消痰散结方对胃癌P16基因甲基化的影响
|
摘要
背景：
胃癌是一种常见的恶性肿瘤,在我国胃癌的发病率居第二位,但死亡率则位居第一,严重危害人类健康。以往认为胃癌的发生是基因遗传学改变的结果。然而,近年来,越来越多的研究表明,表观遗传学改变在肿瘤的发生过程中起了关键的作用。DNA甲基化是目前研究较多的表观遗传机制,在胃癌的发生、发展中起重要作用。现已证实DNA甲基化是基因突变及缺失之外抑癌基因失活的第3种机制,而且在某些情况下是其失活的唯一机制。胃癌发生过程中,有许多抑癌基因因发生高甲基化而失活,导致胃癌的发生。CDKN2A(P16)是已经确认的抑癌基因,该基因的失活在肿瘤细胞增生过程中起着重要作用。P16基因的失活与胃癌、卵巢癌、肺癌等多种肿瘤发生密切相关。在胃癌中已被证实P16基因启动子区域异常甲基化是其失活的主要机制。
基因甲基化可以抑制基因表达,但又是一种可逆的表观遗传学改变。目前,针对抑癌基因甲基化进行有效治疗是肿瘤研究的一个热点。去甲基化剂5-azacytidine和5-Aza-CdR均可使因甲基化而表达受抑制的抑癌基因重新表达,从而抑制肿瘤细胞生长。但是由于这两种药物缺乏特异性阻断效应,这限制了其应用范围,目前只用于骨髓增生异常综合症的治疗,尚未用于胃癌等实体瘤的治疗。探索特异性去甲基化药物,对于肿瘤的治疗有重要的意义。
中医药治疗胃肠道肿瘤早有报道,探讨中医药对胃肠道肿瘤的作用机理,有望为胃癌的治疗提供新的药物以及治疗思路。关于中医药的研究甚多,但目前仅有极少几篇文献报道中医药影响基因甲基化的研究。魏品康教授提出了肿瘤的“痰污染学说”,在此学说的指导下,创立经验方消痰散结方,临床治疗胃癌效果明显。本课题拟从表观遗传学角度着手,初步探索消痰散结方对胃癌中P16基因甲基化的影响,以便阐明消痰散结方的作用机理；同时,为研究中医药与基因甲基化的关系提供理论基础。
目的
通过体外用含药血清干预胃癌细胞系,体内用消痰散结方煎剂给裸鼠人胃癌原位移植瘤模型灌胃,探讨消痰散结方对胃癌细胞系以及裸鼠原位移植瘤模型基因甲基化的影响；运用CCK-8法、RT-PCR；Methylight分子生物学方法,由定性到定量研究消痰方对肿瘤细胞系以及肿瘤组织中抑癌基因P16 mRNA表达以及DNA甲基化水平的影响,从表观遗传学角度探讨消痰散结方抗肿瘤的作用机理。
方法
1、6周龄SD雄性大鼠10只,体重350g±20g,随机分成空白对照组(生理盐水)、消痰散结方组(消痰散结方水煎剂),灌胃给药5日后股动脉取血制备药物血清。
2、人胃癌细胞系MKN45, BGC823随机分成4组,1)药物血清组,每孔加入消痰散结方药物血清20μl; 2) 5-Aza-CdR组：每孔加5-Aza-CdR 20μl,药物浓度为5μmol/L; 3)空白血清组,每孔加入无药血清20μl；4)空白对照组,不加任何药物。每组设5个复孔,置37,5%CO2培养箱中培养24h、48h、72h后,每孔加入CCK-810μl,检测细胞活力
3、分别收集药物作用前和药物作用72小时后的细胞,提取总RNA,紫外分光光度仪测纯度和浓度,RT-PCR测定P16基因mRNA表达；
4、收集药物作用前后的细胞,用DNA Mini kit试剂盒抽提细胞DNA, Methylight法测定P16基因甲基化改变；
5、培养人胃腺癌MKN-45细胞株,裸鼠腋窝皮下注射反复传代形成实体瘤,取第6代瘤块,造胃癌肿瘤原位移植模型；裸鼠原位移植瘤模型随机分成空白对照组、消痰散结方组,每组10只。于造模成功后72小时开始给药：消痰散结方组给予消痰散结方水煎剂(含生药量2.5lg/ml),空白对照组给予生理盐水。每次灌胃0.2ml,每日2次,连续给药6周,每周灌6天休息1天,给药期间观察裸鼠活动状况和瘤体生长状况,给药最后一天脱颈处死；
6、第6周实验结束时取两组动物肿瘤组织称取瘤体重量,测定消痰散结方抑瘤率；
7、第6周实验结束时取两组动物的肿瘤组织,提取总RNA,紫外分光光度仪测总RNA纯度和浓度,RT-PCR测定P16基因mRNA表达；
8、第6周实验结束时,取两组动物的肿瘤组织；提取DNA, Methylight法测定P16基因甲基化改变。
结果
1、血清状况：
实验动物生长良好,两组动物血液分别制备取得20ml无菌药物血清；
2、抑瘤率：
含药血清和5-Aza-CdR均可抑制胃癌细胞株MKN-45及BGC823细胞增殖,与空白对照组有明显差异(P<0.05),药物血清组抑瘤率高于5-Aza-CdR组。含药血清作用72小时后抑瘤作用最强,细胞生长最低降低为76.14%；空白血清组和空白对照组相比较则无明显差别(P>0.05)；
3、RT-PCR结果显示：
胃癌细胞系MKN45、BGC823中P16基因弱表达,通过含药血清以及5-Aza-CdR作用72小时后,P16基因表达明显升高。5-Aza-CdR组P16基因表达量高于中药药物血清组；
4、Methylight测定P16基因甲基化水平：
结果表明胃癌细胞系MKN45、BGC823中P16基因启动子明显甲基化(PMR>4),经过含药血清以及5-Aza-CdR作用72小时后,P16基因甲基化水平均明显降低,与对照组比较有统计学意义(P<0.05)。5-Aza-CdR组P16基因甲基化水平明显低于药物血清组(P<0.05)。在MKN45细胞中,经5-Aza-CdR作用72小时后,无P16基因甲基化条带扩增；
5、动物生长状况：
实验过程中,术后第4天空白对照组动物死亡1只,经解剖发现肠粘连梗阻造成。各组标本数分别为空白对照组9只,消痰散结方组10只。在给药3周左右空白对照组开始出现活动减少、饮食减少,消瘦等现象；而消痰散结方组则在4周左右才出现类似情况。空白对照组在造模2周左右在上腹部触及肿块,而消痰散结方组则较晚。给药6周后,空白对照组出现消瘦,呈弓背,肋骨隐约可见,活动受限遂结束饲养,断颈处死动物；
6、消痰散结方对原位移植瘤的影响：
肿瘤呈实质性圆形或椭圆形,切面呈鱼肉状,表面结节状突起,较大者切开中央有小片浆液样坏死区。瘤体称量结果显示消痰散结方组的抑瘤率为54.82%,与空白对照组相比有统计学意义(P<0.01)；
7、RT-PCR结果显示：
裸鼠人胃癌原位移植瘤模型中P16基因弱表达,通过消痰散结方灌胃6周后,P16基因表达明显升高,与空白对照组比较有明显差异(P<0.01)；
8、Methylight测定P16基因甲基化水平：
裸鼠人胃癌原位移植瘤模型中P16基因启动子明显甲基化(PMR>4),通过消痰散结方灌胃6周后,P16基因甲基化水平均明显降低,与空白对照组比较有统计学差异(P<0.01)
结论
1、消痰散结方含药血清能抑制胃癌肿瘤细胞系的增殖；
2、消痰散结方可抑制裸鼠人胃癌原位移植瘤模型肿瘤的生长；
3、体外及体内实验显示消痰散结方能降低P16基因启动子区域甲基化水平,增加P16基因mRNA的表达；从而达到抑制肿瘤增殖的作用。
Background:
Gastric carcinoma (GC) is the most common cancer in eastern Asia and the third most frequent cancer across the world. Gastric Cancer has long been known to be a genetic disease.However, in recent years it has become increasingly obvious that genetic abnormalities are by no means the only mechanism by which gene expression becomes altered during tumourigenesis. Growing evidence now suggests that epigenetic factors, in particular DNA methylation, play a major role in carcinogenesis and sometimes even act as the only reason for inactivation of tumor suppressor genes in human cancers. CDKN2A (P16),a well-known tumor suppressor gene,was found to be highly methylated on it's promoter region in gastric cancer cells,leading to the transcriptional silence of gene.
Demethylating agents 5-azacytidine and 5-Aza-CdR could inhibit cell proliferation of cancer by reversing DNA hypermethylation,but their usage were limited to myelodysplastic syndrome (MDS). Until now,there is no evidence that 5-azacytidine and 5-Aza-CdR could inhibit the growth of gastric cancer by reversing abnormal DNA hypermethylation without affecting DNA hypomethylation that often occurred to proto-oncogenes.
Traditional Chinese herbal medicine(TCM) has been long used to treated tumours,and compared to the chemotherapeutic agents TCM could improve the patient's life quality with less side-effect. XTSJF is a very popular herbal medicine in china. XTSJF has been used in the treatment of gastrointestinal cancer,such as gastric carcinoma and esophageal cacinoma. It improved subjective symptoms and inhibit the proliferation of cancer cells. Many reports assayed the preventive and therapeutic effects of XTSJF on the experimental gastric carcimoma induced in rats by orthotopic transplantation.
Many studies reported the effect of TCM on tumours,however, untill now there is few articles reported the effect of TCM on DNA methylation in carcinomas.The aim of this study was to assay the therapeutic effects of XTSJF decocton on gastric cancer cells. In order to understand the mechanism of XTSJF to inhibit proliferation of cancer cells, we also tested the drug's effect on the mRNA expression of P16 gene, and The DNA methylation level of P16 gene in gastric cancer cells.
Objective:
To explore the effect to tumorous growth,we study tumor development characteristic and proliferation of gastric cancer cell lines;we study the effect from mRNA level to DNA methylation level about CDKN2A(P16)by RT-PCR method、Methylight method (RT-PCR) from qualitation to quantitation with XiaoTanSanJieRecipe,we explored anti-tumor effect and mechanism from the aspect of epigenetics.
