【作者】 刘泰荣；

【导师】 杨罗艳；

【作者基本信息】 中南大学 ， 外科学， 2007， 博士

【摘要】 肾癌是泌尿系比较常见的恶性肿瘤，仅次于膀胱癌位于第二位，约占成人肿瘤的2％～3％，并且有研究表明其发病率和死亡率均有进一步上升的趋势。除了依赖于早期手术切除外，肾癌对放疗、化疗及内分泌等治疗均不敏感，治疗效果较差，手术后的放疗、化疗并不能减少转移率，免疫治疗也只对15～20％的患者有效。然而有将近50％的肾癌患者首次就诊时已属晚期，失去了最佳的手术时机，并且有约40％的患者术后出现复发或转移，未接受治疗的患者3年存活率低于5％。因此寻找肾癌发生、发展的原因并进行有针对性的治疗一直是学者们研究的目标。虽然目前肾癌的发病机理并不十分明确，但大量的研究结果表明，细胞凋亡机制的缺陷在其发生、发展中具有重要的作用，肿瘤细胞的凋亡受到抑制是肾癌发生的重要机制。化疗是恶性肿瘤综合治疗的一种重要方法，然而肾癌是典型的耐药标本，对大多数化疗药物耐药，因而肾癌发生耐药的机制成为亟需解决的问题。同样有研究发现，肾癌发生耐药的原因除了P糖蛋白、谷胱甘肽转移酶等蛋白的异常表达外，凋亡作用受到抑制也可能是肾癌对化疗药物不敏感甚至失败的重要原因。凋亡抑制蛋白（IAP）是一类在结构上具有同源性的细胞内源性凋亡抑制蛋白家族，XIAP是近来发现的IAP家族中的一员，具有强列的凋亡抑制作用，它能通过有效地抑制天冬氨酸特异性半胱氨酸蛋白酶（caspase）等凋亡途径抑制细胞凋亡。研究表明，XIAP在多种肿瘤如卵巢癌、白血病、前列腺癌等疾病的发生、发展中具有重要的作用，并且与这些肿瘤的化疗耐药密切相关。而通过反义核酸技术设计出与目的基因序列互补的寡核苷酸序列能够封闭该基因的表达，因而为肿瘤耐药的逆转和治疗提供了新的方法和途径。目前关于XIAP在肾癌组织中的表达特点及其与肾癌发生、发展的关系研究极少，而XIAP对肾癌化疗敏感性的影响则未见研究报道。因此，研究XIAP在肾癌组织中的表达情况及其对肾癌化疗敏感性的影响、研究XIAP反义寡核苷酸对肾癌化疗敏感性的作用，对于阐述肾癌的发病及化疗耐药机制，探讨提高肾癌化疗敏感性的方法，进而促进肾癌的治疗疗效，都具有重要的意义。第一部分XIAP和Caspase-3在肾癌中的表达及意义目的：研究XIAP、caspase-3在肾癌组织中的表达情况，探讨XIAP、caspase-3与肾癌临床病理特征之间的关系。方法：采用免疫组织化学方法，检测XIAP、caspase-3蛋白在50例肾癌组织及15例远离肾癌的正常肾组织中的表达情况。结果：（1）免疫组化方法检测显示XIAP在肾癌标本中的阳性表达率明显高于癌旁正常肾组织中的阳性表达率，而caspase-3在肾癌组织中阳性表达明显低于癌旁正常肾组织中的阳性表达率。经比较，XIAP、caspase-3在肾癌中的表达均与癌旁正常肾组织中的表达有非常显著性差异（p＜0.01）。（2）在不同年龄、性别及不同组织学类型的肾癌组织中，XIAP和caspase-3的阳性表达均无显著性差异（p＞0.05）。在不同病理分级、临床分期及有无淋巴结转移的肾癌组织中，XIAP的阳性表达均有显著性差异。组织分化程度越低，XIAP的阳性表达率越高（p＜0.05）；临床分期越高，XIAP的阳性表达率也越高（p＜0.05）；发生淋巴结转移的肾癌组织，其XIAP的表达率显著高于无淋巴结转移的肾癌组织（p＜0.01）。caspase-3的表达程度与肾癌的病理分级有关，在不同的病理分级之间其表达有显著性差异（p＜0.05），组织分化程度越高，caspase-3的阳性表达越高；而在不同临床分期及有无淋巴结转移之间，caspase-3的表达无显著差异（p＞0.05）。结论：（1）XIAP在肾癌组织中的表达明显上调，而caspase-3的表达则相应地下调。（2）XIAP的阳性表达与肾癌的组织学病理分级、临床分期及有无淋巴结转移相关，而与患者的年龄、性别及肾癌的组织学类型无关。caspase-3的阳性表达与肾癌的病理分级相关，与患者的年龄、性别及肾癌的组织学类型、临床分期及有无淋巴结转移无关。（3）XIAP在肾癌组织中的阳性表达与caspase-3的阳性表达呈负相关，两者表达的改变可能在肾癌的发生、发展中起重要作用。第二部分XIAP对肾癌786-0细胞化疗敏感性的影响目的：研究XIAP在肾癌耐药发生中的作用及XIAP反义寡核苷酸对肾癌化疗的增敏作用，探讨肾癌耐药的发生机制并为逆转肾癌耐药探索新的方法和途径。方法：将不同浓度的化疗药物顺铂作用于肾癌786-0细胞，应用RT-PCR、Western blot、流式细胞技术等方法观察XIAP mRNA和蛋白表达的变化及细胞凋亡率的改变；然后将肾癌786-0细胞转染XIAP反义寡核苷酸，应用RT-PCR、Western blot、流式细胞及电镜技术、MTT等方法，检测XIAP mRNA和蛋白表达的变化及caspase-3活性的改变，观察化疗药物顺铂对786-0细胞生长抑制和诱导凋亡作用的改变。结果：（1）10umol／L及以下浓度的化疗药物顺铂不能有效地通过下调XIAP mRNA和蛋白表达而诱导肾癌786-0细胞的凋亡（p＞0.05），高于10umol／L浓度的化疗药物顺铂能下调XIAP mRNA和蛋白表达，但细胞凋亡率并不增加（p＞0.05）。（2）将XIAP反义寡核苷酸成功转入肾癌786-0细胞后，在10umol／L顺铂的作用下，XIAP mRNA和蛋白的表达明显下调，细胞生长受到抑制，并且caspase-3活性增加，细胞凋亡率明显增加，对顺铂的半效抑制浓度IC₅₀也显著降低。与单用顺铂或XIAP反义寡核苷酸比较均有显著性差异（p＜0.05或0.01）。结论：（1）XIAP参与了肾癌耐药的发生，XIAP基因可作为逆转肾癌耐药治疗的有效靶点。（2）XIAP反义寡核苷酸能够下调肾癌细胞XIAP mRNA和蛋白的表达，与化疗药物顺铂联合应用具有显著的增敏作用，它能通过自身并促进顺铂抑制肾癌细胞的增殖并诱导其凋亡，降低肾癌的耐药性，从而逆转肾癌耐药。更多还原

