【作者】 邹瀛波；

【导师】 蒋耀光；

【作者基本信息】 第三军医大学 ， 外科学， 2014， 博士

【摘要】 背景食管癌是常见的恶性肿瘤之一，近些年来发病率有所上升，与其他恶性肿瘤相比其预后较差，纠其主要原因就是门部分食管癌在早期就己经发生远处转移和淋巴转移。近年来，基因治疗成为肿瘤治疗方面的一大热点，有可能成为未来治疗肿瘤的一个有效手段，对于食管癌来说，基因层次的研究较其他肿瘤来说还比较少，因此寻找一个在食管癌发生、移转中能够起到关键作用的分子就显得比较迫切[1]。WIG-1（wild-typep53-induced gene1，野生型p53诱导基因1）是一种由p53基因诱导表达的锌指蛋白，与双链RNA结合后，能够发挥抑制细胞生长的作用，因此有可能是食管癌的一个潜在的基因治疗靶点。本研究通过分析WIG-1在不同食管癌组织的表水平达差异，得到其与食管癌患者的预后可能存在的关系；并通过构建WIG-1不同表达水平的人源性食管鳞癌细胞系EC109，来进一步探讨WIG-1对食管鳞癌细胞主要的生物学行为方面的影响以及可能的作用机制，并进一步筛选出WIG-1可能的作用分子，可望为食管癌的基因治疗方面提供一定的临床应用价值。目的制备WIG-1单克隆抗体，为进一步的研究工作打下坚实的基础。了解WIG-1不同的表达水平与食管癌患者临床表现之间的关系；构建WIG-1高表达与低表达的人源性食管鳞癌细胞系EC109，从而可以进一步研究WIG-1基因对于EC109细胞的生长、凋亡以及侵袭等生物学行为的影响；探讨WIG-1对EC109细胞的化疗药物敏感性、化疗药物的耐药性以及对细胞损伤、修复等能力的影响，并提出其可能的分子机制；通过WIG-1对基因表达谱的影响来筛选出其可能的作用分子。方法应用杂交瘤技术制WIG-1单克隆抗体；应用RT-PCR和免疫组化技术检测食管癌组织及其对应近癌、远癌组织中WIG-1基因的表达；应用脂质体转染法构建了WIG-1基因高表达与低表达的人源性食管鳞癌细胞系EC109，并用RT-PCR和Western blot技术进行鉴定；应用MTT实验绘制WIG-1基因不同表达水平的EC109细胞的生长曲线；应用MTT实验、平板克隆实验、流式细胞仪技术以及DNA断裂实验检测WIG-1基因不同表达水平的EC109细胞的细胞周期和细胞凋亡的变化；应用Transwell侵袭小室实验和细胞损伤刮擦试验来检测WIG-1基因不同表达水平对人源性食管鳞癌细胞系EC109侵袭活性的影响；应用MTT实验进行体外药物敏感性分析，计算细胞对药物的IC50值；应用Western Blot检测WIG-1基因的不同表达水平对部分耐药分子表达水平的影响；应用单细胞凝胶电泳技术检测WIG-1基因不同表达水平对于由紫外线照射造成的不同的DNA损伤情况及修复能力；10、应用基因芯片技术检测WIG-1基因对EC109细胞基因表达谱的影响；11、就用酵母双杂交技术筛选出WIG-1基因可能的作用分子。结果1、成功制备了WIG-1单克隆抗体，并通过Western Blot及免疫组化实验证实；2、WIG-1基因在食管癌肿瘤的不同部位的标本中存在差异性表达，食管癌组织中WIG-1基因的表达水平明显低于近癌及远癌组织，而近癌组织则显著低于远癌组织（p <0.05）；成功构建了WIG-1基因高表达及低表达的人源性食管鳞癌细胞株EC109，并通过半定量RT-PCR及Western blot实验证实；MTT法、平板克隆形成实验、DNA断裂实验、流式细胞仪技术、Transwell侵袭小室实验以及细胞损伤刮擦实验等的结果表示，WIG-1基因的高表达可以抑制EC109细胞的增殖，促进其凋亡，并能够显著降低人源性食管鳞癌细胞系EC109的侵袭、移动能力；5、WIG-1基因的高表达能够显著增强EC109细胞对5-氟尿嘧啶（5-Fu）、顺铂（CDDP）、阿霉素（ADR）等化疗药物的敏感性，对长春新碱（VCR）的敏感性，则无明显影响，WIG-1基因可能通过抑制ERCC1及P-gp的表达进而提高EC109细胞对于化疗药物的敏感性，降低EC109细胞对化疗药物的耐受性；6、WIG-1基因的高表达能够减少紫外线照射对EC109细胞的DNA损伤效应，并显著增强EC109细胞的DNA修复能力；7、基因芯片检测结果表明，上调WIG-1基因的表达水平可以影响419种基因的表达，而上调WIG-1基因的表达水平可能影响301种基因的表达；8、酵母双杂交实验结果筛选出WIG-1基因的可能作用分子为：ILF3（NM₀17620），UTP14A（NM₀06649），Sox3（NM₀05634）。结论制备了WIG-1单克隆抗体，为本实验研究以及进一步的深入研究奠定了一个良好的基础。并构建了WIG-1基因高表达及低表达的人源性食管鳞癌细胞株EC109。WIG-1基因不仅在食管癌组织细胞中有表达，且在正常的组织细胞中亦有表达，其食管癌组织中的表达水平显著低于对应近癌及远癌组织；WIG-1基因的高表达可以抑制EC109细胞的增殖，促进其凋亡，而WIG-1基因的低表达可以促进EC109细胞增殖，抑制其凋亡；WIG-1表达水平增高可以抑制ERCC1和P-gp的表达，进而提高EC109细胞对于化疗药物的敏感性，而WIG-1表达水平下降时，结果恰恰恰相反；WIG-1基因高表达可以增强EC109细胞的紫外线耐受性及DNA修复能力；上调或下调WIG-1基因表达水平可以影响多种基因的表达，希望在进一步的研究中可以探寻到WIG-1基因，作为一个己知抑癌基因的一个调控点，其具体的作用机制，从而为食管癌的诊断与治疗方案提供更多的选择。更多还原

【Abstract】 BackgroundEsophageal cancer is one of the ten most common cancerous growth in today’s world.Compared with others, esophageal cancer is worse in prognostic, because in early stage itmay have distant metastasis and lymphatic spread. In recent years, genetic therapy hasbecome a hot spot in tumor treatment, which is also likely to be an effective measure in thefuture. In the esophageal cancer case, researches on the genetic level are comparatively less.Hence, it is imperative to find the molecules which play a key role in its occurring andmetastasis. WIG-1（wild-type p53-induced gene1） is a kind of zinc finger protein expressedby the induction of p53gene. After combining with double-strand RNA, it can restrain thecells from growing. Therefore, it may be a potential genetic therapy