Methods:
1.10 male SD mouse models were divided into two groups:control group and TCM group,then treated with corresponding medicine respectively;8ml blood was got through arteria cruralis from each mouse after 5-day stomach lavage and then made into serum containing medicine;
2.. Briefly,100μl cells(1×104cells/ml) were seeded per well in a 96-well flat-bottom plate. They were then treated for 24,48 and 72 h with appropriate concentrations of XTSJF pharmacological serum(20μl), normal rat serum (20μl),5-Aza-CdR (5μmol/L) and vehicle(20μl) in the presence of 10% FBS. We then added 10μl CCK-8 to each well to assess cell growth; Cell growth was assessed by CCK8 (cell counting kit 8) according to the manufacturer's instructions;
3. Total RNA was isolated from MKN-45 and BGC823 cells according to the manufacturer's instruction after 72 hours treatment. then RT-PCR was performed to detect mRNA expression of PI 6;
4. Total DNA was isolated from MKN-45 and BGC823 cells with DNA Mini kit according to the manufacturer's instruction after 72 hours treatment. then Methylight was performed to detect DNA methylation level of P16;
5. 20 nude mouse models of human gastric carcinoma cell were made by using orthotopic transplantation. then they were diviede into two groups:control group and TCM group,then treated with corresponding medicine respectively 72 hours after orthotopic transplantation. Each dose was dissolved in 0.4 ml distilled water and administered twice a day intragastrally (i.g.) for 6 weeks, Each animal received one dose of XTSJF or vehicle daily for 6 weeks and then executed. Dietetic state and weight change of the tumor-bearing mice were observed and tumorous size was measured during administration;
6. The experiment was terminated on the 6th week, The mice were sacrificed.we measured weight and volume of tumor taken from the mice and calculate the inhibitory rate;
7. The tumours were taken on the 6th week,then we extract totle RNA from each tumour sample and detect the expression of PI 6 mRNA by RT-PCR.
8. we detect the methylation level of P16 gene by Methylight. Results:
1.Experimental animals had a good animation during the experimental period; and 20 ml pharmacological serum were got from each group of animals;
2. Compared to cell proliferation of control group, those of 5-Aza-CdR group、XTSJF pharmacological serum group was degraded,the difference has statistical significance (P<0.05),and inhibiterary rate of cell proliferation was higher in XTSJF pharmacological serum group than that in 5-Aza-CdR group. Whereas there was no statistical significance between normal rat serum group and control group (P>0.05);
3. We observed expression of P16 mRNA by RT-PCR method, control group is lower than XTSJF pharmacological serum group and 5-Aza-CdR group,the difference has statistical significance (P<0.05); 5-Aza-CdR group is higher than XTSJF pharmacological serum group, the difference has statistically significant (P<0.05);
4.The methylation level of P16 gene is detected by methylight method:hypermethylation were observed in the promoter region of P16 gene in gastric cancer cell line MKN45 and BGC823(PMR>4); the methylation level were degraded after treated for 72hours with XTSJF pharmacological serum or 5-aza-dC; the methylation level of P16 gene is lower in 5-Aza-CdR group than in pharmacological serum group (P<0.05);P16 gene methylated was not observed in the gastric cancer line MKN45 after treated with 5-Aza-CdR for 72 hours;
5. one mouse in the control group died of bowel obstruction on the 4th day of experiment. Emaciation, decreacing of movement and diet were observed in the control group on the 3th week, and in the TCM group the 4th week. The mice of the control group got a tumour lump on their right epigastric zone in earlier time than those of the TCM group, on the 6th week of experiment the mice got emaciation, limitation of movement and camptocormia, then they were sacreficed;
6.The shape of tumours taken from the mice was round or oval, the cut surface looked like fish and had some ecptomas,and zone of necrosis was found in the center of some big tumours. The result of tumour weighing indicate that the TCM group have a higher inhibitory rate (54.82%) than the control group, the difference have a statistical significance(P<0.01)
7. The result of RT-PCR indicated that expession mRNA of P16 was lower in the control group than in the TCM group which was treated with XTSJF for six weeks;the difference has remarkable statistical significance(p<0.01))
8. The methylation level of P16 gene is detected by methylight method:hypermethylation were observed in the promoter region of P16 gene in control group (PMR>4); the methylation level were degraded after treated for 6 weeks with XTSJF decoction; the difference has remarkable statistical significance(p<0.01).
Conclusion:
1. Medical serum of XTSJF could depess the diffrentiation of gastric cancer cell lines;
2. Decoction of XTSJF could depress the tumorous growth in mouse models of human gastric carcinoma cell;
3. Both invivo and invitro study indicated that XTSJF could depress the tumorous growth by reversing DNA methylation and increasing the mRNA expession of P16 gene.
胃癌是一种常见的恶性肿瘤,在我国胃癌的发病率居第二位,但死亡率则位居第一,严重危害人类健康。以往认为胃癌的发生是基因遗传学改变的结果。然而,近年来,越来越多的研究表明,表观遗传学改变在肿瘤的发生过程中起了关键的作用。DNA甲基化是目前研究较多的表观遗传机制,在胃癌的发生、发展中起重要作用。现已证实DNA甲基化是基因突变及缺失之外抑癌基因失活的第3种机制,而且在某些情况下是其失活的唯一机制。胃癌发生过程中,有许多抑癌基因因发生高甲基化而失活,导致胃癌的发生。CDKN2A(P16)是已经确认的抑癌基因,该基因的失活在肿瘤细胞增生过程中起着重要作用。P16基因的失活与胃癌、卵巢癌、肺癌等多种肿瘤发生密切相关。在胃癌中已被证实P16基因启动子区域异常甲基化是其失活的主要机制。
基因甲基化可以抑制基因表达,但又是一种可逆的表观遗传学改变。目前,针对抑癌基因甲基化进行有效治疗是肿瘤研究的一个热点。去甲基化剂5-azacytidine和5-Aza-CdR均可使因甲基化而表达受抑制的抑癌基因重新表达,从而抑制肿瘤细胞生长。但是由于这两种药物缺乏特异性阻断效应,这限制了其应用范围,目前只用于骨髓增生异常综合症的治疗,尚未用于胃癌等实体瘤的治疗。探索特异性去甲基化药物,对于肿瘤的治疗有重要的意义。
中医药治疗胃肠道肿瘤早有报道,探讨中医药对胃肠道肿瘤的作用机理,有望为胃癌的治疗提供新的药物以及治疗思路。关于中医药的研究甚多,但目前仅有极少几篇文献报道中医药影响基因甲基化的研究。魏品康教授提出了肿瘤的“痰污染学说”,在此学说的指导下,创立经验方消痰散结方,临床治疗胃癌效果明显。本课题拟从表观遗传学角度着手,初步探索消痰散结方对胃癌中P16基因甲基化的影响,以便阐明消痰散结方的作用机理；同时,为研究中医药与基因甲基化的关系提供理论基础。
目的
通过体外用含药血清干预胃癌细胞系,体内用消痰散结方煎剂给裸鼠人胃癌原位移植瘤模型灌胃,探讨消痰散结方对胃癌细胞系以及裸鼠原位移植瘤模型基因甲基化的影响；运用CCK-8法、RT-PCR；Methylight分子生物学方法,由定性到定量研究消痰方对肿瘤细胞系以及肿瘤组织中抑癌基因P16 mRNA表达以及DNA甲基化水平的影响,从表观遗传学角度探讨消痰散结方抗肿瘤的作用机理。
方法
1、6周龄SD雄性大鼠10只,体重350g±20g,随机分成空白对照组(生理盐水)、消痰散结方组(消痰散结方水煎剂),灌胃给药5日后股动脉取血制备药物血清。
2、人胃癌细胞系MKN45, BGC823随机分成4组,1)药物血清组,每孔加入消痰散结方药物血清20μl; 2) 5-Aza-CdR组：每孔加5-Aza-CdR 20μl,药物浓度为5μmol/L; 3)空白血清组,每孔加入无药血清20μl；4)空白对照组,不加任何药物。每组设5个复孔,置37,5%CO2培养箱中培养24h、48h、72h后,每孔加入CCK-810μl,检测细胞活力
3、分别收集药物作用前和药物作用72小时后的细胞,提取总RNA,紫外分光光度仪测纯度和浓度,RT-PCR测定P16基因mRNA表达；
4、收集药物作用前后的细胞,用DNA Mini kit试剂盒抽提细胞DNA, Methylight法测定P16基因甲基化改变；
5、培养人胃腺癌MKN-45细胞株,裸鼠腋窝皮下注射反复传代形成实体瘤,取第6代瘤块,造胃癌肿瘤原位移植模型；裸鼠原位移植瘤模型随机分成空白对照组、消痰散结方组,每组10只。于造模成功后72小时开始给药：消痰散结方组给予消痰散结方水煎剂(含生药量2.5lg/ml),空白对照组给予生理盐水。每次灌胃0.2ml,每日2次,连续给药6周,每周灌6天休息1天,给药期间观察裸鼠活动状况和瘤体生长状况,给药最后一天脱颈处死；
6、第6周实验结束时取两组动物肿瘤组织称取瘤体重量,测定消痰散结方抑瘤率；
7、第6周实验结束时取两组动物的肿瘤组织,提取总RNA,紫外分光光度仪测总RNA纯度和浓度,RT-PCR测定P16基因mRNA表达；
8、第6周实验结束时,取两组动物的肿瘤组织；提取DNA, Methylight法测定P16基因甲基化改变。
结果
1、血清状况：
实验动物生长良好,两组动物血液分别制备取得20ml无菌药物血清；
2、抑瘤率：
含药血清和5-Aza-CdR均可抑制胃癌细胞株MKN-45及BGC823细胞增殖,与空白对照组有明显差异(P<0.05),药物血清组抑瘤率高于5-Aza-CdR组。含药血清作用72小时后抑瘤作用最强,细胞生长最低降低为76.14%；空白血清组和空白对照组相比较则无明显差别(P>0.05)；
3、RT-PCR结果显示：
胃癌细胞系MKN45、BGC823中P16基因弱表达,通过含药血清以及5-Aza-CdR作用72小时后,P16基因表达明显升高。5-Aza-CdR组P16基因表达量高于中药药物血清组；
4、Methylight测定P16基因甲基化水平：
结果表明胃癌细胞系MKN45、BGC823中P16基因启动子明显甲基化(PMR>4),经过含药血清以及5-Aza-CdR作用72小时后,P16基因甲基化水平均明显降低,与对照组比较有统计学意义(P<0.05)。5-Aza-CdR组P16基因甲基化水平明显低于药物血清组(P<0.05)。在MKN45细胞中,经5-Aza-CdR作用72小时后,无P16基因甲基化条带扩增；
5、动物生长状况：
实验过程中,术后第4天空白对照组动物死亡1只,经解剖发现肠粘连梗阻造成。各组标本数分别为空白对照组9只,消痰散结方组10只。在给药3周左右空白对照组开始出现活动减少、饮食减少,消瘦等现象；而消痰散结方组则在4周左右才出现类似情况。空白对照组在造模2周左右在上腹部触及肿块,而消痰散结方组则较晚。给药6周后,空白对照组出现消瘦,呈弓背,肋骨隐约可见,活动受限遂结束饲养,断颈处死动物；
6、消痰散结方对原位移植瘤的影响：
肿瘤呈实质性圆形或椭圆形,切面呈鱼肉状,表面结节状突起,较大者切开中央有小片浆液样坏死区。瘤体称量结果显示消痰散结方组的抑瘤率为54.82%,与空白对照组相比有统计学意义(P<0.01)；
7、RT-PCR结果显示：
裸鼠人胃癌原位移植瘤模型中P16基因弱表达,通过消痰散结方灌胃6周后,P16基因表达明显升高,与空白对照组比较有明显差异(P<0.01)；
8、Methylight测定P16基因甲基化水平：
裸鼠人胃癌原位移植瘤模型中P16基因启动子明显甲基化(PMR>4),通过消痰散结方灌胃6周后,P16基因甲基化水平均明显降低,与空白对照组比较有统计学差异(P<0.01)
结论
1、消痰散结方含药血清能抑制胃癌肿瘤细胞系的增殖；
2、消痰散结方可抑制裸鼠人胃癌原位移植瘤模型肿瘤的生长；
3、体外及体内实验显示消痰散结方能降低P16基因启动子区域甲基化水平,增加P16基因mRNA的表达；从而达到抑制肿瘤增殖的作用。
Background:
Gastric carcinoma (GC) is the most common cancer in eastern Asia and the third most frequent cancer across the world. Gastric Cancer has long been known to be a genetic disease.However, in recent years it has become increasingly obvious that genetic abnormalities are by no means the only mechanism by which gene expression becomes altered during tumourigenesis. Growing evidence now suggests that epigenetic factors, in particular DNA methylation, play a major role in carcinogenesis and sometimes even act as the only reason for inactivation of tumor suppressor genes in human cancers. CDKN2A (P16),a well-known tumor suppressor gene,was found to be highly methylated on it's promoter region in gastric cancer cells,leading to the transcriptional silence of gene.
