【作者】 刘斌；

【导师】 刘贤锡；

【作者基本信息】 山东大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 胃癌在全世界很多国家的发病率都很高。在日本,胃癌仍旧是男性最常见的肿瘤。我国也是世界上胃癌的高发国家,全世界约35%的胃癌病例发生在中国。目前,胃癌治疗仍以手术为主,术后根据不同的病理检查结果,辅以药物治疗。近年来随着早期胃癌发现率的提高,手术方法的改进和综合治疗的应用,胃癌的治愈率有所提高,但大多数报道的5年生存率仍徘徊于20%～30%。胃癌的治疗疗效,也并不十分令人满意。因此寻找一种新型非手术干预的治疗方法对于胃癌的预防和治疗具有非常重要意义。多胺是腐胺、精脒和精胺的统称,是一类广泛存在于生物体内的阳离子脂肪族化合物,这些阳离子电荷使得多胺可通过静电作用与细胞内的含有多聚阴离子的大分子化合物如DNA、RNA、蛋白质等发生反应,在正常细胞生长和分化过程中发挥着重要的作用。在迅速增殖的正常细胞以及肿瘤细胞中,多胺的含量明显升高。以多胺代谢通路为靶点,降低细胞内多胺水平,在肿瘤治疗研究中具有非常重要的意义。多胺合成途径有两个关键酶,即鸟氨酸脱羧酶(Ornithine decarboxylase,ODC)和S-腺苷甲硫氨酸脱羧酶(AdeMetDC)。它们一直是研究的重点。我们之前的研究表明,与相应的正常组织相比,在结肠癌组织中ODC和AdeMetDC的表达明显升高,而且通过抑制它们的表达可以下调多胺的合成,从而抑制肿瘤生长。精脒／精胺N1乙酰基转移酶(spermidine/spermine N’-acetyltransferase,SSAT),是多胺分解途径中的第一个限速酶。SSAT将乙酰-CoA上的乙酰基团转移到精脒和精胺的N1位置上。SSAT可以防止多胺在细胞内过高积聚,在保持细胞内多胺的平衡中起到重要作用。SSAT的活性可以被胞内的多胺水平高度诱导和调节,从而维持细胞内的多胺平衡。另外,SSAT可以被多种生长抑制剂及各种有毒刺激诱导产生。SSAT的蛋白水平和酶活性在正常和没有被诱导的细胞中是一般测不到的。而在多胺拟似物的诱导下,活性可以增加1000倍以上。Kristin Kee和Slavoljub Vujcic等人研究发现在SSAT水平上促进多胺降解能更有效的降低细胞内多胺水平。端粒酶是目前所发现的恶性肿瘤最广谱的分子标记,可以在90%的恶性肿瘤中被激活,而其在正常体细胞中一般为阴性表达。人端粒酶的主要成分有人端粒酶RNA(hTR)、人端粒酶相关蛋白(hTP)、人端粒酶逆转录酶(hTERT)。人类端粒酶是一种维持端粒长度的一种逆转录酶,hTERT的表达几乎只限制在肿瘤细胞中,并与端粒酶活性密切相关。而端粒酶的其他组分在正常肿瘤细胞中均表达。因此,我们选择胃癌为目标,以SSAT为靶点,利用hTERT启动子只在肿瘤细胞中表达,而在正常细胞中不表达的特点,构建了一种由hTERT启动子介导的、表达SSAT的重组腺病毒载体。我们利用该表达载体系统在体外研究了SSAT表达增高对胃癌细胞生长的抑制作用,并建立了裸鼠皮下胃癌移植瘤模型,在体内对SSAT表达增高抑制胃癌的作用进行了研究。第一部分腺病毒介导的SSAT表达抑制胃癌细胞增殖的体外研究实验目的：构建hTERT启动子介导的人SSAT表达的腺病毒载体,研究重组腺病毒Ad-SSAT对胃癌细胞多胺合成的作用,观察对细胞增殖的抑制作用。实验方法：1.采用RT-PCR方法克隆人SSAT mRNA翻译区的基因片断；将其插入pMD-18T载体中,经Nco I、XbaI酶切回收,插入到质粒PGL3-hTERT中,然后将重组的pGL3-hTERT-SSAT用Sal I和Hind III双酶切回收,插入穿梭质粒pAdTrack形成重组质粒pAdTrack-hTERT-SSAT。重组质粒经PmeI酶切线性化后,转入Adeasy-1细菌与pAdeasy-1质粒发生同源重组。将重组质粒pAdeasy-hTERT-SSAT,经PacI酶切后,转染293细胞包装成腺病毒颗粒。2.利用荧光显微镜和PCR的方法对重组腺病毒Ad-hTERT-SSAT进行鉴定。3.根据参考文献报道设计SSAT基因的siRNA片段,表达载体pGPU6／GFP／Neo-shSSAT的合成与构建由上海吉玛公司完成。4.采用MTS法测定不同MOI的腺病毒对胃癌MGC803和SGC7901细胞的基因转染效率,寻找不同细胞的最适感染滴度。5.采用Western印迹技术检测重组腺病毒以及RNA干扰载体pGPU6／GFP／Neo-shSSAT感染或转染胃癌细胞后SSAT蛋白表达情况,并利用高效液相(HPLC)法检测其对胃癌细胞中多胺含量的影响。6.采用MTS法检测重组腺病毒Ad-SSAT和RNA干扰载体pGPU6／GFP／Neo-shSSAT对胃癌MGC803和SGC7901细胞增殖的影响。并进一步运用克隆形成实验观察各组细胞克隆形成率。7.采用流式细胞术检测Ad-SSAT对MGC803和SGC7901细胞周期分布的影响。实验结果：1.成功扩增出SSATcDNA片断且测序正确,然后经TA克隆连接到质粒pGL3-hTERT和pAdTrack中,酶切及测序验证基因插入方向和序列都正确。在转入Adeasv-1细菌后获得多个阳性重组克隆。重组质粒pAdeasy-hTERT-SSAT转染293细胞进行包装扩增,荧光显微镜下可见绿色荧光蛋白在293细胞中表达,并且PCR证实基因组中含有目的基因。2.成功合成沉默SSAT的RNAi载体pGPU6／GFP／Neo-shSSAT。3.腺病毒对胃癌细胞MGC803和SGC7901的最适MOI分别是50和25。分别以该MOI的Ad-SSAT感染MGC803和SGC7901细胞可明显抑制其生长增殖,最大抑制率分别为65%和55%,但不会引起细胞毒性作用。4.Ad-SSAT感染MGC803和SGC7901细胞,可明显增强SSAT基因表达,两种细胞SSAT表达分别为对照组的265%和210%；而RNA干扰沉默SSAT表达可明显抑制两株细胞中SSAT的表达,其抑制率分别为85%和73%。