益气化瘀解毒法对慢性萎缩性胃炎伴异型增生大鼠干预的实验研究及中药治疗的系统评价

发布时间：2018-04-25 11:51

  本文选题：甲基化 + p16　； 参考：《北京中医药大学》2014年博士论文

【摘要】：胃癌是一个逐步形成的过程,常由胃癌前病变(PLGC)进一步发展而来。胃癌前病变的早诊断和早干预可以阻断其向胃癌的发展。PLGC分为肠上皮化生(IM)和异型增生(Dys)两种,尤其是中重度异型增生。轻度Dys约15-30%会进展为重度Dys和／或腺癌,而重度Dys的病人有60-80%可发展为胃癌。有效干预PLGC,阻断其向胃癌的发展,对防治胃癌有积极意义。 经课题组长期研究,认为PLGC为本虚标实之证,本虚以脾胃气虚和／或阴虚为主,标实以胃络瘀血、毒损胃络为主。临床以“益气化瘀解毒法”为基本治则,以百合乌药汤化裁,选用党参、炙百合、乌药、香橼皮、丹参、三七粉、莪术、蒲公英、白花蛇舌草,组方为消痞颗粒,共奏补气养阴消滞、活血化瘀、清解热毒之功。该法在前期临床观察中取得较好的疗效,不仅明显改善PLGC患者临床症状,而且对胃黏膜异型增生有一定的阻断逆转作用。 本研究主要分为文献研究和实验研究两部分。文献研究运用Meta分析对中药治疗PLGC的综合疗效和胃黏膜病理变化情况进行系统评价,从而为中药疗法干预PLGC提供较高质量的临床医学证据,并为实验研究提供理论基础；实验研究是在前期临床观察和理论研究的基础上进一步验证以益气化瘀解毒法为组方的消痞颗粒对慢性萎缩性胃炎(CAG)伴Dys大鼠的一般情况、宏观表征及胃黏膜病理改变的干预情况,并充分利用分子生物学等现代科学技术方法,探讨其逆转Dys表观遗传学作用机制。 第一部分文献研究 目的：系统评价中药疗法与单纯西药疗法治疗胃癌前病变的综合疗效和胃黏膜病理变化情况。 方法：通过制定检索策略和对国内外数据库检索及手工查阅(不考虑盲法,语言为中文和英文),两名评价人员对符合纳入标准和排除标准的随机对照试验进行独立筛选,对所纳入研究的文献进行有效的数据提取和统计合并,并对文献质量进行科学评价,根据异质性分析选用固定效应模式或随机效应模式进行系统分析。 结果：共有4篇随机对照试验纳入研究,中药疗法的临床综合疗效为[RR=1.52,95%CI(1.27,1.82)]；中药疗法对中、重度异型增生的改善情况分别为[RR1.58,95%CI(1.09,2.29)]和[RR=2.14,95%CI(0.84,5.41)]；中药疗法对胃癌前病变的改善情况为[RR =1.85,95%CI (1.35,2.54)]. 结论：系统评价分析结果显示中药疗法在胃癌前病变的综合疗效、改善中度异型增生和改善癌前病变方面优于单纯西药治疗,但中药疗法在改善重度异型增生上尚不能认为优于单纯西药疗法。由于纳入研究的文献较少且部分文献为低质量偏倚风险不确定的随机对照研究,尚需高质量和低偏倚风险的研究进一步证实。 第二部分实验研究 目的：建立CAG伴胃黏膜Dys大鼠实验模型,探讨消痞颗粒对CAG伴Dys大鼠一般情况、宏观表征及胃黏膜病理变化的干预情况,进一步探讨消痞颗粒对该模型大鼠p16和Pten抑癌基因启动子区域CpG岛(CpG island)甲基化干预机制的研究。 方法：60只无特定病源体(Specified Pathogen Free, SPF)雄性Wistar大鼠按照体重随机分为空白组10只,造模组50只。空白组予屏障环境下标准饮食饮水,造模组予120gg/ml N-甲基-N'-硝基-N-亚硝基胍(N-methyl-N'-nitro-N-nitrosoguanidine, MNNG)溶液(5ml/kg/24h)灌胃为主要负荷因素配合自由饮用0.05%氨水溶液和进食含0.03%盐酸雷尼替丁清洁级饲料,建立CAG伴Dys实验大鼠模型。32周末造模成功后将造模组剩余的33只大鼠按照体重随机分为自然恢复组、维酶素组、消痞颗粒组,各组分别为11只,均给予屏障环境下标准饮食饮水饲养。自然恢复组予生理盐水3mL/kg/24h灌胃；维酶素组予维酶素混悬液2ml/kg/24h(含维酶素0.3g/kg)灌胃；消痞颗粒组予消痞颗粒混悬液3ml/kg/24h(含生药9g/kg)灌胃,持续12周。动物实验第44周末大鼠全部处死。造模阶段和干预阶段均观察大鼠一般情况(体重、饮食及饮水)、宏观表征情况。干预阶段结束后采用普通HE染色观察各组大鼠胃黏膜病理情况,同时选用甲基化特异性PCR法(Methylation-specific PCR, MSP)、免疫组化、光镜等手段和技术方法检测各组大鼠p16和Pten抑癌基因启动子区域CpG岛甲基化状态及蛋白表达情况。 结果： 1.在造模阶段,模型组大鼠一般情况(体重、饮食及饮水量)及宏观表征(皮毛色泽、反应灵敏程度,大便及舌象等)均明显差于空白组。在干预阶段,消痞颗粒组与维酶素组大鼠的一般情况及宏观表征均优于自然恢复组,其中尤以消痞颗粒组明显,接近空白组。自然恢复组一般情况及宏观表征未见明显恢复。 2.在造模阶段,模型组大鼠于实验第26周出现胃黏膜萎缩,于实验第28周萎缩进一步加重明显,在此基础上出现肠上皮化生,于实验第30、32周萎缩继续加重,IM减少但是程度加重,伴有程度不同的Dys,未见明显恢复现象。