【作者】 胡敏；

【导师】 翁亚光；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2013， 博士

【摘要】 有丝分裂期是细胞周期中的重要一环，其中用于监控染色体的正确排列和分离并保证子代细胞基因组完整性的系统称为纺锤体组装检查点（spindle assembly checkpoint, SAC）。SAC功能的完整性是保证生物体以二倍体细胞不断分裂生长的前提，人类肿瘤常有不同程度的SAC异常。肿瘤发生的早期细胞周期检查点失常是促进肿瘤进程的关键步骤。SAC是由一套相互协调的蛋白组成的信号体系，其中BubR1是这个体系重要的感受者和执行者。在多种肿瘤中都发现有BubR1表达的异常，如在乳腺癌、膀胱癌、胃癌、卵巢癌中BubR1有表达上调，而在结肠癌中BubR1报道低表达。BubR1的异常表达代表了SAC功能的损伤，抗有丝分裂期类药物的药物作用通常依赖于正常的SAC存在。这些肿瘤中BubR1异常表达的内在机制却仍不明了，相关报道也很少。为了检测在食管鳞癌中是否有BubR1的表达异常，与抗微管药物紫杉醇的关系，以及在食管鳞癌中BubR1可能的调控机制，本课题利用了定量PCR的方法在收集的50例临床食管鳞癌标本中对比癌和癌旁组织中BubR1的表达，发现有72%（36/50）的样本中BubR1表达上调；三株食管鳞癌细胞株内BubR1的表达也较高，相对BubR1高表达的细胞株对抗微管药物紫杉醇敏感性较低。为了探讨BubR1在食管鳞癌中的作用，构建了BubR1表达腺病毒和干扰腺病毒。在食管鳞癌细胞株BubR1的表达被干扰后，细胞对紫杉醇的敏感性增加，药物的IC50减低，紫杉醇诱导的细胞死亡增多。虽然BubR1的表达下调在食管鳞癌细胞ECA-109中并不引起细胞增殖的短期变化，但能明显减低ECA-109在免疫缺陷小鼠的皮下成瘤。相反的，BubR1在食管鳞癌细胞过表达后能进一步减低细胞对抗微管药物的敏感性，表现在有丝分裂指数下降。免疫组化检测发现BubR1和C-Myc蛋白表达在食管鳞癌组织中有一致性升高趋势。为了检测是否C-Myc对BubR1有调控作用，构建了BUB1b基因启动子报告质粒，在食管鳞癌中检测是否癌基因C-Myc对BubR1的表达有调控作用。结果发现C-Myc的过表达可以激活BUB1b启动子活性并上调BubR1，而C-Myc抑制剂10058-F4则可减低BubR1的表达并恢复食管鳞癌细胞对紫杉醇诱导的细胞生长抑制，并增强紫杉醇作用下有丝分裂期阻滞。对BubR1在食管鳞癌中表达、药物影响和相关调控机制的研究能进一步了解BubR1在肿瘤中表达异常的原因，为食管鳞癌的有效治疗提供可能的理论依据。第一部分BubR1在食管鳞癌标本及细胞株中的表达及临床意义分析目的：1.检测BubR1在临床食管鳞癌组织及其癌旁组织中表达；2. BubR1的表达与食管鳞癌病人临床病理特征分析；3.检测BubR1在三株食管鳞癌细胞株中表达。方法：1.用定量PCR的方法检测BubR1在50例食管鳞癌病人癌组织和癌旁组织中的表达；2.统计分析BubR1的表达水平与病人临床病理特征的相关性；3.定量PCR和Western Blot方法检测BubR1在食管鳞癌细胞中表达。结果：1.50例食管鳞癌标本中有36例BubR1表达上调，其中23例肿瘤组织表达超过癌旁组织2倍以上；2. BubR1的表达与病人临床病理特征无直接相关性；3. BubR1在食管鳞癌细胞中有较高表达，其中ECA-109细胞中BubR1表达低于KYSE150和KYSE180细胞。结论：1.验证了BubR1在食管鳞癌中的异常表达，但其与病人临床病理特征之间未见直接联系；2.食管鳞癌细胞株中BubR1表达与临床食管鳞癌组织研究结果较为一致，BubR1表达相对较高。第二部分BubR1表达及干扰腺病毒的构建、鉴定和扩增目的：1.构建BubR1表达腺病毒；2.构建BubR1干扰腺病毒；3.验证BubR1表达腺病毒的效果；4.验证BubR1干扰质粒的效果。方法：1.从293细胞中提取总RNA逆转录为cDNA，使用BubR1克隆引物扩增BubR1CDS区对应的片段全长，插入至腺病毒穿梭质粒载体pAdTrack-Tox中；2.设计针对BubR1mRNA的四段siRNA片段，插入至腺病毒穿梭质粒pSES-HUS中；3.测序验证BubR1表达片段和干扰片段序列的正确性；4.验证BubR1干扰质粒的效果；4.腺病毒穿梭质粒与骨架质粒pAdEasy-1在BJ5183菌中发生同源重组，重组正确的腺病毒质粒转染至293细胞中进行病毒的包装和扩增；5.使用PCR和Western Blot方法验证BubR1表达腺病毒的效果。结果：1.从293cDNA中扩增出BubR1CDS区插入至pAdTrack-TOX中，测序验证正确后命名为pAdTrack-Tox-BubR1；2.pAdTrack-Tox-BubR1与骨架质粒pAdEasy-1同源重组后，利用Pac I限制性内切酶，可以得到30kb的大片段和4.5kb的小片段；3.BubR1表达腺病毒在293中包装和扩增，可见绿色荧光蛋白表达，将腺病毒命名为Ad-BubR1;4.Ad-BubR1感染293后，PCR和Western Blot检测有BubR1表达增加；5.将四段干扰片段插入至pSES-HUS质粒中，测序正确后，转染ECA-109细胞中，PCR和Western Blot验证BubR1的干扰效果，将有明显干扰效果的pSES-HUS-siBubR1-4和乱序质粒（Scramble）进行下一步实验；6. pSES-HUS-siBubR1-4与骨架质粒pAdEasy-1同源重组后，利用Pac I限制性内切酶验证，可以得到30kb的大片段和3kb的小片段；7. BubR1干扰腺病毒在293中包装和扩增，可见红色荧光蛋白的表达，命名为Ad-siBubR1-4。结论：成功构建了BubR1表达腺病毒和干扰腺病毒，并通过PCR和Western Blot的方法分别在293细胞和ECA-109细胞验证了其表达BubR1和干扰BubR1的效果。第三部分食管鳞癌中BubR1的表达水平影响紫杉醇药物敏感性目的：1.检测BubR1干扰前后三株食管鳞癌细胞对紫杉醇的敏感性变化；2.观察BubR1干扰后紫杉醇诱导ECA-109细胞死亡的变化；3.观察过表达BubR1后细胞对抗微管药物诱发有丝分裂期阻滞反应；4.BubR1干扰后ECA-109细胞体外短期存活和体内长期存活。方法：1.干扰BubR1和未干扰BubR1的三株食管鳞癌细胞在浓度梯度紫杉醇作用下，MTT检测相应的存活率，并计算药物的IC50；2.流式细胞术检测干扰BubR1后的ECA-109细胞在紫杉醇作用下细胞周期分布；3.台盼蓝染色计数ECA-109细胞干扰BubR1后紫杉醇诱导细胞死亡率变化。4.三株食管鳞癌细胞中感染Ad-BubR1后，Nocodazole处理后不同时间点下，通过吉姆萨染色观察计数处于有丝分裂期细胞比例变化；5.ECA-109细胞干扰BubR1后，MTT检测细胞在0-96h的生长曲线变化；6.ECA-109细胞干扰BubR1后，打入免疫缺陷小鼠皮下，观察体内成瘤的大小和体积。结果：1.