Demethylating agents 5-azacytidine and 5-Aza-CdR could inhibit cell proliferation of cancer by reversing DNA hypermethylation,but their usage were limited to myelodysplastic syndrome (MDS). Until now,there is no evidence that 5-azacytidine and 5-Aza-CdR could inhibit the growth of gastric cancer by reversing abnormal DNA hypermethylation without affecting DNA hypomethylation that often occurred to proto-oncogenes.
Traditional Chinese herbal medicine(TCM) has been long used to treated tumours,and compared to the chemotherapeutic agents TCM could improve the patient's life quality with less side-effect. XTSJF is a very popular herbal medicine in china. XTSJF has been used in the treatment of gastrointestinal cancer,such as gastric carcinoma and esophageal cacinoma. It improved subjective symptoms and inhibit the proliferation of cancer cells. Many reports assayed the preventive and therapeutic effects of XTSJF on the experimental gastric carcimoma induced in rats by orthotopic transplantation.
Many studies reported the effect of TCM on tumours,however, untill now there is few articles reported the effect of TCM on DNA methylation in carcinomas.The aim of this study was to assay the therapeutic effects of XTSJF decocton on gastric cancer cells. In order to understand the mechanism of XTSJF to inhibit proliferation of cancer cells, we also tested the drug's effect on the mRNA expression of P16 gene, and The DNA methylation level of P16 gene in gastric cancer cells.
Objective:
To explore the effect to tumorous growth,we study tumor development characteristic and proliferation of gastric cancer cell lines;we study the effect from mRNA level to DNA methylation level about CDKN2A(P16)by RT-PCR method、Methylight method (RT-PCR) from qualitation to quantitation with XiaoTanSanJieRecipe,we explored anti-tumor effect and mechanism from the aspect of epigenetics.
Methods:
1.10 male SD mouse models were divided into two groups:control group and TCM group,then treated with corresponding medicine respectively;8ml blood was got through arteria cruralis from each mouse after 5-day stomach lavage and then made into serum containing medicine;
2.. Briefly,100μl cells(1×104cells/ml) were seeded per well in a 96-well flat-bottom plate. They were then treated for 24,48 and 72 h with appropriate concentrations of XTSJF pharmacological serum(20μl), normal rat serum (20μl),5-Aza-CdR (5μmol/L) and vehicle(20μl) in the presence of 10% FBS. We then added 10μl CCK-8 to each well to assess cell growth; Cell growth was assessed by CCK8 (cell counting kit 8) according to the manufacturer's instructions;
3. Total RNA was isolated from MKN-45 and BGC823 cells according to the manufacturer's instruction after 72 hours treatment. then RT-PCR was performed to detect mRNA expression of PI 6;
4. Total DNA was isolated from MKN-45 and BGC823 cells with DNA Mini kit according to the manufacturer's instruction after 72 hours treatment. then Methylight was performed to detect DNA methylation level of P16;
5. 20 nude mouse models of human gastric carcinoma cell were made by using orthotopic transplantation. then they were diviede into two groups:control group and TCM group,then treated with corresponding medicine respectively 72 hours after orthotopic transplantation. Each dose was dissolved in 0.4 ml distilled water and administered twice a day intragastrally (i.g.) for 6 weeks, Each animal received one dose of XTSJF or vehicle daily for 6 weeks and then executed. Dietetic state and weight change of the tumor-bearing mice were observed and tumorous size was measured during administration;
6. The experiment was terminated on the 6th week, The mice were sacrificed.we measured weight and volume of tumor taken from the mice and calculate the inhibitory rate;
7. The tumours were taken on the 6th week,then we extract totle RNA from each tumour sample and detect the expression of PI 6 mRNA by RT-PCR.
8. we detect the methylation level of P16 gene by Methylight. Results:
1.Experimental animals had a good animation during the experimental period; and 20 ml pharmacological serum were got from each group of animals;
2. Compared to cell proliferation of control group, those of 5-Aza-CdR group、XTSJF pharmacological serum group was degraded,the difference has statistical significance (P<0.05),and inhibiterary rate of cell proliferation was higher in XTSJF pharmacological serum group than that in 5-Aza-CdR group. Whereas there was no statistical significance between normal rat serum group and control group (P>0.05);
3. We observed expression of P16 mRNA by RT-PCR method, control group is lower than XTSJF pharmacological serum group and 5-Aza-CdR group,the difference has statistical significance (P<0.05); 5-Aza-CdR group is higher than XTSJF pharmacological serum group, the difference has statistically significant (P<0.05);
4.The methylation level of P16 gene is detected by methylight method:hypermethylation were observed in the promoter region of P16 gene in gastric cancer cell line MKN45 and BGC823(PMR>4); the methylation level were degraded after treated for 72hours with XTSJF pharmacological serum or 5-aza-dC; the methylation level of P16 gene is lower in 5-Aza-CdR group than in pharmacological serum group (P<0.05);P16 gene methylated was not observed in the gastric cancer line MKN45 after treated with 5-Aza-CdR for 72 hours;
5. one mouse in the control group died of bowel obstruction on the 4th day of experiment. Emaciation, decreacing of movement and diet were observed in the control group on the 3th week, and in the TCM group the 4th week. The mice of the control group got a tumour lump on their right epigastric zone in earlier time than those of the TCM group, on the 6th week of experiment the mice got emaciation, limitation of movement and camptocormia, then they were sacreficed;
6.The shape of tumours taken from the mice was round or oval, the cut surface looked like fish and had some ecptomas,and zone of necrosis was found in the center of some big tumours. The result of tumour weighing indicate that the TCM group have a higher inhibitory rate (54.82%) than the control group, the difference have a statistical significance(P<0.01)
7. The result of RT-PCR indicated that expession mRNA of P16 was lower in the control group than in the TCM group which was treated with XTSJF for six weeks;the difference has remarkable statistical significance(p<0.01))
8. The methylation level of P16 gene is detected by methylight method:hypermethylation were observed in the promoter region of P16 gene in control group (PMR>4); the methylation level were degraded after treated for 6 weeks with XTSJF decoction; the difference has remarkable statistical significance(p<0.01).
Conclusion:
1. Medical serum of XTSJF could depess the diffrentiation of gastric cancer cell lines;
2. Decoction of XTSJF could depress the tumorous growth in mouse models of human gastric carcinoma cell;
3. Both invivo and invitro study indicated that XTSJF could depress the tumorous growth by reversing DNA methylation and increasing the mRNA expession of P16 gene.
引文
[1].杨玲,李连弟.中国2000年及2005年恶性肿瘤发病死亡的估计与预测[J].中国卫生统计,2005；22(4),218-221.
[2].孙秀娣,牧人,周有尚等.中国胃癌死亡率20年变化情况分析及发展趋势预测[J].中华肿瘤杂志,2004；26(1)：4-9.
[3]. VanSpeybreeckL.From epigenesis to epigenetics:the case of C.H.Waddington[J]. Ann NY Acad Sci,2002,981:7-49.
[4]. Bird A. Perceptions of epigenetics[J]. Nature,2007,447(7143):396-398
[5]. Jones PA, Baylin SB. The fundamental role of epigenetic events incancer[J]. Nat Rev Genet, 2002;3(6):415-428.
[6]. Fuks F. DNA methylation and histone modifications:teaming up to silence genes[J]. Curt Opin Genet Dev,2005;15(5):490-495.
[7]. Robertson KD. DNA methylation and human disease[J]. Nat Rev Genet,2005;6(8): 597-610.
[8]. Wilson AS,Power BE,Molloy PL. DNA hypomethylation and human diseases[J]. Biochim Biophys Acta,2007;1775(1):138-162.
[9]. Gyeong Hoon Kang,Sun Lee,Nam-Yun Cho,et al.DNA methylation profiles of gastric carcinoma characterized by quantitative DNA methylation analysis[J]. Laboratory Investigation,2008;88:161-170.
[10]. Lund AH,van Lohuizen M.Epigenetics and cancer Genes [J]. Dev,2004;18:2315-2335.
[11]. Song SH, Jong HS,Choi HH, etc.Methylation of specific CpG sites in the promoter region could significantly down-regulate P16INK4a exprdssion in gastric adenomcarcinoma[J]. Int J Cancer,2000;87:236-240.
[12]. Shim YH,KangG H,RoJ Y. Correlation of P16 hypermethylation with P16 protein loss in sporadic gastric carcinomas[J].Lab Invest,2000;80:689-695.
[13]. SUN DanFeng, TIAN XiaoQing, ZHANG YanJie,etc. Mammalian target of rapamycin pathway inhibition enhances the effects of 5-aza-dC on suppressing cell proliferation in human gastric cancer cell lines[J]. Sci China SerC-Life Sci,2008;51 (7):640-647.
[14].栾菊,覃扬,徐丹等.中药成分CDP抑制肿瘤细胞生长及其去甲基化作用的研究[J].四川大学学报(医学版),2005；36(6)：834-838.
[1].朱曾柏.因毒致癌及解毒治癌探析[J].辽宁中医杂志,1994,21(3)：105.
[2].赵智强,李嘉.略论周仲瑛教授的“癌毒”学说及其临床运用[J].新中医,1998,30(10)：6.
[3].沈克平,郑坚,赵海磊.有瘤体必虚有虚首健脾(邱佳信治疗胃癌的研究思路)[J].医学理论与实践,1999,12(11)：625-629.
[4].伍全海,许延发.山西医结合治疗胃癌56例[J].陕西中医,1995,16(11)：498-499.
[5].陈四清.周仲瑛教授从癌毒辨治肿瘤经验[J].新中医,2004,36(2)：7.
[6].刘启泉,杜艳茹,王志坤等.解毒活血法治疗胃癌前病变临床研究[J].辽宁中医杂志,2005,32(6)：543.
[7].查雪良.中西医结合治疗晚期胃癌34例[J].山东中医药大学学报,1997,21(5)：376-377.
