【作者】 魏玥；

【导师】 杨晋翔；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2012， 博士

【摘要】 胃癌是中国恶性肿瘤中最常见的类型,其发病率和死亡率居我国消化系统恶性肿瘤之首,严重威胁着人们的身体健康和生命安全。胃癌的发生是一个多因素、多步骤、多基因参与的复杂生物学过程,慢性浅表性胃炎→慢性萎缩性胃炎→肠上皮化生→异型增生→胃癌的Correa模式已得到全世界的广泛认同。肠上皮化生和异型增生又被统称为胃癌前病变,是较易转变为癌组织的病理学变化。尤其是异型增生,重度异型增生患者有60%-80%的可能发展为胃癌。然而目前关于异型增生的治疗手段仍然比较有限。因此,如何有效干预异型增生,逆转胃癌前病变,阻断其向胃癌的发展,是目前防治胃癌的研究关键。本研究建立了慢性萎缩性胃炎伴异型增生大鼠模型,选用以益气化瘀解毒法组方的消痞颗粒(党参、炙百合、乌药、香橼皮、丹参、三七粉、莪术、蒲公英、白花蛇舌草)为干预药物,研究益气化瘀解毒法治疗慢性萎缩性胃炎伴异型增生、逆转胃癌前病变、阻断胃癌发生发展的分子作用机制。目的：研究益气化瘀解毒法对慢性萎缩性胃炎伴异型增生过程中大鼠的一般情况、死亡率和病理学变化的影响,及其对抑癌基因p53、原癌基因c-Myc和EGFR/MAPK细胞信号转导通路的干预作用。方法：60只SPF级健康雄性Wi star大鼠随机分为空白组10只,造模组50只。空白组给予SPF级动物标准饮食喂养,持续至实验结束。造模组采用以120μ/mL的MNNG溶液灌胃,每只5mL/kg,1次/d为主,配合自由饮用0.1%氨水溶液及进食含0.03%雷尼替丁的颗粒状饲料,三种因素联合建立慢性萎缩性胃炎伴异型增生大鼠模型。28周末造模成功后将造模组剩余的30只大鼠随机分为模型组、维酶素组、消痞颗粒组各10只,均给予SPF级动物标准饮食喂养。模型组予生理盐水3mL/kg灌胃,1次/d；维酶素组予维酶素悬浊液2mL/kg(即维酶素0.3g/kg)灌胃,1次/d；消痞颗粒组予消痞颗粒制备药液3mL/kg(含生药9g/kg)灌胃,1次/d。持续治疗12周后即实验第40周末处死全部大鼠。选用光镜观察、Real-time PCR及Wes tern blot等方法检测各组大鼠病理变化、p53. c-Myc.EGFR和ERK等指标。结果：1消痞颗粒组与维酶素组大鼠的一般情况均好于模型组,其中尤以消痞颗粒组明显。消痞颗粒组与空白组大鼠治疗阶段均无死亡,死亡率最低；模型组死亡3只(3/10,30%)；维酶素组死亡2只(2/10,20%)。造模组大鼠体重明显轻于空白组,两组比较有显著差异(P=0.000,P<0.01)；造模组大鼠造模阶段的体重增长值也明显低于空白组,两组比较有显著差异(P=0.000,P<0.01)。2各组病理切片比较,模型组有3例出现胃腺癌(3/7,43%)；消痞颗粒组和维酶素组胃黏膜异型增生程度较模型组均有所逆转,其中尤以消痞颗粒组明显,5例已接近正常胃黏膜(5/10,50%)。3野生型p53mRNA表达量比较,模型组较空白组显著降低(P=0.007,P<0.01)；消痞颗粒组较模型组和维酶素组均有所增高(P=0.039和0.049,P<0.05),较空白组无差异(P=0.411,P>0.05)；维酶素组较空白组显著降低(P=0.009,P<0.01),较模型组无差异(P=0.911,P>0.05)。突变型p53蛋白表达量比较,模型组较空白组显著增高(P=0.000,P<0.01)；消痞颗粒组较模型组和维酶素组均显著降低(P=0.000和0.006,P<0.01),较空白组无差异(P=0.374,P>0.05)；维酶素组较模型组显著降低(P=0.000,P<0.01),较空白组显著增高(P=0.002,P<0.01)。4c-Myc mRNA表达量比较,模型组较空白组显著增高(P=0.000,P<0.01)；消痞颗粒组较模型组显著降低(P=0.001,P<0.01),较维酶素组也有所降低(P=0.025,P<0.05),较空白组无差异(P=0.886,P>0.05)；维酶素组较空白组有所增高(P=0.019,P<0.05),与模型组比较无差异(P=0、066, P>0.05)。 c-Myc蛋白表达量比较,模型组较空白组显著增高(P=0.001,P<0.01)；消痞颗粒组较模型组显著降低(P=0.001,P<0.01),较空白组和维酶素组无差异(P=0.927和0.059,P>0.05)；维酶素组较模型组有所降低(P=0.032,P<0.05),较空白组有所增高(P=0.049,P<0.05)。5EGFR蛋白表达量比较,模型组较空白组显著增高(P=0.000,P<0.01)；消痞颗粒组较模型组显著降低(P=0.000,P<0.01),较维酶素组也有所降低(P=0.032,P<0.05),较空白组无差异(P=0.064,P>0.05)；维酶素组较模型组有所降低(P=0.034,P<0.05),较空白组显著增高(P=0.001,P<0.01)。6ERK1/2蛋白表达量比较,模型组较空白组显著增高(P=0.000,P<0.01)；消痞颗粒组较模型组和维酶素组均显著降低(P均为0.000,P<0.01),较空白组显著增高(P=0.003,P<0.01)；维酶素组较模型组显著降低(P=0.001,P<0.01),较空白组显著增高(P=0.000,P<0.01). p-ERK1/2蛋白表达量比较,模型组较空白组显著增高(P=0.000,P<0.01)；消痞颗粒组较模型组和维酶素组均显著降低(P均为0.000,P<0.01),较空白组无差异(P=0.616,P>0.05)；维酶素组较模型组有所降低(P=0.010,P<0.05),较空白组显著增高(P=0.000,P<0.01)。结论：以益气化瘀解毒法组方的消痞颗粒可有效改善慢性萎缩性胃炎伴异型增生大鼠的一般情况,降低其死亡率,逆转胃癌前病变的病理变化,防治胃癌的发生发展。其有效作用机制可能为：通过逆转p53基因的突变、促进p53发挥其原有抑制肿瘤的作用,抑制原癌基因c-Myc mRNA及蛋白的过表达,降低EGFR蛋白的高表达及阻断EGFR/MAPK细胞信号转导通路的异常激活,从而达到治疗慢性萎缩性胃炎伴异型增生、逆转胃癌前病变、阻断胃癌发生发展的目的。更多还原

【Abstract】 Gastric cancer is the most common type of Chinese malignant tumors, the incidence and mortality rate are at the first place of digestive malignant tumors in China. Based on that, gastric cancer is a serious threat to people’s health and life safe. Gastric carcinogenesis is a complex biological process that multiple factors, steps and genes involved in. The Correa model (chronic superficial gastritis→chronic atrophic gastri t is→intestinal metaplasia→dysplasia→gastric cancer) has been got widespread recognition all over the world. Intestinal metaplasia and dysplasia are called gastric precancerous lesions, which can more easily transform as cancer tissues, especially the