【Abstract】 Renal cell carcinoma (RCC) is one of the most common seenmalignant tumors which is secondary to bladder cancer in the urinarysystem carcinoma. Study showed that the morbidity and mortality ofRCC are increasing gradually. Except the reliable treatment of surgicalresection, RCC is insensitive to radiotherapy, chemiotherapy and internalincretion therapy, and therapeutic effect is bad.Immunotherapy hastherapeutic effect only to 15～20% patients.About 50% members of thosepatients are in their late stage when they were firstly diagnosed of thiscancer and have lost their best opportunity for treatment, about 40% willbe recurrenced and metabasised after radical renal resection, the survivalrate is lower than 5% within 3 years who had not received any treatment.So to find the tumorigenesis and seek perfect therapies of RCC are thegoals of researcher.Although pathogenesis of RCC is not very clear, but a great deal ofstudy has shown that inhibition of tumor cell apoptosis is an importantmechanism. Chemiotherapy is one of the important comprehensivetherapeutic methods of malignant tumors, but RCC is a typical sampleof drug resistance and resists many chemotherapeutic drugs. So to finddrug resistance mechanism of RCC become the urgent thing. Study alsoindicated that except the abnormal expression of P-glycoprotein (P-gp)and Glutathione (GSH), the tolerance of tumor cells to apoptosis is animportant cause of RCC be insensitive even irresponsive tochemotherapeutic drugs.x-chromosome-linked inhibitor of apoptosis(XIAP) is a newmember of inhibitors of apoptosis protein (IAP)family.It has strong effectof inhibiting apoptosis by exerting inhibitory effect on the activities ofcaspase, and so on. XIAP play an important role in the occurrence andprogress in many kind of tumors such as ovarian cancer, myeloidleukemias, prostate cancer, it is also close related with drug resistance ofthese tumors.Antisense oligonucleotide(ASODN) can well act as a geneblocker to inhibition of expression of specific gene, so this made a newway to reverse cancer drug resistance to chemotherapeutic drugs. At present, the assocition of XIAP expression characteristics with RCCdevelopment was rarely studied, and there is no report on the relationbetween XIAP and drug-sensitive of RCC cells yet. So as to studyexpression characteristics and the effect to drug-sensitive of XIAP inRCC, to study the reverse drug resistance of XIAP ASODN are veryimportant. It is significant to illustrate the mechanism of devepment anddrug resistance, and explore the methods of enhancing therapeuticsensitivity of RCC.PART ONEEpression of XIAP and caspase-3 in RCC tissuesObjective: To investigate the expression of XIAP and caspase-3 inRCC tissues, and to explore their correlations with RCC.Methods: XIAP and caspase-3 expression were detected byimmunohisto-chemistry in 50 cases RCC tissues and 15 cases of normalrenal tissuses.Results:(1)Imunohistochemical analysis showed that positiveexpression of XIAP protein was higher in RCC tissues than in normalrenal tissuses, but positive expression of caspase-3 protein is lower.Positive expression of XIAP and caspase-3 there is significant differencebetween RCC and normal renal tissuses (p＜0.01).