HPLC结果显示,MGC803和SGC7901细胞感染Ad-SSAT后细胞内精胺和精脒含量明显降低,而RNAi沉默SSAT表达后细胞内精胺和精脒含量明显升高。5.细胞生长曲线显示Ad-SSAT感染MGC803和SGC7901细胞,可以明显抑制细胞的增殖,而RNAi沉默SSAT表达则使细胞增殖加快。细胞克隆形成实验进一步验证了这个观点。6.流式细胞DNA含量分析显示,Ad-SSAT可引起MGC803和SGC7901细胞周期山东大学博士学位论文S期阻滞,但并未引发凋亡。实验结论：成功构建SSAT腺病毒表达载体以及SSAT RNA干扰表达载体,为进一步研究SSAT作为胃癌的基因治疗和预防研究提供了必要的工具。Ad-SSAT可显著上调胃癌细胞中SSAT的表达,使细胞周期阻滞于S期,抑制细胞增殖,提示其有抑胃癌作用。第二部分腺病毒介导的SSAT表达引起胃癌细胞周期阻滞于S期的分子机理研究实验目的：观察Ad-SSAT对S期主要的细胞周期调节蛋白的调控作用,研究Ad-SSAT引起胃癌细胞周期阻滞于S期的分子机制。实验方法：1. Western blotting检测Ad-SSAT对细胞周期调节蛋白cyclin A和Cdk2蛋白水平的影响作用。2.半定量RT-PCR法检测Ad-SSAT对细胞内cyclin A和Cdk2的mRNA水平的影响作用。3.采用Western blotting和RT-PCR法检测Ad-SSAT对核转录因子E2F-1表达的影响。4.双荧光素酶活性测定检测Ad-SSAT对E2F启动子活性的影响。实验结果：1. Western blot结果显示,与对照组相比,Ad-SSAT感染分别使MGC803和SGC7901细胞中cyclin A蛋白表达下调80%和60%左右,但Cdk2蛋白的表达无明显变化。2.RT-PCR结果显示,Ad-SSAT处理组MGC803细胞cyclinA mRNA表达水平降低了80%, SGC7901细胞cyclinA mRNA表达水平降低了70%。而三组中的Cdk2mRNA没有明显变化。3. Western blotting和RT-PCR结果显示Ad-SSAT抑制核转录因子E2F-1的表达,E2F1蛋白表达下调70%和60%左右,E2F1的mRNA也分别有90%,60%的降低。4.双荧光素酶活性测定结果显示Ad-SSAT可抑制E2F启动子活性,使其活性下调60%以上。实验结论：Ad-SSAT抑制S期主要的细胞周期蛋白cyclin A的转录和翻译,同时抑制其上游核转录因子E2F-1的表达,并且抑制核转录因子E2F启动子的活性。提示Ad-SSAT可能是通过下调E2F启动子活性,抑制其基因表达,从而下调下游S期主要细胞周期蛋白cyclin A的表达,引起细胞周期阻滞,起到抑制细胞增殖的作用。第三部分Ad-SSAT对胃癌细胞生长抑制作用的体内研究实验目的：通过裸鼠皮下胃癌移植瘤试验,观察腺病毒Ad-SSAT介导的SSAT基因上调对胃癌的抑制作用。实验方法：1.移植瘤模型的建立：将SGC7901, SGC7901／GFP和SGC7901／Ad-SSAT细胞分别接种在4周龄的裸鼠皮下。2.移植瘤及裸鼠的监测：从注射后的第八天开始,每隔4天测量肿瘤大小,制备各组裸鼠肿瘤的生长曲线,并称取裸鼠体重,观察动物的一般状况。接种后33天处死裸鼠,称取肿瘤重量。实验结果：裸鼠皮下胃癌移植瘤模型显示SGC7901／Ad-SSAT组裸鼠的肿瘤出现较晚,生长速度明显低于对照组,致死裸鼠时SGC7901／Ad-SSAT组肿瘤的体积不足对照组的一半,肿瘤重量约为对照组肿瘤的40%。实验结论：Ad-SSAT能有效抑制裸鼠皮下胃癌细胞的生长,提示SSAT基因过表达可起到抑制肿瘤的作用。更多还原

【Abstract】 Gastric cancer is the fourth most common cancer and the second leading cause of cancer-related death, with 700000 deaths annually. Now the first-line therapy is radical surgery with adjuvant chemotherapy. But overall 5 year survival rate for this disease is 20%～30%. Because of lacking targeting, these traditionary treatments may also damage the normal tissues and cells. Therefore, it is important to find a novel non-surgical targeting intervention for treating gastric cancer.Polyamines include spermidine, spermine and their diamine precursor, putrescine. Their positive charges enable polyamines to interact electrostatically with polyanionic macromolecules within the cell, such as DNA, RNA and proteins. They are critical for cell growth and differentiation.The level of polyamine is high in cancer cell and tissues, and rapid tumor growth has been associated with remarkable elevation of polyamine biosynthesis and accumulation. Therefore, polyamine pathway becomes an important target for preventing and treating