【作者】 赵晔；

【导师】 李建生；

【作者基本信息】 郑州大学 ， 内科学， 2010， 博士

【摘要】 胃癌是常见的恶性肿瘤之一,在我国的死亡率居各种癌症的首位,全国每年死于胃癌的人数约为15万,我国胃癌的死亡率在世界范围内属于较高的水平。随着现代分子生物学理论和技术的发展,认为胃癌的发生是涉及到多基因、多分子水平变化、多阶段的一个复杂过程,而癌基因的激活和抑癌基因的失活是该过程中的重要事件之一。近年来提出的表观遗传学说,使人们注意到DNA甲基化在肿瘤发生、发展中起着重要的作用。DNA甲基化是具有可逆性与遗传性的一种基因修饰方式,是指DNA双螺旋中,胞嘧啶核苷的嘧啶环第5位碳原子在DNA甲基转移酶(DNMT)介导下以S-腺苷蛋氨酸(SAM)为甲基供体发生甲基化。甲基化状态的改变是引起肿瘤的一个重要因素,这种变化包括基因组整体甲基化水平降低和CpG岛局部甲基化水平的异常升高,从而导致癌基因的激活和抑癌基因的沉默,以及基因组的不稳定。S-腺苷蛋氨酸,又名S-腺苷甲硫氨酸,是人体内所有甲基化反应的甲基供体,由S-腺苷蛋氨酸合成酶催化合成。S-腺苷蛋氨酸被认为是一种去甲基化抑制剂,能够逆转基因的低甲基化和诱导基因高甲基化。近年来许多研究显示S-腺苷蛋氨酸有预防和治疗肿瘤的作用。通过对肝癌、结肠癌、乳腺癌等体内外研究发现,S-腺苷蛋氨酸可以抑制肿瘤细胞的生长、促进细胞凋亡,并可使存在低甲基化的基因发生甲基化,达到抑制肿瘤的作用。但是在目前众多的文献中,尚无S-腺苷蛋氨酸对胃癌细胞体内外作用的报导,因此我们对S-腺苷蛋氨酸对体内外胃癌细胞的影响进行了研究,探讨S-腺苷蛋氨酸对胃癌的抑制作用。癌基因c-myc的异常表达与肿瘤的发生、发展密切相关,并且研究发现其在肝癌、胃癌和肺癌等肿瘤的发生过程中出现基因甲基化水平的降低。uPA(尿激酶型纤溶酶原激活剂)由于具有促进肿瘤细胞生长、侵袭、转移及肿瘤新生血管形成的能力,被看作是一个与转移有关的肿瘤促进基因,近年来发现结肠癌、乳腺癌和前列腺癌等肿瘤中uPA基因启动子区呈低甲基化。因此我们对胃癌中c-myc和uPA基因的表达和启动子区甲基化状态进行了研究,以期探讨S-腺苷蛋氨酸作用可能的机制。实验方法1.S-腺苷蛋氨酸对体外胃癌细胞系增殖能力的影响使用MTT方法检测最终浓度为0.5mmol/L、1mmol/L、2mmol/L、4mmol/L、8mmol/L、16mmol/L和32mmol/L的S-腺苷蛋氨酸对胃癌细胞系MKN-28、SGC-7901、BGC-823和MKN-45作用24h、48h和72h的影响,检测细胞吸光度值(A492值),计算细胞的生长抑制率和IC5。并绘制细胞增殖抑制曲线。2.S-腺苷蛋氨酸对体外胃癌细胞系细胞周期和凋亡率的影响使用流式细胞仪检测不同浓度的S-腺苷蛋氨酸(0mmol/L、2mmol/L和4mmol/L)对4种胃癌细胞系作用72h后细胞周期和凋亡率的影响。3. S-腺苷蛋氨酸对体外胃癌细胞系侵袭力和迁移力的影响收集不同浓度的S-腺苷蛋氨酸(0mmol/L、2mmol/L和4mmol/L)作用72h后的4种胃癌细胞,在聚碳酯膜覆盖有Matrigel的Transwell小室内加入各组细胞(1×105个/孔)孵育24h,观察侵袭力。或在Transwell小室内直接加入各组细胞(1×106个/孔)孵育20h,观察迁移力。检测穿膜细胞数,计算侵袭抑制率和迁移抑制率。4.S-腺苷蛋氨酸对体外胃癌细胞中c-myc和uPA基因mRNA和蛋白表达及启动子区甲基化状态的影响收集不同浓度的S-腺苷蛋氨酸(0mmol/L、2mmol/L和4mmol/L)作用72h后的4种胃癌细胞,使用半定量RT-PCR法和Western Blot方法检测各组细胞中c-myc和uPA基因mRNA和蛋白的表达,使用甲基化特异性PCR(MSP)方法检测c-myc和uPA基因启动子区的甲基化状态。5.建立人胃癌裸鼠移植瘤模型及药物处理将SGC-7901细胞(1×107个/只)接种于裸鼠左侧腋窝皮下,建立移植瘤模型。随机分为低浓度SAM组[192μmol/(kg·d)]、高浓度SAM组[768μmol/(kg·d)]和对照组(NS),每组5只,腹腔给药15天。6.S-腺苷蛋氨酸对体内胃癌生长及c-myc和uPA基因的影响每3天测量一次裸鼠体质量及移植瘤体积,绘制移植瘤生长曲线。HE染色观察移植瘤组织病理学改变和肿瘤细胞凋亡指数,观察肝脏和肺部的转移灶。使用免疫组化SP法和Western Blot方法检测各组移植瘤中c-myc基因和uPA基因蛋白的表达,使用半定量RT-PCR法检测基因mRNA的表达,使用甲基化特异性PCR方法检测基因启动子区的甲基化状态。统计学分析采用SPSS 11.1软件进行统计学分析。计量资料数据以均数±标准差(x±5)表示。细胞体外侵袭结果、细胞体外迁移结果和移植瘤免疫组化结果使用多个独立样本的秩和检验,移植瘤MSP结果使用列联表x2检验,其余结果使用单因素方差分析和成组t检验,以α=0.05为检验水准。结果1.随着S-腺苷蛋氨酸浓度和作用时间的增加,MKN-28、SGC-7901、BGC-823和MKN-45这4种细胞均逐渐出现生长抑制,凋亡细胞增多。MTT结果显示这4种细胞随S-腺苷蛋氨酸浓度和作用时间的增加,细胞的增殖活性逐渐受到抑制(P均<0.05),细胞的生长抑制率也逐渐加大。根据生长抑制率得到72h的半数抑制率(IC50)分别为：MKN-28细胞IC50 6.37mmol/L、SGC-7901细胞IC50 5.40mmol/L、BGC-823细胞IC50 4.01mmol/L和MKN-45细胞IC504.77mmol/L.2.随SAM浓度增加,SGC-7901和BGC-823细胞G1期细胞比例逐渐增加(P分别<0.05和<0.01)；细胞增殖指数PI明显降低(P分别<0.05和<0.01)；而MKN-28和MKN-45细胞无明显变化(P均>0.05)；4种细胞凋亡率均显著升高(P均<0.01)3.4种细胞的侵袭细胞数和迁移细胞数均随S-腺苷蛋氨酸浓度的增加而逐渐减少(P均<0.01),侵袭抑制率和迁移抑制率逐渐增大。4.SGC-7901和BGC-823细胞中c-myc基因,4种细胞中uPA基因mRNA和蛋白的表达随S-腺苷蛋氨酸浓度增加而逐渐减弱(P分别<0.05和<0.01),基因启动子区的低甲基化逐渐改善；而MKN-28和MKN-45细胞中c-myc基因mRNA和蛋白的表达无明显变化(P均>0.05),基因启动子区的低甲基化状态也无改善。5.S-腺苷蛋氨酸对裸鼠体质量无影响,低浓度组和高浓度组的移植瘤体积分别为(618.51±149.27)mm3和(444.32±118.51)mm3,与对照组[(1018.22±223.07)mm3]相比,体积明显减小(P均<0.01),S-腺昔蛋氨酸对移植瘤的生长有明显的抑制作用,低浓度SAM组和高浓度SAM组抑瘤率分别为39.26%和56.36%。6.3组移植瘤组织均呈人中分化胃腺癌的表现。实验组移植瘤组织中有较多的凝固性坏死灶和凋亡细胞,肿瘤细胞凋亡指数随S-腺苷蛋氨酸浓度增大而逐渐增加(P均<0.01)。3组裸鼠均发现未有肝脏和肺部的转移灶。7.