target for the treatmentof esophageal cancer.In this research, by analyzing the expression level difference of WIG-1in differentesophageal cancer tissues, its possible relationship with esophageal cancer patients’prognostic is found; by constructing human esophageal squamous carcinoma cell lineEC109of different WIG-1expression levels, possible functional mechanism of theinfluence of WIG-1on the biological behavior of esophageal cancer cell such as growthcycle, apoptosis, invasion and DNA damage repair, etc. is further studied; then possiblefunctional molecules are selected, hopefully clinical application significance can beprovided in the genetic therapy of esophageal cancer.ObjectivesMaking monoclonal antibody of WIG-1to lay a solid foundation for further researchwork. Understanding the relationship between different expression levels of WIG-1andesophageal cancer patients’ clinical pathological features and prognostic. Constructinghuman esophageal squamous carcinoma cell line EC109of high and low WIG-1expressionlevels to further study the influence of WIG-1gene on the biological behavior of EC109cell such as its growth, apoptosis, and invasion, etc. Discussing the influence of the expressionlevel of WIG-1gene on the chemotherapy drug sensitivity and tolerance of EC109cell andits influence on DNA damage repair as well as its possible molecule mechanism. Selectingpossible functional molecules based on the influence of WIG-1on gene expression profile.Methods1. Make monoclonal antibody of WIG-1by using hybridoma technology;2. Check the expression of WIG-1gene in esophageal cancer tissue and itscorresponding near and far cancer tissues by using RT-PCR and immunohistochemistrytechnology;3. Construct human esophageal squamous carcinoma cell line EC109of high and lowWIG-1expression levels by using lipofection, and appraise by using RT-PCR and WesternBlot technology;4. Draw the growth curve of EC109cell when WIG-1gene is in different expressionlevel by using MTT experiment;5. Check the change of EC109cell in cell cycle and apoptosis when WIG-1gene is indifferent expression level by using MTT experiment, colony formation assay, flowcytometry technology, and DNA fragmentation experiment;6. Examine the influence of different expression level of WIG-1gene on the invasionactivity of esophageal squamous carcinoma cell by using Transwell experiment and celldamage and scratch experiment;7. Calculate the IC50of cells on drugs, and analyze vitro drug sensitivity by usingMTT experiment;8. Test the influence of different WIG-1gene expression levels on the expression levelsof some drug resistant molecules by using Western Blot;9. Check the different DNA damage condition and repair ability caused by ultravioletradiation when WIG-1gene is in different expression level by using single cell gelelectrophoresis technology;10. Examine the influence of WIG-1gene on the gene expression profile of EC109byusing gene chip technology;11. Select possible functional molecules of WIG-1gene by using yeast twohybridization technology. Results1. Monoclonal antibody of WIG-1has been successfully made and affirmed byWestern Blot and