cancer.The intracellular polyamine biosynthetic pathway is mainly regulated by the actions of two rate-limiting enzymes, Ornithine decarboxylase （ODC） and Sadenosylmethionine decarboxylase （AdoMetDC）. Studies in our laboratory have demonstrated that ODC mRNA and ODC protein levels in colorectal cancer were higher than that were found in the surrounding normal tissues, the down regulation of ODC and AdoMetDC could inhibit colorectal cancer. Spermidine/spermine N1-acetyltransferase （SSAT） is the rate limiting step in polyamine catabolism,which is primarily responsible for regulation of intracellular polyamine concertrations in mammalian cells. SSAT catalyzes the transfers of the acetyl group from acetyl-CoA to the N1 positions of spermidine or spermine. SSAT is thought to prevent accumulation of higher polyamines from becoming toxic to the cell, and so maintains a balanced ration of intracellular polyamines according to cellular needs.SSAT activity is highly regulated and is induced rapidly in response to high intracellular levels of natural polyamine. Additionally, SSAT activity can be induced by a number of other stimuli, including hormones, physiological stimuli, drugs and toxic agents. In polyamine analogue-responsive cells, the super-induction of SSAT expression can result in a> 1000-fold increase in protein activity. Depletion of intracellular polyamine pools invariably inhibits cell growth. Although this is usually accomplished by inhibiting polyamine biosynthesis, Kristin Kee and Slavoljub Vujcicthis have found that this might be more effectively achieved by activating polyamine catabolism at the level of SSAT.Telomerase is an enzyme involved in the synthesis and maintenance of chromosome termini known as telomeres that conventional polymerases are unable to replicate fully. Telomerase activation is observed in approximately 90%of human cancers, irrespective of tumor type, while most normal tissues contain inactivated telomerase. Numerous studies have demonstrated that hTERT promoter expression is highly specific to cancer cells and tightly associated with telomerase activity, while the other subunits are constitutively expressed both in normal and cancer cells. Therefore, based on the specialty of hTERT promoter, we constructed an adenovirus which can express SSAT mediated by hTERT promoter, valued its inhibitory effects on gastric cancer. We also investigated the in vivo growth inhibitory effect using a SGC7901 xenograft model in nude mice.Part 1 Adenovirus vector-mediated upregulation of spermidine/spermine N1-acetyltransferase