相对高表达的食管鳞癌细胞KYSE150和KYSE180对紫杉醇的敏感性较低，而干扰了BubR1后，三株食管鳞癌细胞对紫杉醇的敏感性都相应增加，药物的IC50减低；2.ECA-109干扰BubR1后，紫杉醇诱导下细胞周期中sub-G1期比例增加，台盼蓝染色细胞死亡比率增加；3.在三株食管鳞癌中过表达BubR1后会进一步减低Nocodazole诱发的有丝分裂期阻滞，表现为有丝分裂指数下降，处于有丝分裂期细胞减少。4.ECA-109细胞中干扰BubR1后，体外检测细胞增殖无明显差异，而体内成瘤实验的肿瘤重量和体积明显减低。结论：1.相对高表达BubR1的细胞株对紫杉醇敏感性更差，对Nocodazole诱导的有丝分裂期细胞比例低；2.干扰BubR1后可提高食管鳞癌细胞对紫杉醇的敏感性，表现为紫杉醇诱导的细胞死亡比例增加。3.食管鳞癌细胞中干扰BubR1可抑制体内成瘤。第四部分食管鳞癌中C-myc对BubR1的调控作用间接影响紫杉醇敏感性目的:1.检测食管鳞癌组织中BubR1与C-Myc的表达趋势；2.验证C-Myc在三株食管鳞癌中的表达及对BubR1调控的影响；3.C-Myc对食管鳞癌细胞紫杉醇敏感性的影响。方法：1.免疫组化的方法检测BubR1和C-Myc在食管鳞癌组织中的表达；2.PCR和Western Blot方法检测C-Myc在三株食管鳞癌中的表达水平；3.检测pSEAP2BubR1-P2000在三株食管鳞癌细胞中的碱性磷酸酶活性，293细胞中过表达C-Myc后对pSEAP2BubR1-P2000的激活；4.PCR和Western Blot方法检测过表达C-Myc和C-Myc抑制剂10058-F4对BubR1mRNA和蛋白水平表达的影响。5.MTT检测C-Myc抑制后对紫杉醇作用下食管鳞癌细胞存活率变化；6.DAPI染色检测C-Myc抑制剂联用低浓度紫杉醇后细胞有丝分裂期指数。结果：1.食管鳞癌组织中BubR1和C-Myc表达有正相关性；2.C-Myc在食管鳞癌细胞中的表达趋势与BubR1表达相似；3.C-Myc能激活BubR1启动子报告质粒活性并上调BubR1mRNA和蛋白表达。4.干扰C-Myc后能增强紫杉醇对食管鳞癌细胞的生长抑制，加强紫杉醇诱导的有丝分裂期阻滞。结论：1.食管鳞癌中C-Myc的表达上调可能是BubR1表达上调的上游诱导因子；2.C-Myc在食管鳞癌中的表达可间接影响紫杉醇的药物敏感性。更多还原

【Abstract】 Mitosis is a very important phase in cell cycle progression. Spindleassembly checkpoint (SAC) is the system which monitors chromosomalproper arrangement and separation in mitosis and further guaranteegenomic integrity in posterity cells. The intact SAC function is prerequisitecondition for diploid cells’ division and growth. Abnormal SAC isfrequently observed in human tumors. Weaken cell cycle checkpoint in theearly stage of tumorigenesis is the crucial step to promote tumor growth.SAC is a signal pathway composed by a set of coordinated proteinsand BubR1is the most important sensor and executor of this system.Disordered BubR1expression has been found in several kinds of clinicaltumor reports, including overexpression of BubR1in breast, bladder,gastric and ovarian cancer, and down-regulation in colorectal cancer.Abnormal BubR1expression represents impaired SAC function andanti-mitosis drugs always depend on normal SAC for their drug-inducedcytotoxic effects. This study is aimed to investigate whether BubR1isup-regulated in esophageal squamous cancer and its’ relationship withanti-microtubule drug paclitaxel effect, and further explore the underlying mechanism of BubR1regulation. Real-time quantitative PCR assay wasemployed to compare BubR1mRNA expression in fifty collected clinicalesophageal squamous cancer tissues and adjacent non-cancerous tissues.Seventy two percentages of those samples (36of50) were found BubR1up-regulation. BubR1expression was high in three esophageal squamouscancer cell lines and relatively high BubR1expression cells showed lowresponse to anti-microtubule drug paclitaxel. To determine the role ofBubR1in esophageal squamous cancer, BubR1expression adenovirus andBubR1interference adenovirus were constructed using adenovirus basedtechnique. After BubR1knockdown in esophageal cancer cells by usingBubR1interference adenovirus, increased cells sensitivity to paclitaxel wasfound, represented by decreased drug IC50and augmentedpaclitaxel-induced cell death. Although down-regulated expression ofBubR1in ECA-109cells