[8].李和根.刘嘉湘治疗胃癌经验述要[J].辽宁中医杂,2005,32(7)：642.
[9].胡国庆.藏象学说在胃癌辨治中的地位[J].河南中医药学刊,1996,11(1)：5-7
[10].宋恩峰,张新一,孙桂芝.防治胃癌复发转移的经验[J].湖北中医杂,1995,17(6)：4-8.
[11].魏品康,许玲,秦志丰,等.胃癌从痰论治的机理与临床研究[J].中国中医基础医学杂志,2002,8(3)：18-20.
[12].崔儒涛,谢建群,吴绚邦.胃癌前病变从痰瘀论治[J].浙江中医杂志,1998,33(10)：458-462.
[13].周岱翰.临床中医肿瘤学[M].北京：人民卫生出版社,2003：162.
[14].黄学武,罗群带.胃癌的中医辩治探析[J].新中医,2004,36(3)：5.
[15].代国平,杨艳霞.晚期胃癌的中西医姑息疗法[J].黑龙江中医药,2000,5：59.
[16].王琦,等.肥胖人痰湿型体质血液流变学和甲皱微循环研究[J].中国中医基础医学杂志,1995,1(1)：52
[17].方永奇,等.痰证的血液循环特征初探[J].湖北中医杂志,1992,(6)：33
[18].孙建芝,等.痰浊证微观辨证指标的实验研究[J].河南中医,1996,16(2)：21
[19].苏庆民,等.肥胖人痰湿型体质血脂、血糖、胰岛素及红细胞Na-K-ATP酶活性的检测及特征[J].中国中医基础医学杂志,1995,1(2)：39
[20].熊尚全,等.冠心病中医辨证与载脂蛋白关系的初步研究[J].福建中医学院学报,1994,4(3)：
[21].方永奇.痰证研究思路探讨[J].中国中医基础医学杂志,1996,2(6)：20
[22].潘桂娟.中医痰病研究与临床[c].北京：中国中医药出版社,1995.94
[23].李小兵,等.心脑血管病痰证患者免疫功能特点初探[J].中国中医基础医学杂志,1997,3(6)： 50
[24].徐伯平.痰瘀同源与癌瘤,中医杂志,1997,38(6)：378
[25].魏品康,许玲,秦志丰,等,胃癌从痰论治的机理与临床研究,中国中医基础医学杂 志,2002,(3)：18-20.
[26].李相勇,魏品康,金龙蛇口服液治疗晚期胃癌的疗效观察,湖北中医杂志,2001,23(11)3-5
[27].郭晓冬,魏品康,许玲,消痰散结方对裸鼠MKN-45胃腺癌组织中PCNA表达的影响,广州中医药大学学报,2000,17(2)：152-154
[28].王建平,李毅华,魏品康,许玲,消痰散结方对MKN-45人胃癌细胞黏附分子CD44V6表达的影响,山东中医药大学学报,2002,26(1)：69-70
[29].王建平,魏品康,许玲,耿怀成,消痰散结方对裸鼠MKN-45人胃腺癌组织中E-Cad表达的影响,中医研究,2002,15(2)：18-20
[30].肖艳,魏品康,尤清波,许玲,秦志丰,消痰散结方对裸鼠MKN-45人胃癌组织中MTl-MMP表达的影响,辽宁中医学院学报,2003,5(3)：271-272
[31].肖艳,魏品康,许玲,秦志丰,消痰散结方对胃癌基质金属蛋白酶表达的影响,中华实用中西医杂志,2005,11(18)：1035-1037
[32].苏晓妹,李峻,许玲,陈亚琳,郑国静,消痰散结方对鸡胚绒毛尿囊膜血管生成及胃癌组织中VEGF表达的影响,山东中医药大学学报,2003,27(4)：296-297
[33].陈亚琳,魏品康,许玲,苏晓妹,边莉,消痰散结方对胃癌组织中P21ras及P185蛋白表达的影响,山东中医药大学学报,2003,27(5)：374-376
[34]#12
[35].陈奇.中药药理研究方法.北京：人民卫生出版社,1993.75
[36].刘成海,刘平,刘成,等.抗肝纤维化有效中药复方血清药理学方法
[37].崔晓兰,贺玉琢,高英杰,等.中药复方血清药理研究方法学的探讨-11.中国实验方剂学杂志.1998,4(3)：45
[38].吴建宇,李仪毫符胜光等补阳还五汤保护自由墓损伤内皮细胞的血清药理实脸方法的建立[J).中药药理与临床,1999,15(1)：45-46
[30].蒙一纯,丁靛,责长恩,等.中药血清药理学应用研究展望.北京中医药大学学报,1999：22(4)：42-43
[40].李仪奎中药血清药理学实脸方法的若干问题田.中药断药与临床药理,1999,10(2)：95-98
[41].刘成海,刘平,刘成等.抗肝细胞纤维化有效中药复方血清药理学方法探讨[J].中国实脸方剂学杂志,1998,4(2)：16-19
[42].Wu MS, Shun CT, Sheu JC, et al. Overexpression of mutant p53 and cerbB-2 proteins and mutations of the p15 and P16 genes in human gastric carcinoma:with respect to histological subtypes and stages[J]. J Gastroenteml Hepatol,1998,13:305.
[43].KambA, GruisNA, Feldhaus WJ, et al. Acell cycle regulator potentially involved in genesis of many tumor types[J]. Science,1994,264:436.
[44].Gregory JH, David B. PI51NK4B is a potential effector of TGF-β-induced cell cyclear rest[J]. Nature,1994,371:257.
[45].Mannel S, Grego JH, David B. A new regulatory motif incelleyele control causing specific inhibition of cyclin D/CDK4. Nature,1993,366:704.
[46].Leung WK, Yu J, Ng EK, et.Concurrent hyperrnethylation of multiple tumor-related genes in gastric carcinoma and adjacent normal tissues[J]. Cancer,2001,91(12):2294-2301
[47]. Chen YT, Shih LS, Chen YM. Quantitative analysis ofCDKN2, p53 and retinoblastoma mRNA in human gastric carcinoma[J]. Int J Oncol。1998,13(2):249.
[48].王天宝,高鹏,曲延刚等.P15、P16在胃癌中表达及其意义[J].中国肿瘤临床与康复,2001,8(2)：23.
[49].缪林,赵志泉,季国忠等.胃癌前病变及胃癌组织中抑癌基因P16、Rb及周期素D-蛋白表达的研究[J].南京医科大学学报(自然科学版),2003,23(3)：272-274
[50].Kanai Y. Ushijima S, Nakanishi Y, et al. Mutation of the DNA methyltransrase(DNMT)1 gene in human colorectal cancers. Cancer Lett,2003,192(1):75-82.
[51]. Pogribny I, Raiche J. Slovack M, et al. Dose-dependence, sex and tissue-specificity, and persistence of radiation-induced genomic DNA methylation changes. Biochem Biophys Res Commun,2004,320:1253 1261
[52]. Gupta A, Godwin AK, Vanderveer 1, et al. Hypomethylation of the synucleingamma gene CpG island promotes its aberrant expression inbreast carcinoma and ovarian carcinoma. Cancer Res, 2003,63(3):664.673.
[53].Brooks JD, W einstein M, IinX, et al. CG island methylationchanges near the GSTP1 gene in prostatic intraepithelial neopla-sia. Cancer Epidemiol. Biom arkers Prev,1998,7:531-536
[54]. Chan AO, Rashid A. CpG island methylation in precursors ofgastrointestjnal malignancies. Curr MolMed,2006,6:401-408
[55].Anupam K, Tusharkant C, Gupta SD, et al. I OSS of disabled 2expression is an early event in esophageal squamous tumorigenesis. World J Gastroenterol,2006,12:6041-6045
[56].郭晓青,王士杰,张健慧.食管癌前病变组织P16及FHIT基因甲基化探讨.中国肿瘤临床,2005,32：554-557
[57].Kim TY, Lee HJ, Hwang KS, et al. Methylation of RUNX3 invarious types of human cancers and prem alignant stages of gas-tric carcinoma. Lab Invest.2004,84:479-484
[58].TsuchiyaT,Ta muraqSatoK,E ndohY,et al.Me ltz er S J.D istinctm ethylationp attemso ft woA
PC genep romotersi nn ormala ndcan ce rou s g as trice pithelia.O ncogene.2000; 19:3642-3646.
[59].F leisherA S,Esteller M,Wang S,et al. Hypermethylation of the hMLHl gene promoter in human gastric cancers with microsatelliteinstability.Cancer Res.1999;59:1090-1095.
[60].Machado JC, Oliveira C, CarvalhoR,et al.E-cadherin gene(CDHI)promoter methylation as the second hit insporadic difuse gastric carcinoma.Oncogene.2001;20:1525-1528.
[61].Satoshi Yamashita, Yoshimi Tsujino, Kazuki Moriguchi, et al. Chemical genomic screening for methylationsilencedgenes in gastric cancer cell lines using 5-aza-2'-deoxycytidine treatment andoligonucleotide microarray[J]. Cancer Sci.2006; 97 (1):64-71
[62].Gyeong Hoon Kang, Sun Lee, Nam-Yun Cho, et al.DNA methylation profiles of gastric carcinoma characterized byquantitative DNA methylation analysis.Laboratory Investigation.2008;88:161-170
[63].Hiranuma C, Kawakami K.Oyama K.et al. Hypermethylationof the MYOD1 gene is a novel prognostic factor in patients with colorectal cancer[J]. Int J Mol Med.2004,13(3):413-417.
[64].SUN DanFeng, TIAN XiaoQing, ZHANG YanJie, CHEN YingXuan, LU Rong, WANG Xia, FANG JingYuan. Mammalian target of rapamycin pathway inhibition enhances the effects of 5-aza-dC on suppressing cell proliferation in human gastric cancer cell lines. Sci China Ser C-Life Sci.2008;51[7]:640-647
[65].LeeYY,Kang SH,SeoJY,etal.Alterations of P16INK4a and p15INK4B genes ingastric carcinoma cancer,1997;80:1889.
[66].Shim YH, KangGH, RoJ (?) Correlation of P16 hypermetbylation with P16 Protein loss in sporatic gastric carcinoams.Labslnvest2000;80:689.
[67].BaiH, Gu L, ZhouJ, DengD.P16 hypermethylation during gastricCarcinomgenesis of Wistar rats by N-methyl-N-nitrosoguanidinc.Mutat Res2003;535(1):73.
[68]齐舯,李建芳,瞿颖等.胃癌及癌旁组织多个肿瘤相关基因超甲基化.上海交通大学学报(医学版)2007；27(5)：555-557
[69].栾菊,覃扬,徐丹等.中药成分CDP抑制肿瘤细胞生长及其去甲基化作用的研究.四川大学学报(医学版),2005；36(6)：834-836
[70].林一萍,陈比特,陈玉春.DNA甲基化酶与中医抗衰老机理的关系中国中医药信息杂志.1999,6(6)：18-19
[1]欧阳弘.中医药治疗中晚期胃癌99例临床疗效观察[J].中华实用中西医杂志,2009：22(5)：302-305
[2]朱曾柏.因毒致癌及解毒治癌探析[J].辽宁中医杂志,1994：21(3)：105.