免疫组化、Western Blot方法及半定量RT-PCR方法检测均显示裸鼠移植瘤中c-myc和uPA基因蛋白和mRNA的表达强度随S-腺苷蛋氨酸浓度增大而明显减弱(P分别<0.05和<0.01)；对照组中c-myc和uPA基因启动子区未甲基化,随着S-腺苷蛋氨酸浓度增高,逐渐恢复甲基化(c-mycx2值=10.179,P<0.01；uPA x2值=11.667,P<0.01)。结论1、S-腺苷蛋氨酸能够抑制体外胃癌细胞的增殖、侵袭力和迁移力,诱导凋亡,使细胞阻滞在G1期,且作用与浓度和时间成正相关。S-腺苷蛋氨酸可逆转体外胃癌细胞中c-myc和uPA基因启动子区的低甲基化,抑制其表达。胃癌细胞的增殖、周期、凋亡、侵袭力和迁移力的改变可能与S-腺苷蛋氨酸改善c-myc和uPA基因启动子区的低甲基化状态,抑制其表达有关。2、S-腺苷蛋氨酸可抑制人胃癌裸鼠移植瘤的生长,促进细胞凋亡,并能逆转移植瘤中c-myc和uPA基因启动子区的低甲基化状态,使其表达受到抑制,正是这种作用可能导致了S-腺苷蛋氨酸对体内胃癌生长的抑制。3、我们研究并证实了S-腺苷蛋氨酸对体内外胃癌的增殖及其肿瘤特性有抑制作用；其作用机制可能是SAM通过提供甲基基团,逆转了癌基因的低甲基化状态,使其失活。更多还原

【Abstract】 Gastric cancer is one of the most prevalent malignant tumors, and the number of death from it is more than 150 thousand per year in China. Gastric cancer continue to be the most common fatal cancer recently in China. The carcinogenesis is known as a complicated process involved in polygene, poly-molecular change and multistage. And the most important events are the activation of oncogene and inactivation of tumor suppressor gene in the process. Recently, there are more and more attention to epigenetics, such as DNA methylation. DNA methylation, the covalent addition of a methyl group to the C-5 position of cytosine in the context of the CpG dinucleotide, is catalysised by the DNA methyltransferase enzyme, and has the significant role in the process of carcinogenesis and growth. DNA methylation is a process modified by genes, which is reversible and hereditary. The changes of methylation status, including the decrease of global genome methylation level and the abnomaly raise of CpG island partly methylation level, are important factors in carcinogenesis. These changes can lead to the activation of oncogene and inactivation of tumor suppressor gene, as well as the instability of genome.S-adenosylmethionine (SAM) is synthetized through S-adenosylmethionine synthetase, and is the primary methyl group donor for most biological methylation reactions. SAM is known as a demethylation suppressor and can reverse gene hypomethylation, induce gene hypermethylation. In recent studies, SAM has been found having the effect on chemopreventing and treating on cancers of liver, colon and breast, because it could promote apoptosis and suppress the growth of cancer cells. So far there were no reports on gastric cancer. For the reason, we investigated the potential benefits of SAM in inhibiting the growth of gastric cancer in vivo and in vitro. Abnomal expression of c-myc is closely correlated with carcinogenesis. And the methylation level of c-myc gene is decreased in the process of carcinogenesis such as liver cancer, gastric cancer, lung cancer. Urokinase-type plasminogen activator (uPA) is related with tumor metastasis. uPA is regard as a tumor trigger gene, for promoting the growth, invasion, transfer, and neovascularity of tumor. We studied the methylation status of gene promoter region and the expression of c-myc and uPA genes, trying to identify the anti-tumor mechanisms of SAM in gastric cancer.Methods1 Effects of SAM on proliferation of gastric cancer cell linesFor dose-response experiments, MKN-28, SGC-7901, BGC-823 and MKN-45 cells were incubated