did not impact cell growth curve in vitro for shortterm, it largely limited tumor growth of ECA-109cells in nude mice.Conversely, overexpression of BubR1in three esophageal cancer cell linesfurther reduce mitotic index under anti-microtubule drug treatment. HighBubR1and oncogene C-Myc expression were found simultaneously high inesophageal squamous cancer tissues. To investigate whether C-Mycpotentially regulates BubR1, BUB1b gene promoter reporter vector wasconstructed. The result showed that C-Myc expression could activateBUB1b gene promoter and promote BubR1expression. Otherwise, C-Myc inhibition by10058-F4can suppress BubR1expression and recover cells’response to paclitaxel-induced cell growth inhibition, and further boostmitotic arrest. The study of BubR1expression in esophageal squamouscancer, its’ impact on drug effects and related regulation mechanism canhelp us understand why abnormal BubR1exists in tumors, and can providetheoretical foundation for effective esophageal squamous cancer therapy.PART ITHE EXPRESSION OF BUBR1IN ESOPHAGEAL SQUAMOUSCANCER AND CELL LINES AND ANALYSIS OF CLINICALMEANINGSObjective:1. Detect BubR1expression in clinical esophageal squamous cancer tissuesand adjacent no-tumor tissues;2.Analysis the relationship between BubR1expression and patients’clinical characteristics；3. Detect BubR1expression in three esophageal squamous cancer celllines.Methods:1. BubR1relative mRNA expression was detected in fifty pairedesophageal squamous cancer tissues and adjacent no-tumor tissues byreal-time quantitative PCR; 2. Statistically analysis the correlation of BubR1expression and clinicaltraits.3. Real-time PCR and Western Blot assays were used to detect BubR1expression in esophageal squamous cancer cell lines.Results:1. Thirty six of fifty esophageal squamous cancer samples showed BubR1up-regulation, twenty three of which BubR1expression exceed two foldcompared with adjacent no-tumor tissues.2. There is no significantly correlation between BubR1expression andclinical pathological traits.3. High BubR1expression can be observed in esophageal squamous cancercells, in which BubR1expression in ECA-109was lower than that inKYSE150and KYSE180cells.Conclusion:1. Abnormal BubR1expression was confirmed in esophageal squamouscancer tissues but it’s up-regulation was not related with patients’ clinicaltraits.2. BubR1expression level in esophageal squamous cell lines wasconsistent with the result of clinical study. PART ⅡTHE CONSTRUCTION, VERIFICATION AND AMPLIFICATIONOF BUBR1EXPRESSION AND INTERFERENCE ADENOVIRUSObjective:1. Construct BubR1expression adenovirus;2. Construct BubR1interference adenovirus;3. Verify the effect of BubR1expression adenovirus;4. Verify the effect of BubR1interference adenovirus.Methods:1. Total RNA was extracted from293cells and reversed transcript intocDNA and BubR1CDS district fragment was cloned by PCR andinserted into shuttle vector pAdTrack-Tox;2. Four siRNA fragments target to BubR1mRNA were designed andinserted into shuttle vector pSES-HUS.3. DNA sequencing technique