impairs human gastric cancer growth in vitroObjective:To construct a recombinant adenovirus that can expression human SSAT mediated by hTERT promoter, and study the inhibitory effects of Ad-SSAT on growth of SGC7901 and MGC803 cells. To construct the shRNA expression vector pGPU6/ GFP/Neo-shSSAT.Methods:1. Human SSAT cDNA was amplified by RT-PCR and was cloned into pGL3-hTERT plasmid. The pGL3-hTERT-SSAT construction was digested, and the small fragments were cloned into pAdTrack. Then the pAdTrack-hTERT-SSAT plasmids recombined with pAdEasy-1 vectors in AdEasy-1 cells. pAdeasy-hTERT-SSAT were linearized by Pac I, and transfected into the HEK293 to package. Green fluorescent protein expression and PCR technique was used to confirm the process.2. The siRNA duplex was designed to SSAT （NM₀02970）. The shRNA expression vector pGPU6/GFP/Neo-shSSAT was commercially obtained from GenePharma （Shanghai, China）.3. MTS assay was used to analyze adenovirus-mediated gene transduction efficiency of different MOI in SGC7901 and MGC803 cells.4. The SSAT protein levels were determined by Western blot, and intracellular polyamine content was detected by reverse-phase high performance liquid chromatography.5. MTS assay and Colony-Forming assay were used to analyze the effect of the the growth of SGC7901 and MGC803 cells.6. Cell cycle progression was detected by flow cytometry analysis.Results:1. A 516bp cDNA was amplified by RT-PCR and the sequencing result showed it was the domain of SSAT gene. Several positive recombinant clones were identified after transformation of Adeasy-1 cells with linearized pAdTrack-hTERT-SSAT. Fluorescent microscope observation and PCR conformed that pAd-hTERT-SSAT （abbreviate as Ad-SSAT） could infect 293 cell and replicate in the cell.2. The shRNA expression vector pGPU6/GFP/Neo-shSSAT was successfully constructed. 3. MTS assay results showed that Ad-SSAT could significantly inhibit the growth of SGC7901 and MGC803 about 65%and 55%, respectively, by 50MOI and 25MOI without cell toxicity.4. Western blot results showed that Ad-SSAT could increase the expression of SSAT about 265%and 210%, respectively, in SGC7901 and MGC803; pGPU6/ GFP/Neo-shSSAT could lower the expression of SSAT to 15%and 27% respectively. Spermidine and spermine were changed correspondingly.5. MTS assay and Colony-Forming assay results showed that Ad-SSAT could significantly inhibit the growth of SGC7901 and MGC803 cells.While silence of SSAT by pGPU6/GFP/Neo-shSSAT could accelerate the growth of gastric cells.6. Cell flow cytometry analysis showed that upregulated