with increasing concentrations (0.5-32mmol/L) of SAM for 24h,48h and 72h. Cell growth was detected by MTT colorimetric method. The cell growth inhibition ratio and IC50 were calculated, and the restrain curves were ploted.2 Effects of SAM on cell cycle and apoptosis of gastric cancer cell linesThe four cell lines were incubated with different concentrations(0,2 and 4 mmol/L) of SAM for 72h. Cell cycle distribution and cell apoptosis were calculated by using of flow cytometry system.3 Effects of SAM on invasiveness and migration of gastric cancer cell linesThe four cell lines were incubated with different concentrations(0,2 and 4mmol/L) of SAM for 72h. Cells were incubated in the transwell caves at a density of 1×105/well for 24h, to investigate the invasiveness, after the polycarbonate membrane of transwell cave was covered by Matrigel. Or, cells were incubated at a density of 1×106/well for 20h, to investigate the migration. The number of cell which going through the membrane was counted.4 Effects of SAM on expression and methylation status of c-myc and uPA of gastric cancer cell linesThe four cell lines were incubated with different concentrations(0,2 and 4mmol/L) of SAM for 72h. RT-PCR analysis of c-myc and uPA mRNA expression in cells. Western blot analysis of c-myc and uPA protein expression in cells. MSP analysis of c-myc and uPA gene promoter region methylation status in cells.5 Establishment of nude mice model of tumor xenografts and treatment by SAMTumor xenografts were established by inoculation of SGC-7901 cells subcutaneously in BALB/c nude mice. The mice were randomized separated into three groups:low concentration group(192μmol/(kg-d) SAM), high concentration group(768μmol/(kg-d) SAM) and control group(NS), and treated by peritoneal injection for 15 days.6 Effects of SAM on growth and c-myc,uPA genes of xenograftsTumor volume, the weight of nude mice were recorded every three days. After gotten the samples,the changes of histopathology and apoptotic index were observed by HE. The metastasis of liver and lung of nude mice were observed. Immunohistochemistry, Western Blot and RT-PCR were used to analysis c-myc and uPA protein and mRNA expression. MSP analysis of methylation status of gene promoter regions in xenografts.Statistical AnalysisData were expressed as mean±SD. The results of the invasiveness and migration of cells, the immunohistochemistry of xenografts were evaluated by rank-sum test, The results of MSP of xenografts were evaluated by contingency table x2 test. The results of the rest were evaluated by ANOVA with SPSS 11.1. The statistical level of significance was set at P<0.05.Results1 The growths of MKN-28,SGC-7901,BGC-823 and MKN-45 cell were inhibited and the apoptosis cells were increasing with increase of SAM concentration and action time. The MTT assay showed that the proliferation of the four cell lines were restrained gradually (P<0.05) and the growth inhibition ratio were increasing after treated with SAM. The IC50 of cells of 72h were: MKN-28 IC50 6.37 mmol/L, SGC-7901 IC50 5.40 mmol/L, BGC-823 