was used to verify the accuracy of BubR1expression and interference fragments.4. Homologous recombination happened in BJ5183bacteria betweenadenoviral shuttle vector and backbone vector. Correct recombinedadenoviral vector was transfected into293cells for adenovirus packingand amplification;5. PCR and Western Blot assays were employed to verify the effect ofBubR1expression and interference adenovirus. Results:1. BubR1CDS district fragment was inserted into pAdTrack-Tox from293cDNA and the correct vector was named pAdTrack-Tox-BubR1;2. After homogeneous recombination between pAdTrack-Tox-BubR1andbackbone vector pAdEasy-1, Pac I enzyme cut recombined vector toconfirm right recombination.30kb large fragment and4.5kb smallfragment can be seen from enzyme cut result;3. BubR1expression adenovirus packing and amplification wereprogressed in293cells. GFP expression can be observed. Thisadenovirus was named Ad-BubR1;4. PCR and Western Blot detected elevated BubR1expression afterAd-BubR1infection;5. Four siRNA fragments were inserted into pSES-HUS vector. Aftercorrect sequencing, four BubR1interference vectors were transfectedinto ECA-109cells and BubR1expression was detected by PCR andWestern Blot. Result showed that pSES-HUS-siBubR1-4had significantffect. pSES-HUS-siBubR1-4vector and scramble vector were used forfurther experiment;6. After homogeneous recombination between pSES-HUS-siBubR1-4andbackbone vector pAdEasy-1, Pac I enzyme cut recombined vector toconfirm right recombination.30kb large fragment and3kb smallfragment can be seen from enzyme cut result; 7. BubR1interference adenovirus packing and amplification wereprogressed in293cells. RFP expression can be observed. Thisadenovirus was named Ad-siBubR1-4.Conclusion: BubR1expression and interference adenovirus weresuccessfully constructed and their effects were verified in293cells andECA-109cells, respectively, by PCR and Western Blot assays. PART IIITHE EFFECTS OF BUBR1EXPRESSION IN ESOPHAGEALSQUAMOUS CANCER ON PACLITAXEL SENSITIVITYObjective:1. Three esophageal cancers’ response to paclitaxel before and after BubR1knockdown;2. The difference of paclitaxel-induced cell death was compared before andafter BubR1knockdown;3. Mitotic index under paclitaxel treatment change was observed afterBubR1knockdown;4. Short-term growth of ECA-109cells with BubR1suppression in vitroand long-term tumorigenesis in nude mice was observed. Methods:1. Three cell lines with or without BubR1suppression were treated bygradient concentration of paclitaxel. MTT assay was employed to detectcell viability and drug IC50was calculated.2. FACS assay was carried out to detect cell cycle distribution of ECA-109cells with BubR1suppression under paclitaxel treatment;3. Trypan blue staining was used to count the number of dead cellpercentage of ECA-109cells with BubR1expression under paclitaxeltreatment;4. Mitotic cell percentages of three cell lines with BubR1overexrepssionby Ad-BubR1infection of different time points were observed afterNocodazole treatment;5. MTT assay was used to detect cell