[3]赵智强,李嘉.略论周仲瑛教授的“癌毒”学说及其临床运用[J].新中医,1998,
[4]何立丽.孙桂芝教授治疗胃癌的经验[J].北京中医药,2008：27(9)：689-691.
[5]查雪良.中西医结合治疗晚期胃癌34例[J].山东中医药大学学报,1997,21(5)：376-377.
[6]李和根.刘嘉湘治疗胃癌经验述要[J].辽宁中医杂,2005,32(7)：642.
[7]魏品康,许玲,秦志丰,等.胃癌从痰论治的机理与临床研究[J].中国中医基础医学杂志,2002,8(3)：18-20.
[8]许玲,孙大志,施俊,陈亚琳,魏品康.胃癌中医证型分类的文献研究.世界华人消化杂志2004：12：1457-1458
[9]吴水生,郑东海,林求诚.胃癌转移状态血液流变学变化与中医证型关系的临床研究.中国中西医结合杂志2000；20：583-585
[10]陈郭君.中医辨证分型治疗胃癌.湖北中医杂志2000；15：19
[11]陶炼.辨证治疗晚期胃癌40例Ⅰ临Ⅰ观察.湖南中医杂志2002；18：5-6
[12]钱心兰,钱力兰,钱伯文.攻补兼施治疗胃癌113例.上海中医药杂志1996；2：7.9
[13]张宗岐.临床肿瘤综合治疗大全.北京：奥林匹克出版社,1995：182-184
[14]许玲,魏品康,秦志丰等.胃癌中医证候特点分析767例.世界华人消化杂志.2005：13(15)：1804-1808
[15]陶丽,杨金坤.胃癌中医辨证分型的文献分析.中医药学刊.2006：24(4)：684-686
[16]赵爱光,蔡泳,杨金坤,郑坚,沈克平.健脾法为基础的中医药干预对胃癌预后的影响.世界华人消化杂志.2005：13(9)：1055-1058.
[17]曹雯,赵爱光.中药治疗胃癌的用药规律.中西医结合学报.2009：7(1)：1-7
[18]陶炼.辨证治疗晚期胃癌40例临床观察[J].湖南中医杂志,2002,18(2)：5
[19]魏品康,许玲,秦志丰,等.胃癌从痰论治的机理与临床研究[J].中国中医基础医学杂志,2002,8(3)：18-20
[20]陈翠花,刘爱学.中药在肿瘤治疗中的应用[J].河南中医,2004,24(3)：74-75
[21]田非,贾英杰,陈军,等.温针灸对于恶性肿瘤患者的免疫生物调控[J].针灸临床杂志,1999,15(5)：46.
[22]李俊,葛书翰.芒针提胃针法临床应用举隅[J].针灸临床杂志,2008,24(4)：14-15.
[23]李慧吉,武成.情志医学的研究[J].天津中医,2000.17(6)：37.
[24]杨振淮,袁小东,李关宁.参附注射液联合ELFP方案治疗晚期胃癌40例[J].中医研究,2005,18(7)：26.
[25]王纪东,王丽亚,李夏昀.中西医结合治疗中晚期胃癌92例临床分析[J].中国中西医结合杂志,2004,13(17)：2281.
[26]徐长朴.慢性胃炎、肠化不典型增生随访观察.中华消化病杂志,1984：4(1)：10
[27 FarinatioF el al, Follow-up in gastric dyspla-sia patients. Am j Surg Pathol.1989:13(2):173
[28]李志民.消痞汤治疗慢性萎缩性胃炎(胃癌前期病变)105例临床观察.新中医,1998：30(10)：18
[29]李俊柳,胡冬菊。黄梅淑.清热养阴降浊冲剂治疗胃癌前病变60例[J].陕西中医。2005,26(9)：885-886.
[30]胡玲,劳绍贤,王建华.胃炎消对胃癌癌前病变bcl_2癌基因mRNA表达的影响[J].上海中医药杂志,2006,40(1)：27-28.
[31]崔儒涛等.蔡诠逆转胃癌前病变的经验,中医杂志,1999：40(7)：398
[32]张文尧.中药对慢性萎缩性胃炎伴胃粘膜不典型增生患者的临床及实验研究.中医杂志,1998：39(8)：481
[33]张旭晨.细胞逆转丸治疗胃癌前期病变的临床与实验研究,新消化病杂志,1997：4：216
[34]刘兴山,杨会香,方红.根幽愈疡胶囊治疗幽门螺杆菌相关性消化性溃疡100例临床研究[J].中医杂志,2005,46(7)：514.
[35]周留勇,单珍珠,尤建良.六君子汤配伍藤梨根治疗晚期胃癌107例[J].四川中医,2005,23(11)：41-42.
[36]尤建良,周留勇,徐明,等.中药微调三号合剂治疗晚期胃癌的临床研究[J].湖北中医杂药卫生志,2004,26(6)：8-9.
[37]宋家驹.健脾中药抗胃癌术后转移的研究[J].现代中西医结合杂志,2004,13(22)：2956-2957.
[38]李相勇,魏品康.金龙蛇口服液治疗晚期胃癌的疗效观察[J].湖北中医杂志,2001,23(11)：3-4.
[39]陈培丰,孙维刚.清热消积冲剂治疗胃癌的临床疗效观察[J].辽宁中医杂志,2003,30(1)： 39-40.
[40]李财宝,龚航军.149例胃癌术后化疗与中药综合治疗临床观察[J].上海中医药杂志,2002(2)：15.
[41]王纪东,王丽亚,李夏昀.中西医结合治疗中晚期胃癌92例临床分析[J].现代中西医结合杂志,2004。13(17)：2281.
[42]陈郭君,薛曼朗,王先菊.脾肾方对晚期胃癌化疗中减毒作用的临床研究[J].湖北中医杂志,2001,23(7)：9-10.
[43]宁廷禄.平消胶囊联合化疗治疗进展期胃癌121例[J].中国中西医结合杂志,1998,18(6)：370.
[44]李敏.大承气汤加味治疗胃癌术后合并粘连性肠梗阻100例临床观察[J].医用放射技术杂志,2004,2：22.
[45]俞企望,李世宏.升阳益胃汤治疗胃癌术后消化不良39例观察[J].实用中医药杂志,2005,21(70)：397.
[46]李斌,宋明全.四君子汤含药血清对SGC-7901细胞株凋亡和相关蛋白的影响[J]山东中医杂志,2008,27(4)：262-265
[47]庄严,董青.新加良附颗粒含药血清体外诱导细胞凋亡凋亡研究[J].中国实验方剂学杂志,2007,13(8)：23
[48]赵爱光,杨金砷,尤圣富,等.中药胃肠安诱导裸鼠胃癌移植瘤细胞凋亡的途径和基因调控的研究[J].中西医结合学报,2007,5(3)：287-297
[49]Kerbel RS. Tumor angiogenesis past,present and the near future[J] Carcinogenesis,2000,21 (3):505-515
[50]颜大海,朱树林.鸡胚法筛选具有抗血管生成作用的中药[J].黑龙江医药,1998,11(2)：94
[51]许玲,魏品康,程亚琳,等.中药消痰散结方抑制人胃癌裸鼠原位移植瘤的人胃癌SGC7901的生长转移[J].世界华人消化杂志,2004,12(5)：1015-1020
[52]孙波,吴云林,王升年,等.鸦胆子油乳抗人胃癌增殖作用的初步研究[J].上海医学,2001,24(8)：481-483
[53]王加保,俞建平,章晓.参麦注射液对抗胃肠道恶性肿瘤术后化疗白细胞水平下降的研究[J].浙江中医学院学报,2003,27(4)：59
[1].Chen RZ, Pettersson U. Beard C, et al. DNA hypomethylationleads to elevated mutation rates. Nature,1998; 395:89-93
[2].Amir E. Francois G, Alpana W, et al. Chromosomal Instability and Tumors Promoted by DNA Hypomethylation. Science,2003; 56(18):455.
[3].Gupta A, Godwin AK, Vandmweer L, et al. Hypomethylation of the synuelein gene CpG island promotes its aberrant expression in breast carcinoma and ovarian carcinoma Cancer Res,2003,63:664-673.
[4].Shim YH,KangG H,RoJ Y. Correlation of P16 hypermethylation with P16 protein loss in sporadic gastric carcinomas.Lab Invest.2000;80:689-695.
[5].TsuchiyaT,Ta muraqSatoK,E ndohY,et al.Me ltz er S J.D istinct methylationp at temsoft wo APC gene promoters in noral and cancerous gastric epithelia. Oncogene. 2000;19:3642-3646.
[6].F leisherA S,Esteller M,Wang S,et al. Hypermethylation of the hMLH1 gene promoter in human gastric cancers with microsatelliteinstability.Cancer Res.1999;59:1090-1095.
[7].Machado JC, Oliveira C, CarvalhoR,et al.E-cadherin gene(CDHI)promoter methylation as the second hit insporadic difuse gastric carcinoma.Oncogene.2001;20:1525-1528.
[8].KangG H,L eeS,Kim JS,JungH Y.Profile of aberrant CpG island methylation along theMultistep pathway of gastric carcinogenesis.Lab Invest,2003;83(5):635
[9].HongSH,KimHG,ChungWB,et al.DNA hypermethylation of tumor-related genes in gastriccarcinoma.J Korean Med Sci,2005;20(2):236
[10].Satoshi Yamashita, Yoshimi Tsujino, Kazuki Moriguchi, et al. Chemical genomic screening for methylationsilencedgenes in gastric cancer cell lines using5-aza-2'-deoxycytidine treatment andoligonucleotide microarray[J]. Cancer Sci.2006; 97 (1):64-71
[11].Gyeong Hoon Kang, Sun Lee, Nam-Yun Cho, et al.DNA methylation profiles of gastric carcinoma characterized byquantitative DNA methylation analysis.Laboratory Investigation.2008;88:161-170
[12].Costello JF, Fruhwald MC, Smiraglia DJ et al. Aberrant CpG-island methylation has non-random and tumour-type-specific patterns. NatGenet 2000; 24:132-8.
[13].Eden A, Gaudet F, Waghmare A, et al. Chromosomal instability andtumors promoted by DNA hypomethylation. Science,2003,300(5618):455.
[14].Florl AR,Steinhof C, Muller M,et al. Coordinate hypermethylation atspecific genes in prostate carcinoma precedes LINE-1 hypomethylation. Br J Cancer,2004,91(5):985-994
[15].Feinberg AP, Tycko B. The history of cancer epigenetics. Nat RevCancer,2004, 4(2):143-153
[16].Seog-Yun Park,l Eun J Yoo,2 Nam-Yun Cho,et al.Comparison of CpG island hypermethylation andrepetitive DNA hypomethylation in remalignant stagesof gastric cancer, stratified for Helicobacter pylori infection. Journal of Pathology,2009
[17].Scelfo RA, Schwienbacher C, Veronese A, et al. Loss of methylation at chromosome 11p15.5 is common in human adult tumors. Oncogene,2002,21(16):2564-2572.