IC50 4.01mmol/L and MKN-45 IC50 4.77 mmol/L.2 The apoptosis cells were all significantly increased (P<0,01) after treated with SAM. In SGC-7901 and BGC-823, the cell percentages of G0/G1 phase were significantly increased (P<0.05 and P<0.01), whereas the cell proliferation indexes (PI) were significantly decreased (P<0.05 and P<0.01). But in MKN-28 and MKN-45, the cell cycle and the PI did not show any difference (P>0.05).3 The number of invasion and migration cell of the four cell lines were decreasing(P<0.01) and the invasion and migration inhibition ratio were all increasing, with increase of SAM concentration.4 The expressions of c-myc mRNA and protein in SGC-7901 and BGC-823, and the expressions of uPA mRNA and protein in the four cell lines were all significantly decreasing(P<0.05 and P<0.01) with increase of SAM concentration, and the hypomethylation of gene promoter regions were reversed. But the the expressions of c-myc mRNA and protein in MKN-28 and MKN-45, and the hypomethylation of gene promoter regions were no marked change(P>0.05).5 When nude mice were puting to death, the body weights of three groups were no difference (P>0.05). Compared with control group[(1018.22±223.07)mm3], the tumor volume of low concentration group[(618.51±149.27)mm3] and high concentration group[(444.32±118.51)mm3] were obviousily diminished(P<0.01), and the inhibitory rate of tumor growth of low and high concentration group were 39.26% and 56.36%, respectively.6 The xenografts pathology conformed with the characteristics of moderately differentiated human gastric carcinoma. The xenografts had more coagulative necrosis and apoptosis after treated with SAM. Apoptotic index had positive correlation with SAM concentration (P<0.01). The metastases of liver and lung of three groups were no found.7 In the xenografts, the expressions of c-myc and uPA mRNA and protein were all significantly weakening(P<0.05 and P<0.01), and the hypomethylation of gene promoter regions were improving (c-mycχ2=10.179, uPAχ2=11.667, all P<0.01), with increase of SAM concentration.Conclusions1 SAM can suppress the proliferation, invasion and migration, induce apoptosis, blocke at G1 phase of the gastric cancer cell lines, in a dose-and time-dependent manner in vitro. And SAM can reverse the hypomethylation of c-myc, uPA gene promoter regions, reduce their expression. The changes of proliferation, cell cycle, apoptosis, invasion and migration of gastric cancer cells may correlate with the effects of SAM on hypomethylation of c-myc and uPA gene promoter region and their expression.2 SAM can inhibit the growth, promote apoptosis, reverse the hypomethylation of c-myc and uPA gene promoter region, and reduce genes’expression of human gastric carcinoma xenografts. May be these effects result the inhibition of human gastric carcinoma in vivo.3 Our data demonstrate the inhibition of SAM on human gastric carcinoma in vivo and in vitro. The mechanisms of the effects may be SAM can reverse the hypomethylation of oncogenes and re-inactivate them by providing methyl groups.更多还原