在干预阶段,自然恢复组大鼠胃黏膜持续萎缩,伴有IM及Dys,未见明显组织修复现象,消痞颗粒组大鼠的病理变化情况优于自然恢复组(P0.05),维酶素组大鼠的病理变化情况不优于自然恢复组(P0.05)。 3.干预阶段结束后,经两变量相关分析大鼠体重与病理变化情况关系：Spearman相关系数=-0.155,P=0.3520.05,不能认为体重与大鼠胃黏膜病理变化情况有关。 4.经MSP结果显示空白组、自然恢复组、维酶素组及消痞颗粒组均可检测到大鼠p16抑癌基因启动子区域CpG岛甲基化阳性条带,甲基化阳性表达率分别为16.7%、85.7%、50.0%、40.0%。经目的条带图像半定量分析计算得出,消痞颗粒组与自然恢复组甲基化水平比较均有显著差异(P0.01),维酶素组p16抑癌基因CpG岛甲基化水平与自然恢复组比较差异,有统计学意义(P0.05),消痞颗粒组p16抑癌基因启动子区域CpG岛甲基化水平优于维酶素组(P0.05)。 5.空白组、自然恢复组、维酶素组及消痞颗粒均可检测到大鼠p16蛋白表达,阳性表达率分别为100%、25%、55.6%和72.7%。经免疫组化图像半定量分析计算得出,消痞颗粒组p16阳性蛋白表达情况优于维酶素组(P0.05)和自然恢复组(P0.01),但是与空白组p16蛋白表达比较仍然有显著差异(P0.01)。 6.14例大鼠p16抑癌基因启动子区域CpG岛甲基化大鼠中有2例p16蛋白表达阳性；而16例大鼠p16抑癌基因启动子区域CpG岛非甲基化大鼠中仅有1例蛋白表达阴性表达。经两变量相关分析检验,Spearman系数-0.800,P0.01,可以认为p16抑癌基因CpG岛甲基化与p16蛋白阴性表达有关。 7.进行二分类Logistic回归分析,可以认为p16抑癌基因启动子区域CpG岛高甲基化(P0.01)及p16蛋白阴性表达(P0.05)与大鼠胃黏膜病理情况有关。 8.经MSP结果显示,各组均未出现大鼠Pten抑癌基因启动子区域CpG岛甲基化现象,但是阳性标准品可扩增出目的条带。空白组、自然恢复组、维酶素组及消痞颗粒均可检测到Pten蛋白表达,阳性表达率分别为100%、50%、66.7%和81.8%。经免疫组化图像半定量分析计算得出,消痞颗粒组大鼠Pten蛋白表达情况明显优于维酶素组(P0.01)和自然恢复组(P0.01),同时与空白组大鼠Pten蛋白表达情况没有差异,差异无统计学意义(P0.05)。 9.经两变量相关分析大鼠病理变化与Pten抑癌基因蛋白表达关系：Spearman系数-0.619,P0.01,可以认为大鼠胃黏膜病理变化情况与Pten抑癌基因蛋白表达有关。 结论： 1.建立以MNNG溶液灌胃为主要负荷因素的CAG伴Dys大鼠模型； 2.以益气化瘀解毒法为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、宏观表征,逆转大鼠胃黏膜Dys的病理变化,防治胃癌的发生发展； 3.以益气化瘀解毒法为组方的消痞颗粒通过降低大鼠p16抑癌基因启动子区域CpG岛甲基化水平及其上调p16蛋白表达,逆转异型增生,从而达到阻断胃癌发生发展； 4.各组大鼠Pten抑癌基因启动子区域CpG岛未出现甲基化产物,不能认为大鼠Pten抑癌基因启动子区域CpG岛高甲基化与胃癌发生发展有关； 5.以益气化瘀解毒法为组方的消痞颗粒可通过提高Pten蛋白表达来逆转异型增生,从而阻断胃癌发生发展。
[Abstract]:Gastric cancer is a progressive process, which is often developed by the precancerous precancerous lesion (PLGC). Early diagnosis and early intervention of gastric precancerous lesions can block the development of gastric cancer, which can be divided into two kinds of.PLGC: intestinal metaplasia (IM) and dysplasia (Dys), especially moderate to severe dysplasia. Mild Dys about 15-30% will advance to severe Dys and / or glands. Cancer and severe Dys patients can develop 60-80% into gastric cancer. Effective intervention of PLGC, blocking the development of gastric cancer, has positive significance in the prevention and treatment of gastric cancer.