SSAT arrests gastric cancer cells in S phase.Conclusions:The recombinant adenovirus Ad-SSAT and shRNA expression vector pGPU6/GFP/Neo-shSSAT were successfully constructed. Ad-SSAT has significant inhibitory effects on gastric cancer cells and induces S arrest in SGC7901 and MGC803 cells.Part 2 Molecular mechanism of Adenovirus-mediated expression of SSAT expression induces S arrestObjective:To investigate underlying regulatory responses of Adenovirus-mediated expression of SSAT induces S arrest in MGC803and SGC7901 cells.Methods:1.The effect of Ad-SSAT on protein levels of cell cycle regulated proteins was measured by Western blotting analysis.2.The effect of Ad-SSAT on mRNA levels of cell cycle regulated proteins was measured by RT-PCR.3.The effect of Ad-SSAT on expression level of E2F-1 was measured by Western blotting and RT-PCR.4.Observe the effect of Ad-SSAT on E2F promoter activity.Results: 1. Ad-SSAT could decrease protein expression of cyclin A about 80%and 60%in SGC7901 and MGC803 cells, but there were no obvious changes in Cdk2 protein level.2. The mRNA level of cyclin A decreased significantly in Ad-SSAT-treated cells, but no changes in Cdk2 mRNA level.3. Ad-hTERT-SSAT could decrease protein and mRNA expression level of nuclear factor E2F-1.4. The E2F promoter activity measured by luciferase reporter decreased about 80% and 60%by Ad-SSAT infection in SGC7901 and MGC803 cells.Conclusions:SSAT expression mediated by Ad-SSAT induces S arrest in MGC803 and SGC7901 cells and the arrest was associated with suppression of cyclin A expression and inhibition of nuclear factor E2F-1.Part 3 Adenovirus vector-mediated up-regulation of spermidine/spermine N1-acetyltransferase suppress the gastric tumor growth in vivo.Objective:To investigate the antitumorigenicity of Ad-SSAT in vivo.Methods:1. Tumor cells injection:SGC7901, SGC7901/GFP and SGC7901/Ad-SSAT cells were injected subcutaneously in 4 week-old nude mice for tumor implantation studies.2. Tumor growth observation:Tumor growth and animal weight were monitored every 4 days since the eighth day after injection. On day 33day after inoculation, all the mice were sacrificed, and the tumor masses were weighted.Results:1. Visible tumors in SGC7901 and SGC7901/GFP groups were detectable 8 days after implantation and grew rapidly in the next days, while the cell treated with Ad-SSAT cells remarkably delayed tumor growth.2. In tumors from SSAT-up-regulated groups, size and weight were significantly decreased by 68.3%.Conclusions:Up-regulation of SSAT has an in vivo growth-inhibitory effect.更多还原