[18].Nishigaki M, Aoyagi K, Danjoh I, et al. Discovery ofaberrant expression of R-RAS by can cer-linked DNA hypomethylation in gastric cancer using microarrays. Cancer Res,2005, 65(6):2115-2124
[19].ToK F,LeungW K,LeeT L,Yu J,TongJ H,ChanM W,NgEK,ChungSC,SugJJ.Promoter hypermethylation of tumor-related genes in gastric intestinal metaplasia of patients with and without gastric cancer.intJ Cancer.2002; 102:623-628.
[20].Leung WK, YuJ, NgEK,et al.. Concurrent hypermethylation of multiple tumor-related genes in gastric carcinoma and adjacent normal tissues. Cancer.2001; 91:2294-2301.
[21].Rintaro Mikata, Osamu Yokosuka, et al. Analysis of genes upregulated by the demethylating agent 5-aza-2-deoxycytidine in gastric cancer cell linesInt. J. Cancer:2006; 119:1616-1622.
[22].Eun Joo Yoo,Seog-Yun Park,Nam-Yun Cho,Nayoung Kim,Hye Seung Lee,Gyeong Hoon Kang. Helicobacter pylori-infection-associated CpG island hypermethylation in the stomach and its possibleassociation with Polycomb repressive marks. Virchows Arch 2008;452:515-524
[23].Kang GH, Lee S, Kim WH et al. Epstein-Barr virus-positive gastriccarcinoma demonstrates frequent aberrant methylation of multiple genes and constitutes CpG island methylator phenotype-positive gastric carcinoma. Am J Pathol 2002; 160:787-94.
[24].Enomoto S, Maekita T, Tsukamoto T,et al. Lack of association between CpG island methylator phenotype in human gastric cancers and methylation in their background non-cancerous gastric mucosae. Cancer Sci 2007; 98:1853-61.
[25].Kaneda A, Kaminishi M, Yanagihara K, Sugimura T, Ushijima T. Identification of silencing of nine genes in human gastric cancers. Cancer Res 2002; 62:6645-50.
[26].Ushiku T, Chong JM, Uozaki H et al.p73 gene promoter methylation in Epstein-Barr virus-associated gastric carcinoma. Int J Cancer 2007; 120:60-6.
[27].Sudo M, Chong JM, Sakuma K et al. Promoter hypermethylation of Ecadherin and its abnormal expression in Epstein-Barr virus-associated gastric carcinoma. Int J Cancer 2004; 109:194-9.
[28].Li H, Minarovits J. Host cell-dependent expression of latent Epstein-Barr virus genomes: regulation by DNA methylation. Adv Cancer Res 2003; 89:133-56.
[29].Lieberman PM. Chromatin organization and virus gene expression. J Cell Physiol 2008; 216: 295-302.
[30].Hiranuma C, Kawakami K.Oyama K.et al. Hypermethylationof the MYOD1 gene is a novel prognostic factor in patients with colorectal cancer[J]. Int J Mol Med.2004,13(3):413-417.
[31].Chan M W, Chu E S, To K F. et al. Quantitative detection of methylated SOCk-1, a tumor suppressor gene, by a modified protocol of quantitative real time methylation-specific PCR using SYBR green and its use in early gastric cancer detection[J]. Biotechnol Lett.2004.26(16):1289-1293.
[32].House M G , Guo M, Efron D T, et al. Tumor suppressor gene hyperm ethylation as a predictor of gastric stromal tumor behavior[J]. J Gastrointest Surg,2003, 7(8):1004 1014
[33].SUN DanFeng, TIAN XiaoQing, ZHANG YanJie, CHEN YingXuan, LU Rong, WANG Xia, FANG JingYuan. Mammalian target of rapamycin pathway inhibition enhances the effects of 5-aza-dC on suppressing cell proliferation in human gastric cancer cell lines. Sci China Ser C-Life Sci.2008;51[7]:640-647
[2].孙秀娣,牧人,周有尚等.中国胃癌死亡率20年变化情况分析及发展趋势预测[J].中华肿瘤杂志,2004；26(1)：4-9.
[3]. VanSpeybreeckL.From epigenesis to epigenetics:the case of C.H.Waddington[J]. Ann NY Acad Sci,2002,981:7-49.
[4]. Bird A. Perceptions of epigenetics[J]. Nature,2007,447(7143):396-398
[5]. Jones PA, Baylin SB. The fundamental role of epigenetic events incancer[J]. Nat Rev Genet, 2002;3(6):415-428.
[6]. Fuks F. DNA methylation and histone modifications:teaming up to silence genes[J]. Curt Opin Genet Dev,2005;15(5):490-495.
[7]. Robertson KD. DNA methylation and human disease[J]. Nat Rev Genet,2005;6(8): 597-610.
[8]. Wilson AS,Power BE,Molloy PL. DNA hypomethylation and human diseases[J]. Biochim Biophys Acta,2007;1775(1):138-162.
[9]. Gyeong Hoon Kang,Sun Lee,Nam-Yun Cho,et al.DNA methylation profiles of gastric carcinoma characterized by quantitative DNA methylation analysis[J]. Laboratory Investigation,2008;88:161-170.
[10]. Lund AH,van Lohuizen M.Epigenetics and cancer Genes [J]. Dev,2004;18:2315-2335.
[11]. Song SH, Jong HS,Choi HH, etc.Methylation of specific CpG sites in the promoter region could significantly down-regulate P16INK4a exprdssion in gastric adenomcarcinoma[J]. Int J Cancer,2000;87:236-240.
[12]. Shim YH,KangG H,RoJ Y. Correlation of P16 hypermethylation with P16 protein loss in sporadic gastric carcinomas[J].Lab Invest,2000;80:689-695.
[13]. SUN DanFeng, TIAN XiaoQing, ZHANG YanJie,etc. Mammalian target of rapamycin pathway inhibition enhances the effects of 5-aza-dC on suppressing cell proliferation in human gastric cancer cell lines[J]. Sci China SerC-Life Sci,2008;51 (7):640-647.
[14].栾菊,覃扬,徐丹等.中药成分CDP抑制肿瘤细胞生长及其去甲基化作用的研究[J].四川大学学报(医学版),2005；36(6)：834-838.
[1].朱曾柏.因毒致癌及解毒治癌探析[J].辽宁中医杂志,1994,21(3)：105.
[2].赵智强,李嘉.略论周仲瑛教授的“癌毒”学说及其临床运用[J].新中医,1998,30(10)：6.
[3].沈克平,郑坚,赵海磊.有瘤体必虚有虚首健脾(邱佳信治疗胃癌的研究思路)[J].医学理论与实践,1999,12(11)：625-629.
[4].伍全海,许延发.山西医结合治疗胃癌56例[J].陕西中医,1995,16(11)：498-499.
[5].陈四清.周仲瑛教授从癌毒辨治肿瘤经验[J].新中医,2004,36(2)：7.
[6].刘启泉,杜艳茹,王志坤等.解毒活血法治疗胃癌前病变临床研究[J].辽宁中医杂志,2005,32(6)：543.
[7].查雪良.中西医结合治疗晚期胃癌34例[J].山东中医药大学学报,1997,21(5)：376-377.
[8].李和根.刘嘉湘治疗胃癌经验述要[J].辽宁中医杂,2005,32(7)：642.
[9].胡国庆.藏象学说在胃癌辨治中的地位[J].河南中医药学刊,1996,11(1)：5-7
[10].宋恩峰,张新一,孙桂芝.防治胃癌复发转移的经验[J].湖北中医杂,1995,17(6)：4-8.
[11].魏品康,许玲,秦志丰,等.胃癌从痰论治的机理与临床研究[J].中国中医基础医学杂志,2002,8(3)：18-20.
[12].崔儒涛,谢建群,吴绚邦.胃癌前病变从痰瘀论治[J].浙江中医杂志,1998,33(10)：458-462.
[13].周岱翰.临床中医肿瘤学[M].北京：人民卫生出版社,2003：162.
[14].黄学武,罗群带.胃癌的中医辩治探析[J].新中医,2004,36(3)：5.
[15].代国平,杨艳霞.晚期胃癌的中西医姑息疗法[J].黑龙江中医药,2000,5：59.
[16].王琦,等.肥胖人痰湿型体质血液流变学和甲皱微循环研究[J].中国中医基础医学杂志,1995,1(1)：52
[17].方永奇,等.痰证的血液循环特征初探[J].湖北中医杂志,1992,(6)：33
[18].孙建芝,等.痰浊证微观辨证指标的实验研究[J].河南中医,1996,16(2)：21
[19].苏庆民,等.肥胖人痰湿型体质血脂、血糖、胰岛素及红细胞Na-K-ATP酶活性的检测及特征[J].中国中医基础医学杂志,1995,1(2)：39
[20].熊尚全,等.冠心病中医辨证与载脂蛋白关系的初步研究[J].福建中医学院学报,1994,4(3)：
[21].方永奇.痰证研究思路探讨[J].中国中医基础医学杂志,1996,2(6)：20
[22].潘桂娟.中医痰病研究与临床[c].北京：中国中医药出版社,1995.94
[23].李小兵,等.心脑血管病痰证患者免疫功能特点初探[J].中国中医基础医学杂志,1997,3(6)： 50
[24].徐伯平.痰瘀同源与癌瘤,中医杂志,1997,38(6)：378
[25].魏品康,许玲,秦志丰,等,胃癌从痰论治的机理与临床研究,中国中医基础医学杂 志,2002,(3)：18-20.
[26].李相勇,魏品康,金龙蛇口服液治疗晚期胃癌的疗效观察,湖北中医杂志,2001,23(11)3-5
[27].郭晓冬,魏品康,许玲,消痰散结方对裸鼠MKN-45胃腺癌组织中PCNA表达的影响,广州中医药大学学报,2000,17(2)：152-154
[28].王建平,李毅华,魏品康,许玲,消痰散结方对MKN-45人胃癌细胞黏附分子CD44V6表达的影响,山东中医药大学学报,2002,26(1)：69-70
[29].王建平,魏品康,许玲,耿怀成,消痰散结方对裸鼠MKN-45人胃腺癌组织中E-Cad表达的影响,中医研究,2002,15(2)：18-20
[30].肖艳,魏品康,尤清波,许玲,秦志丰,消痰散结方对裸鼠MKN-45人胃癌组织中MTl-MMP表达的影响,辽宁中医学院学报,2003,5(3)：271-272
[31].肖艳,魏品康,许玲,秦志丰,消痰散结方对胃癌基质金属蛋白酶表达的影响,中华实用中西医杂志,2005,11(18)：1035-1037
[32].苏晓妹,李峻,许玲,陈亚琳,郑国静,消痰散结方对鸡胚绒毛尿囊膜血管生成及胃癌组织中VEGF表达的影响,山东中医药大学学报,2003,27(4)：296-297
[33].陈亚琳,魏品康,许玲,苏晓妹,边莉,消痰散结方对胃癌组织中P21ras及P185蛋白表达的影响,山东中医药大学学报,2003,27(5)：374-376
[34]#12
[35].陈奇.中药药理研究方法.北京：人民卫生出版社,1993.75
[36].刘成海,刘平,刘成,等.抗肝纤维化有效中药复方血清药理学方法
[37].崔晓兰,贺玉琢,高英杰,等.中药复方血清药理研究方法学的探讨-11.中国实验方剂学杂志.1998,4(3)：45
[38].吴建宇,李仪毫符胜光等补阳还五汤保护自由墓损伤内皮细胞的血清药理实脸方法的建立[J).中药药理与临床,1999,15(1)：45-46
[30].蒙一纯,丁靛,责长恩,等.中药血清药理学应用研究展望.北京中医药大学学报,1999：22(4)：42-43
[40].李仪奎中药血清药理学实脸方法的若干问题田.中药断药与临床药理,1999,10(2)：95-98
[41].刘成海,刘平,刘成等.抗肝细胞纤维化有效中药复方血清药理学方法探讨[J].中国实脸方剂学杂志,1998,4(2)：16-19
[42].Wu MS, Shun CT, Sheu JC, et al. Overexpression of mutant p53 and cerbB-2 proteins and mutations of the p15 and P16 genes in human gastric carcinoma:with respect to histological subtypes and stages[J]. J Gastroenteml Hepatol,1998,13:305.