dysplasia,60%to80%severe dysplasia patients may be developed into gastric cancers. However, the treatment of dysplasia is still relatively limited. Therefore, how to effectively intervene dysplasia, reverse gastric precancerous lesions, block the development of gastric cancer, these are hot spots for prevention and treatment of gastric cancer study at present.This study established the rat model of chronic atrophic gastritis with dysplasia, choiced Xiaopi particle (Lanceolata, Lily, Combined spicebush, Citrus medica, Salvia, Panax notoginseng powder, Zedoary turmeric, Dandelion, Hedyotis diffusa willd), which was prescripted by Yiqi Huayu Jiedu principle, as therapeutic drug and researched molecular mechanisms of Yiqi Huayu Jiedu principle treating chronic atrophic gastritis with dysplasia, reversing gastric precancerous lesions and blocking the development of gastric cancer.Objective:To observe the effects of Yiqi Huayu Jiedu principle on general situations, mortality and pathological changes of chronic atrophic gastritis with dysplasia rats. And to study the functions of this principle for tumor suppressor gene p53, proto-oncogene c-Myc and the EGFR/MAPK cell signal transduction pathway.Methods:60Wistar rats were randomly divided into making model group (50rats) and blank group (10rats). The blank group rats were given standard diet of SPF animals until the end of the experiment. Three factors were used to make model of chronic atrophic gastritis with dysplasia for the making model group rats, mainly by120μg/mL MNNG,5mL/kg,1time/d, assisted by0.1%ammonia for drinking water and0.03%ranitidine in feed. After the model was succeeded at the28th weekend, residual30rats in the making model group were randomly divided into model group, Weimeisu group and Xiaopi particle group, each group included10rats. Each group was given the corresponding medicine once daily:3mL/kg saline for the model group,2mL/kg Weimeisu suspension liquid (0.3g/kg Weimeisu) for the Weimeisu group and3mL/kg Xiaopi particle Solution (containing9g/kg crude drug). All the rats were sacrificed after treatment for12weeks. The pathological changes, p53, c-Myc, EGFR, ERK and other indexes were detected with light microscope, real-time PCR, Western blot and other means.Results:1The general situations of Xiaopi particle group and Weimeisu group rats were better than the model group, especially Xiaopi particle group. Xiaopi particle group and the blank group had no death and the lowest mortality in treatment stage;3rats died in the model group (3/10,30%);2rats died in Weimeisu group (2/10,20%). The rat body weight of the making model group was significantly lighter than the blank group (P=0.000, P<0.01); the increase of body weight of the making model group was significantly lower than the blank group (P=0.000, P<0.01).2The pathological results showed that3gastric cancers in the model group (3/7,43%), dysplasia degree in Xiaopi particle group and Weimeisu group were lighter than the model group, especially Xiaopi particle group. Five cases were already close to normal gastric mucosae (5/10,50%).3The wild-type p53mRNA expression of the model group was significantly lower than the blank group (P=0.007, P<0.01); Xiaopi particle group was higher than the model group and Weimeisu group (P=0.039and0.049, P<0.05) and had no difference with the blank group (P=0.411, P>0.05); Weimeisu