After a long study of the project group, PLGC was considered as a syndrome of the deficiency of the spleen and stomach. The deficiency of the spleen and stomach qi and / or yin deficiency was the main basis. The main treatment was the stomach collateral blood stasis and the toxic damage to the stomach collateral. The clinical use of "Yiqi Huayu detoxification" was the basic treatment. This method has achieved good effect in the early clinical observation, not only obviously improving the clinical symptoms of PLGC patients, but also blocking and reversing the dysplasia of gastric mucosa.
This research is divided into two parts: literature research and experimental research. The literature research uses Meta analysis to systematically evaluate the comprehensive curative effect of PLGC and the pathological changes of gastric mucosa by Chinese medicine, so as to provide high quality clinical medical evidence for the intervention of PLGC by Chinese medicine therapy, and provide a theoretical basis for the actual study. On the basis of early clinical observation and theoretical study, we further verify the general situation, macro characterization and pathological changes of gastric mucosa in the patients with chronic atrophic gastritis (CAG) with Dys rats, and to explore the reversal of the Dys view by using the modern scientific and technical methods such as molecular biology. Mechanism of genetic action.
The first part of the literature study
Objective: to systematically evaluate the comprehensive effect of Chinese medicine therapy and Western medicine alone in the treatment of gastric precancerous lesions and the pathological changes of gastric mucosa.
Methods: through the formulation of the retrieval strategy and the database retrieval and manual inspection at home and abroad (without considering the blind law, the language is Chinese and English), two evaluators screened the randomized controlled trials that met the inclusion criteria and exclusion criteria, and made effective data extraction and statistical consolidation for the literature included in the study and the literature. The quality is scientifically evaluated, and the fixed effect mode or random effect pattern is selected for systematic analysis based on heterogeneity analysis.
Results: a total of 4 randomized controlled trials were included in the study. The clinical efficacy of traditional Chinese medicine therapy was [RR=1.52,95%CI (1.27,1.82)]; the improvement of Chinese medicine therapy for moderate and severe dysplasia was [RR1.58,95%CI (1.09,2.29)] and [RR=2.14,95%CI (0.84,5.41), respectively. The improvement of traditional Chinese medicine therapy for precancerous lesions was [RR
=1.85,95%CI (1.35,2.54)].
Conclusion: the results of systematic evaluation show that traditional Chinese medicine therapy is superior to pure western medicine in the comprehensive effect of precancerous lesions, improving moderate dysplasia and improving precancerous lesions. However, traditional Chinese medicine therapy can not be considered better than pure western medicine in the improvement of severe dysplasia. A randomized controlled study with low quality bias risk is still needed for further studies with high quality and low bias risk.
The second part experimental study
Objective: to establish an experimental model of CAG with gastric mucosa Dys rats, to explore the general situation of CAG with Dys rats, macro characterization and the intervention of pathological changes of gastric mucosa, and to further explore the mechanism of the intervention mechanism of the methylation of CpG Island (CpG Island) in the promoter region of p16 and Pten suppressor genes in the model rats.
Methods: 60 male Wistar rats without specific source (Specified Pathogen Free, SPF) were randomly divided into 10 blank groups and 50 rats in the model group. The blank group was given the standard diet drinking water under the barrier environment, and the module was given to 120gg/ml N- methyl -N'- nitro -N- nitroguanidine (N-methyl-N'-nitro-N-nitrosoguanidine, MNNG) solution (5ml/kg/24h). The stomach as the main load factor combined with free 0.05% ammonia solution and 0.03% ranitidine containing clean grade feed, the model of CAG with Dys experimental rat model was established after the model of.32 weekend. The remaining 33 rats were randomly divided into the natural recovery group, the vitamin a group, the elimination of the ruffian granule group, each group were 11, all were given. The natural recovery group was fed with the standard diet and drinking water under the barrier environment. The natural saline group was given the physiological saline 3mL/kg/24h for gastric perfusion. The vitamin enzyme group was given the vitamin 2ml/kg/24h 0.3g/kg (vitamin 0.3g/kg) in the stomach, and the Xiao PI granule group was given the suspension of the ruffian granule 3ml/kg/24h (containing 9g/kg) for the gastric perfusion for the last 12 weeks. The animal experiment was all executed at the end of the forty-fourth weekend. The general condition of rats (weight, diet and drinking water) was observed during the period and intervention stage. The pathological conditions of gastric mucosa in the rats were observed by ordinary HE staining after the intervention stage, and the methylation specific PCR (Methylation-specific PCR, MSP), immunohistochemistry and light microscopy were used to detect the rats in each group. Methylation status and protein expression of CpG island in promoter region of p16 and Pten.
Result锛，

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