[43].KambA, GruisNA, Feldhaus WJ, et al. Acell cycle regulator potentially involved in genesis of many tumor types[J]. Science,1994,264:436.
[44].Gregory JH, David B. PI51NK4B is a potential effector of TGF-β-induced cell cyclear rest[J]. Nature,1994,371:257.
[45].Mannel S, Grego JH, David B. A new regulatory motif incelleyele control causing specific inhibition of cyclin D/CDK4. Nature,1993,366:704.
[46].Leung WK, Yu J, Ng EK, et.Concurrent hyperrnethylation of multiple tumor-related genes in gastric carcinoma and adjacent normal tissues[J]. Cancer,2001,91(12):2294-2301
[47]. Chen YT, Shih LS, Chen YM. Quantitative analysis ofCDKN2, p53 and retinoblastoma mRNA in human gastric carcinoma[J]. Int J Oncol。1998,13(2):249.
[48].王天宝,高鹏,曲延刚等.P15、P16在胃癌中表达及其意义[J].中国肿瘤临床与康复,2001,8(2)：23.
[49].缪林,赵志泉,季国忠等.胃癌前病变及胃癌组织中抑癌基因P16、Rb及周期素D-蛋白表达的研究[J].南京医科大学学报(自然科学版),2003,23(3)：272-274
[50].Kanai Y. Ushijima S, Nakanishi Y, et al. Mutation of the DNA methyltransrase(DNMT)1 gene in human colorectal cancers. Cancer Lett,2003,192(1):75-82.
[51]. Pogribny I, Raiche J. Slovack M, et al. Dose-dependence, sex and tissue-specificity, and persistence of radiation-induced genomic DNA methylation changes. Biochem Biophys Res Commun,2004,320:1253 1261
[52]. Gupta A, Godwin AK, Vanderveer 1, et al. Hypomethylation of the synucleingamma gene CpG island promotes its aberrant expression inbreast carcinoma and ovarian carcinoma. Cancer Res, 2003,63(3):664.673.
[53].Brooks JD, W einstein M, IinX, et al. CG island methylationchanges near the GSTP1 gene in prostatic intraepithelial neopla-sia. Cancer Epidemiol. Biom arkers Prev,1998,7:531-536
[54]. Chan AO, Rashid A. CpG island methylation in precursors ofgastrointestjnal malignancies. Curr MolMed,2006,6:401-408
[55].Anupam K, Tusharkant C, Gupta SD, et al. I OSS of disabled 2expression is an early event in esophageal squamous tumorigenesis. World J Gastroenterol,2006,12:6041-6045
[56].郭晓青,王士杰,张健慧.食管癌前病变组织P16及FHIT基因甲基化探讨.中国肿瘤临床,2005,32：554-557
[57].Kim TY, Lee HJ, Hwang KS, et al. Methylation of RUNX3 invarious types of human cancers and prem alignant stages of gas-tric carcinoma. Lab Invest.2004,84:479-484
[58].TsuchiyaT,Ta muraqSatoK,E ndohY,et al.Me ltz er S J.D istinctm ethylationp attemso ft woA
PC genep romotersi nn ormala ndcan ce rou s g as trice pithelia.O ncogene.2000; 19:3642-3646.
[59].F leisherA S,Esteller M,Wang S,et al. Hypermethylation of the hMLHl gene promoter in human gastric cancers with microsatelliteinstability.Cancer Res.1999;59:1090-1095.
[60].Machado JC, Oliveira C, CarvalhoR,et al.E-cadherin gene(CDHI)promoter methylation as the second hit insporadic difuse gastric carcinoma.Oncogene.2001;20:1525-1528.
[61].Satoshi Yamashita, Yoshimi Tsujino, Kazuki Moriguchi, et al. Chemical genomic screening for methylationsilencedgenes in gastric cancer cell lines using 5-aza-2'-deoxycytidine treatment andoligonucleotide microarray[J]. Cancer Sci.2006; 97 (1):64-71
[62].Gyeong Hoon Kang, Sun Lee, Nam-Yun Cho, et al.DNA methylation profiles of gastric carcinoma characterized byquantitative DNA methylation analysis.Laboratory Investigation.2008;88:161-170
[63].Hiranuma C, Kawakami K.Oyama K.et al. Hypermethylationof the MYOD1 gene is a novel prognostic factor in patients with colorectal cancer[J]. Int J Mol Med.2004,13(3):413-417.
[64].SUN DanFeng, TIAN XiaoQing, ZHANG YanJie, CHEN YingXuan, LU Rong, WANG Xia, FANG JingYuan. Mammalian target of rapamycin pathway inhibition enhances the effects of 5-aza-dC on suppressing cell proliferation in human gastric cancer cell lines. Sci China Ser C-Life Sci.2008;51[7]:640-647
[65].LeeYY,Kang SH,SeoJY,etal.Alterations of P16INK4a and p15INK4B genes ingastric carcinoma cancer,1997;80:1889.
[66].Shim YH, KangGH, RoJ (?) Correlation of P16 hypermetbylation with P16 Protein loss in sporatic gastric carcinoams.Labslnvest2000;80:689.
[67].BaiH, Gu L, ZhouJ, DengD.P16 hypermethylation during gastricCarcinomgenesis of Wistar rats by N-methyl-N-nitrosoguanidinc.Mutat Res2003;535(1):73.
[68]齐舯,李建芳,瞿颖等.胃癌及癌旁组织多个肿瘤相关基因超甲基化.上海交通大学学报(医学版)2007；27(5)：555-557
[69].栾菊,覃扬,徐丹等.中药成分CDP抑制肿瘤细胞生长及其去甲基化作用的研究.四川大学学报(医学版),2005；36(6)：834-836
[70].林一萍,陈比特,陈玉春.DNA甲基化酶与中医抗衰老机理的关系中国中医药信息杂志.1999,6(6)：18-19
[1]欧阳弘.中医药治疗中晚期胃癌99例临床疗效观察[J].中华实用中西医杂志,2009：22(5)：302-305
[2]朱曾柏.因毒致癌及解毒治癌探析[J].辽宁中医杂志,1994：21(3)：105.
[3]赵智强,李嘉.略论周仲瑛教授的“癌毒”学说及其临床运用[J].新中医,1998,
[4]何立丽.孙桂芝教授治疗胃癌的经验[J].北京中医药,2008：27(9)：689-691.
[5]查雪良.中西医结合治疗晚期胃癌34例[J].山东中医药大学学报,1997,21(5)：376-377.
[6]李和根.刘嘉湘治疗胃癌经验述要[J].辽宁中医杂,2005,32(7)：642.
[7]魏品康,许玲,秦志丰,等.胃癌从痰论治的机理与临床研究[J].中国中医基础医学杂志,2002,8(3)：18-20.
[8]许玲,孙大志,施俊,陈亚琳,魏品康.胃癌中医证型分类的文献研究.世界华人消化杂志2004：12：1457-1458
[9]吴水生,郑东海,林求诚.胃癌转移状态血液流变学变化与中医证型关系的临床研究.中国中西医结合杂志2000；20：583-585
[10]陈郭君.中医辨证分型治疗胃癌.湖北中医杂志2000；15：19
[11]陶炼.辨证治疗晚期胃癌40例Ⅰ临Ⅰ观察.湖南中医杂志2002；18：5-6
[12]钱心兰,钱力兰,钱伯文.攻补兼施治疗胃癌113例.上海中医药杂志1996；2：7.9
[13]张宗岐.临床肿瘤综合治疗大全.北京：奥林匹克出版社,1995：182-184
[14]许玲,魏品康,秦志丰等.胃癌中医证候特点分析767例.世界华人消化杂志.2005：13(15)：1804-1808
[15]陶丽,杨金坤.胃癌中医辨证分型的文献分析.中医药学刊.2006：24(4)：684-686
[16]赵爱光,蔡泳,杨金坤,郑坚,沈克平.健脾法为基础的中医药干预对胃癌预后的影响.世界华人消化杂志.2005：13(9)：1055-1058.
[17]曹雯,赵爱光.中药治疗胃癌的用药规律.中西医结合学报.2009：7(1)：1-7
[18]陶炼.辨证治疗晚期胃癌40例临床观察[J].湖南中医杂志,2002,18(2)：5
[19]魏品康,许玲,秦志丰,等.胃癌从痰论治的机理与临床研究[J].中国中医基础医学杂志,2002,8(3)：18-20
[20]陈翠花,刘爱学.中药在肿瘤治疗中的应用[J].河南中医,2004,24(3)：74-75
[21]田非,贾英杰,陈军,等.温针灸对于恶性肿瘤患者的免疫生物调控[J].针灸临床杂志,1999,15(5)：46.
[22]李俊,葛书翰.芒针提胃针法临床应用举隅[J].针灸临床杂志,2008,24(4)：14-15.
[23]李慧吉,武成.情志医学的研究[J].天津中医,2000.17(6)：37.
[24]杨振淮,袁小东,李关宁.参附注射液联合ELFP方案治疗晚期胃癌40例[J].中医研究,2005,18(7)：26.
[25]王纪东,王丽亚,李夏昀.中西医结合治疗中晚期胃癌92例临床分析[J].中国中西医结合杂志,2004,13(17)：2281.
[26]徐长朴.慢性胃炎、肠化不典型增生随访观察.中华消化病杂志,1984：4(1)：10
[27 FarinatioF el al, Follow-up in gastric dyspla-sia patients. Am j Surg Pathol.1989:13(2):173
[28]李志民.消痞汤治疗慢性萎缩性胃炎(胃癌前期病变)105例临床观察.新中医,1998：30(10)：18
[29]李俊柳,胡冬菊。黄梅淑.清热养阴降浊冲剂治疗胃癌前病变60例[J].陕西中医。2005,26(9)：885-886.