(2)The positiveexpression of XIAP and caspase-3 showed no significant differencebetween age and gender and pathological type, but a significantdifference between pathological grades (p＜0.05); XIAP expression therewas significant difference between clinical stage(p＜0.05) and with orwithout lymph node metastasis(p＜0.01), but caspase-3 expression therewas no significant difference (p＞0.05).Conclusions:(1)XIAP protein expression up-regulated but caspase-3protein expression down-regulated in RCC tissues.(2) Positive expressionof XIAP in RCC tissues there were correlation with pathological grades,clinical stage and with or without lymph node metastasis, but positive expression of caspase-3 there was correlation with pathological gradesonly.(3)There were a negative correlation between positive expression ofXIAP and caspase-3, their change may play a important role in RCCdevelopment.PART TWOEffect of XIAP on chemotherapeutic sensitivityof RCC cell line786-0Objective: To investigate the effect of XIAP on drug resistance ofRCC and XIAP ASODN on enhancing the sensitivity of RCC tochemotherapy.To explore the drug resistance mechanism and newmethods of reversing drug resistance for RCC.Methods: Through RT-PCR, Western blot, FCM to detect the effectof different concentration chemotherapeutic drug DDP on XIAP mRNA,protein and cell apoptosis rate of RCC cell line 786-0.Then RCC cell line786-0 was transfected with different concentration of XIAP ASODN andtreated with DDP,RT-PCR, Western blot, FCM,electron microscope,MTT and so on were utilized to assay the expression of XIAP mRNA,XIAP protein and the change of cell apoptosis rate,cell proliferationactivity and caspase-3 activity in 786-0 cells.Results:(1)10umol/L or lower than 10umol/L DDP could not buthigher than 10umol/L DDP could down-regulated the expression of XIAPmRNA and protein, different concentration DDP could not induce theapoptosis of 786-0 cells significantly (p＞0.05).(2) RCC cell line 786-0was transfected by XIAP ASODN and then treated with 10umol/L DDP,the expression of XIAP mRNA and protein down-regulated significantly;the cell growth inhibition,the caspase-3 activity and cell apoptosis rateincreased significantly; the half-maximum inhibitory concentration ofDDP was decreased significantly.There were significant differences withcells treated with DDP or XIAP ASODN alonely (p＜0.05 or 0.01).Conclusions:(1)XIAP contributes to the occurrence of drug resistance of RCC, XIAP gene can be an appropriate and effective target to reversedrug resistance.(2) XIAP ASODN can down-regulated the expression ofXIAP mRNA and protein of RCC and enhance DDP sensitivity to RCC,it can by itself and promoting DDP to induce apoptosis significantly andreverse drug resistance of RCC.更多还原