immunohistochemistry experiments;2. The expression of WIG-1gene in esophageal cancer tumor tissue and itscorresponding near and far cancer tissues is different: the expression level of WIG-1gene inesophageal cancer tissue is obviously lower than it is in near and far cancer tissues, whilethe expression level of WIG-1gene in near cancer tissue is greatly lower than it is in farcancer tissue （p <0.05）;3. Human esophageal squamous carcinoma cell line EC109of high and low WIG-1expression levels has been successfully constructed and appraised by semi-quantitativeRT-PCR and Western Blot experiments;4. Judging from the results of MTT experiment, colony formation assay, DNAfragmentation experiment, flow cytometry technology, Transwell experiment and celldamage and scratch experiment, etc., the high expression of WIG-1gene can restrain humanesophageal squamous carcinoma cell line EC109from growing, accelerate its apoptosis,and can notably reduce its invasion and spread ability;5. The high expression of WIG-1gene can remarkably enhance the sensitivity ofEC109to chemotherapy drugs, such as5-Fu, CDDP, ADR. As for VCR, the sensitivity isnot obviously influenced. WIG-1gene may enhance the sensitivity of EC109tochemotherapy drugs and reduce its resistance to them by restraining the expression ofERCC1and P-gp.6. The high expression of WIG-1gene can reduce the damage effect of ultravioletradiation on EC109, and markedly improve its DNA repair ability;7. Gene chip examination results indicate that raising the expression level of WIG-1can influence the expression of419kinds of genes and it perhaps may also influence theexpression of301kinds of genes;8. Through the results of yeast two hybridization experiments, the possible functionalmolecules of WIG-1gene are selected as being ILF3（NM₀17620），UTP14A（NM₀06649），Sox3（NM₀05634）. ConclusionsMonoclonal antibody of WIG-1is made, hence a good foundation is laid for thisexperiment study and further deeper researches. Human esophageal squamous carcinomacell line EC109of high and low WIG-1expression levels is constructed. WIG-1gene is notonly expressed in esophageal cancer tissue cells, but also in normal tissue cells. Theexpression level of WIG-1gene in esophageal cancer tissue is obviously lower than it is innear and far cancer tissues. The high expression of WIG-1can restrain the growth of EC109cell and accelerate its apoptosis while the low expression of WIG-1can accelerate thegrowth of EC109cell and restrain its apoptosis. Raising the expression level of WIG-1canrestrain the expression of ERCC1and P-gp, then enhance the sensitivity of EC109cell tochemotherapy drugs, while the expression level is lowered, the result is exactly the opposite.The high expression of WIG-1gene can improve the ultraviolet radiation tolerance andDNA repair ability of EC109cell. Now it is known that raising or lowering the expressionlevel of WIG-1gene can influence the expression of many genes, hopefully WIG-1genecan be detected in further study. Being a control point of known anti-oncogene, its specificfunctional mechanism may offer more options in the diagnosis and treatment of esophagealcancer.更多还原