[30]胡玲,劳绍贤,王建华.胃炎消对胃癌癌前病变bcl_2癌基因mRNA表达的影响[J].上海中医药杂志,2006,40(1)：27-28.
[31]崔儒涛等.蔡诠逆转胃癌前病变的经验,中医杂志,1999：40(7)：398
[32]张文尧.中药对慢性萎缩性胃炎伴胃粘膜不典型增生患者的临床及实验研究.中医杂志,1998：39(8)：481
[33]张旭晨.细胞逆转丸治疗胃癌前期病变的临床与实验研究,新消化病杂志,1997：4：216
[34]刘兴山,杨会香,方红.根幽愈疡胶囊治疗幽门螺杆菌相关性消化性溃疡100例临床研究[J].中医杂志,2005,46(7)：514.
[35]周留勇,单珍珠,尤建良.六君子汤配伍藤梨根治疗晚期胃癌107例[J].四川中医,2005,23(11)：41-42.
[36]尤建良,周留勇,徐明,等.中药微调三号合剂治疗晚期胃癌的临床研究[J].湖北中医杂药卫生志,2004,26(6)：8-9.
[37]宋家驹.健脾中药抗胃癌术后转移的研究[J].现代中西医结合杂志,2004,13(22)：2956-2957.
[38]李相勇,魏品康.金龙蛇口服液治疗晚期胃癌的疗效观察[J].湖北中医杂志,2001,23(11)：3-4.
[39]陈培丰,孙维刚.清热消积冲剂治疗胃癌的临床疗效观察[J].辽宁中医杂志,2003,30(1)： 39-40.
[40]李财宝,龚航军.149例胃癌术后化疗与中药综合治疗临床观察[J].上海中医药杂志,2002(2)：15.
[41]王纪东,王丽亚,李夏昀.中西医结合治疗中晚期胃癌92例临床分析[J].现代中西医结合杂志,2004。13(17)：2281.
[42]陈郭君,薛曼朗,王先菊.脾肾方对晚期胃癌化疗中减毒作用的临床研究[J].湖北中医杂志,2001,23(7)：9-10.
[43]宁廷禄.平消胶囊联合化疗治疗进展期胃癌121例[J].中国中西医结合杂志,1998,18(6)：370.
[44]李敏.大承气汤加味治疗胃癌术后合并粘连性肠梗阻100例临床观察[J].医用放射技术杂志,2004,2：22.
[45]俞企望,李世宏.升阳益胃汤治疗胃癌术后消化不良39例观察[J].实用中医药杂志,2005,21(70)：397.
[46]李斌,宋明全.四君子汤含药血清对SGC-7901细胞株凋亡和相关蛋白的影响[J]山东中医杂志,2008,27(4)：262-265
[47]庄严,董青.新加良附颗粒含药血清体外诱导细胞凋亡凋亡研究[J].中国实验方剂学杂志,2007,13(8)：23
[48]赵爱光,杨金砷,尤圣富,等.中药胃肠安诱导裸鼠胃癌移植瘤细胞凋亡的途径和基因调控的研究[J].中西医结合学报,2007,5(3)：287-297
[49]Kerbel RS. Tumor angiogenesis past,present and the near future[J] Carcinogenesis,2000,21 (3):505-515
[50]颜大海,朱树林.鸡胚法筛选具有抗血管生成作用的中药[J].黑龙江医药,1998,11(2)：94
[51]许玲,魏品康,程亚琳,等.中药消痰散结方抑制人胃癌裸鼠原位移植瘤的人胃癌SGC7901的生长转移[J].世界华人消化杂志,2004,12(5)：1015-1020
[52]孙波,吴云林,王升年,等.鸦胆子油乳抗人胃癌增殖作用的初步研究[J].上海医学,2001,24(8)：481-483
[53]王加保,俞建平,章晓.参麦注射液对抗胃肠道恶性肿瘤术后化疗白细胞水平下降的研究[J].浙江中医学院学报,2003,27(4)：59
[1].Chen RZ, Pettersson U. Beard C, et al. DNA hypomethylationleads to elevated mutation rates. Nature,1998; 395:89-93
[2].Amir E. Francois G, Alpana W, et al. Chromosomal Instability and Tumors Promoted by DNA Hypomethylation. Science,2003; 56(18):455.
[3].Gupta A, Godwin AK, Vandmweer L, et al. Hypomethylation of the synuelein gene CpG island promotes its aberrant expression in breast carcinoma and ovarian carcinoma Cancer Res,2003,63:664-673.
[4].Shim YH,KangG H,RoJ Y. Correlation of P16 hypermethylation with P16 protein loss in sporadic gastric carcinomas.Lab Invest.2000;80:689-695.
[5].TsuchiyaT,Ta muraqSatoK,E ndohY,et al.Me ltz er S J.D istinct methylationp at temsoft wo APC gene promoters in noral and cancerous gastric epithelia. Oncogene. 2000;19:3642-3646.
[6].F leisherA S,Esteller M,Wang S,et al. Hypermethylation of the hMLH1 gene promoter in human gastric cancers with microsatelliteinstability.Cancer Res.1999;59:1090-1095.
[7].Machado JC, Oliveira C, CarvalhoR,et al.E-cadherin gene(CDHI)promoter methylation as the second hit insporadic difuse gastric carcinoma.Oncogene.2001;20:1525-1528.
[8].KangG H,L eeS,Kim JS,JungH Y.Profile of aberrant CpG island methylation along theMultistep pathway of gastric carcinogenesis.Lab Invest,2003;83(5):635
[9].HongSH,KimHG,ChungWB,et al.DNA hypermethylation of tumor-related genes in gastriccarcinoma.J Korean Med Sci,2005;20(2):236
[10].Satoshi Yamashita, Yoshimi Tsujino, Kazuki Moriguchi, et al. Chemical genomic screening for methylationsilencedgenes in gastric cancer cell lines using5-aza-2'-deoxycytidine treatment andoligonucleotide microarray[J]. Cancer Sci.2006; 97 (1):64-71
[11].Gyeong Hoon Kang, Sun Lee, Nam-Yun Cho, et al.DNA methylation profiles of gastric carcinoma characterized byquantitative DNA methylation analysis.Laboratory Investigation.2008;88:161-170
[12].Costello JF, Fruhwald MC, Smiraglia DJ et al. Aberrant CpG-island methylation has non-random and tumour-type-specific patterns. NatGenet 2000; 24:132-8.
[13].Eden A, Gaudet F, Waghmare A, et al. Chromosomal instability andtumors promoted by DNA hypomethylation. Science,2003,300(5618):455.
[14].Florl AR,Steinhof C, Muller M,et al. Coordinate hypermethylation atspecific genes in prostate carcinoma precedes LINE-1 hypomethylation. Br J Cancer,2004,91(5):985-994
[15].Feinberg AP, Tycko B. The history of cancer epigenetics. Nat RevCancer,2004, 4(2):143-153
[16].Seog-Yun Park,l Eun J Yoo,2 Nam-Yun Cho,et al.Comparison of CpG island hypermethylation andrepetitive DNA hypomethylation in remalignant stagesof gastric cancer, stratified for Helicobacter pylori infection. Journal of Pathology,2009
[17].Scelfo RA, Schwienbacher C, Veronese A, et al. Loss of methylation at chromosome 11p15.5 is common in human adult tumors. Oncogene,2002,21(16):2564-2572.
[18].Nishigaki M, Aoyagi K, Danjoh I, et al. Discovery ofaberrant expression of R-RAS by can cer-linked DNA hypomethylation in gastric cancer using microarrays. Cancer Res,2005, 65(6):2115-2124
[19].ToK F,LeungW K,LeeT L,Yu J,TongJ H,ChanM W,NgEK,ChungSC,SugJJ.Promoter hypermethylation of tumor-related genes in gastric intestinal metaplasia of patients with and without gastric cancer.intJ Cancer.2002; 102:623-628.
[20].Leung WK, YuJ, NgEK,et al.. Concurrent hypermethylation of multiple tumor-related genes in gastric carcinoma and adjacent normal tissues. Cancer.2001; 91:2294-2301.
[21].Rintaro Mikata, Osamu Yokosuka, et al. Analysis of genes upregulated by the demethylating agent 5-aza-2-deoxycytidine in gastric cancer cell linesInt. J. Cancer:2006; 119:1616-1622.
[22].Eun Joo Yoo,Seog-Yun Park,Nam-Yun Cho,Nayoung Kim,Hye Seung Lee,Gyeong Hoon Kang. Helicobacter pylori-infection-associated CpG island hypermethylation in the stomach and its possibleassociation with Polycomb repressive marks. Virchows Arch 2008;452:515-524
[23].Kang GH, Lee S, Kim WH et al. Epstein-Barr virus-positive gastriccarcinoma demonstrates frequent aberrant methylation of multiple genes and constitutes CpG island methylator phenotype-positive gastric carcinoma. Am J Pathol 2002; 160:787-94.
[24].Enomoto S, Maekita T, Tsukamoto T,et al. Lack of association between CpG island methylator phenotype in human gastric cancers and methylation in their background non-cancerous gastric mucosae. Cancer Sci 2007; 98:1853-61.
[25].Kaneda A, Kaminishi M, Yanagihara K, Sugimura T, Ushijima T. Identification of silencing of nine genes in human gastric cancers. Cancer Res 2002; 62:6645-50.
[26].Ushiku T, Chong JM, Uozaki H et al.p73 gene promoter methylation in Epstein-Barr virus-associated gastric carcinoma. Int J Cancer 2007; 120:60-6.
[27].Sudo M, Chong JM, Sakuma K et al. Promoter hypermethylation of Ecadherin and its abnormal expression in Epstein-Barr virus-associated gastric carcinoma. Int J Cancer 2004; 109:194-9.
[28].Li H, Minarovits J. Host cell-dependent expression of latent Epstein-Barr virus genomes: regulation by DNA methylation. Adv Cancer Res 2003; 89:133-56.
[29].Lieberman PM. Chromatin organization and virus gene expression. J Cell Physiol 2008; 216: 295-302.
[30].Hiranuma C, Kawakami K.Oyama K.et al. Hypermethylationof the MYOD1 gene is a novel prognostic factor in patients with colorectal cancer[J]. Int J Mol Med.2004,13(3):413-417.
[31].Chan M W, Chu E S, To K F. et al. Quantitative detection of methylated SOCk-1, a tumor suppressor gene, by a modified protocol of quantitative real time methylation-specific PCR using SYBR green and its use in early gastric cancer detection[J]. Biotechnol Lett.2004.26(16):1289-1293.
[32].House M G , Guo M, Efron D T, et al. Tumor suppressor gene hyperm ethylation as a predictor of gastric stromal tumor behavior[J]. J Gastrointest Surg,2003, 7(8):1004 1014
[33].SUN DanFeng, TIAN XiaoQing, ZHANG YanJie, CHEN YingXuan, LU Rong, WANG Xia, FANG JingYuan. Mammalian target of rapamycin pathway inhibition enhances the effects of 5-aza-dC on suppressing cell proliferation in human gastric cancer cell lines. Sci China Ser C-Life Sci.2008;51[7]:640-647