, which suggested that TCTPcould probably become a new promising marker in clinical prognosis or classificationof tumor.2. The ratio of SLCCs increased significantly when tumor cells experienced thecontinual treatment of chemotherapeutic drugs. The chemoresistant tumor cellsshowed SLCCs biological characteristics, including much higher abilities of cellproliferation, migration and colony-forming, and up-regulation of OCT4. Strikingly, TCTP plays an important role in chemoresistant tumor cells by maintaining theSLCCs biological characteristics, which may relate with the regulation of OCT4.3. Chemoresistant tumor cells also showed significant radio-resistance, TCTP may beinvolved in which through both anti-apoptosis and anti-autophagy functions.更多还原