growth curve after BubR1knockdown in ECA-109;6. ECA-109cells with BubR1suppression were injected into nude mice fortumorigenesis observation. Tumor’s weight and volume was detected.Results:1. Esophageal squamous cancer cell lines KYSE150and KYSE180withrelatively high BubR1expression showed lower sensitivity towardpaclitaxel treatment. After BubR1knockdown, paclitaxel drug IC50isdecreased in all three cell lines;2. After BubR1was down-regulated in ECA-109, sub-G1phase of cell cycle increased and cell death correspondingly increased underpaclitaxel treatment;3. Overexpression of BubR1in three cell lines can further suppress mitoticcell percentage induced by Nocodazole, which represented as decreasedmitotic index;4. ECA-109cell growth in vitro was not affected by BubR1knockdown,but tumorigenesis in vivo was largely inhibited. Tumor weight andvolume was significantly smaller than control group.Conclusion:1. Relatively high BubR1expression cells showed low response topaclitaxel and mitotic index was also low in those cells;2. BubR1interference can promote esophageal squamous cancer cellsresponse to paclitaxel, represented as cell death induced by paclitaxelelevated;3. BubR1interference can inhibit tumor formation of esophageal squamouscancer cells in vivo. Part ⅣTHE REGULATION OF C-MYC ON BUBR1IN ESOPHAGEALSQUAMOUS CANCER INDIRECTLY AFFECT PACLITAXELSENSITIVITYObjective:1. Confirm the correlation of BubR1and C-Myc expression in esophagealcancer tissues；2. Verify C-Myc expression in three esophageal squamous cancer cell linesand its influence on BubR1expression;3. Determine the effect of C-Myc on paclitaxel sensitivity.Methods:1. Immunohistochemistry was employed to detected BubR1and C-Mycprotein expression in esophageal squamous cancer tissues;2. PCR and Western Blot assays were carried out to detect C-Mycexpression in three esophageal squamous cancer cell lines;3. The activity of alkaline phosphatase was detected in three esophagealsquamous cancer cell lines after pSEAP2BubR1-P2000transfection.pSEAP2-BubR1-P2000activation was confirmed in293cells infectedby Ad-C-Myc; 4. PCR and Western Blot assays were used to detect BubR1mRNA androtein expression under the condition of C-Myc overexpression andC-Myc suppression by10058-F4, respectively;5. MTT assay was used to detect cell viability under paclitaxel treatmentafter C-Myc suppression;6. DAPI staining was carried out to demonstrate cell mitotic index changesunder combination of C-Myc inhibitor and low concentration ofpaclitaxel.Results:1. BubR1and C-Myc expression showed close correlation in esophagealsquamous cancer tissues.2. C-Myc expression in three cell lines showed similar results with BubR1expression;3. C-Myc can activate BubR1promoter vector and up-regulate BubR1expression;4. C-Myc suppression can promote paclitaxel-induced cell growthinhibition and help increase mitotic index.Conclusion:1. C-Myc could be upstream regulator of BubR1in esophageal squamouscancer;2. C-Myc expression in esophageal squamous cancer indirectly affects cellsresponse to paclitaxel.更多还原