group was significantly lower than the blank group (P=0.009, P<0.01) and had no difference with the model group{P=0.911, P>0.05). The protein express ion of mutant p53in the model group was significantly higher than the blank group (P=0.000, P<0.01); Xiaopi particle group was significantly lower than the model group and Weimeisu group (P=0.000and0.006, P<0.01) and had no difference with the blank group (P=0.374, P>0.05); Weimeisu group was significant ly lower than the model group (P=0.000, P<0.01) and significantly higher than the blank group (P=0.002, P<0.01).4The c-Myc mRNA expression of the model group was significantly higher than the blank group (P=0.000, P<0.01); Xiaopi particle group was significantly lower than the model group{P=0.001, P<0.01) and also lower than Weimeisu group (/"=0.025, P<0.05), compared with the blank group, they had no difference (P=0.886,P>0.05); Weimeisu group was higher than the blank group(P=0.019, P<0.05) and had no difference with the model group (P=0.066, P>0.05). The c-Myc protein expression of the model group was significantly higher than the blank group (P=0.001, P<0.01); Xiaopi part icle group was significantly lower than the model group (P=0.001, P<0.01) and compared with the blank group and Weimeisu group, they had no difference (P=0.927and0.059, P>0.05); Weimeisu group was lower than the model group (P=0.032, P<0.05) and higher than the blank group (P=0.049, P<0.05).5The EGFR protein expression of the model group was significantly higher than the blank group (P=0.000,P<0.01); Xiaopi particle group was significantly lower than the model group (P=0.000, P<0.01) and also lower than Weimeisu group (P=0.032, P<0.05), compared with the blank group, they had no difference (P=0.064, P>0.05); Weimeisu group was lower than the model group (P=0.034, P <0.05) and significantly higher than the blank group (P=0.001, P<0.01).6The ERK1/2protein expression of the model group was significantly higher than the blank group (P=0.000, P<0.01); Xiaopi particle group was significantly lower than the model group and Weimeisu group (both P=0.000, P <0.01) and significantly higher than the blank group (P=0.003, P<0.01); Weimeisu group was significantly lower than the model group (P=0.001, P<0.01) and significantly higher than the blank group (P=0.000, P<0.01). The p-ERK1/2protein expression of the model group was significantly higher than the blank group (P=0.000,P<0.01); Xiaopi particle group was significantly lower than the model group and Weimeisu group (both P=0.000, P<0.01), compared with the blank group, they had no difference (P=0.616, P>0.05); Weimeisu group was lower than the model group (P=0.010, P<0.05) and significantly higher than the blank group (P=0.000, P<0.01).Conclusion:Xiaopi particle, prescripted by Yiqi Huayu Jiedu principle, can effectively improve the general situations of chronic atrophic gastritis with dysplasia rats, reduce the mortality, reverse the pathological changes of gastric precancerous lesions and block the development of gastric cancer. Its effective mechanism may reverse mutation of p53gene, promote p53to play its original role for tumor suppression, inhibite over-expression of proto-oncogene c-Myc mRNA and its protein, reduce high expression of EGFR protein and block abnormal activation of the EGFR/MAPK cellular signal transduction pathway.更多还原