本文選題：甲基化 + p16　； 參考：《北京中醫(yī)藥大學(xué)》2014年博士論文

【摘要】：胃癌是一個(gè)逐步形成的過程,常由胃癌前病變(PLGC)進(jìn)一步發(fā)展而來。胃癌前病變的早診斷和早干預(yù)可以阻斷其向胃癌的發(fā)展。PLGC分為腸上皮化生(IM)和異型增生(Dys)兩種,尤其是中重度異型增生。輕度Dys約15-30%會(huì)進(jìn)展為重度Dys和／或腺癌,而重度Dys的病人有60-80%可發(fā)展為胃癌。有效干預(yù)PLGC,阻斷其向胃癌的發(fā)展,對(duì)防治胃癌有積極意義。 經(jīng)課題組長期研究,認(rèn)為PLGC為本虛標(biāo)實(shí)之證,本虛以脾胃氣虛和／或陰虛為主,標(biāo)實(shí)以胃絡(luò)瘀血、毒損胃絡(luò)為主。臨床以“益氣化瘀解毒法”為基本治則,以百合烏藥湯化裁,選用黨參、炙百合、烏藥、香櫞皮、丹參、三七粉、莪術(shù)、蒲公英、白花蛇舌草,組方為消痞顆粒,共奏補(bǔ)氣養(yǎng)陰消滯、活血化瘀、清解熱毒之功。該法在前期臨床觀察中取得較好的療效,不僅明顯改善PLGC患者臨床癥狀,而且對(duì)胃黏膜異型增生有一定的阻斷逆轉(zhuǎn)作用。 本研究主要分為文獻(xiàn)研究和實(shí)驗(yàn)研究兩部分。文獻(xiàn)研究運(yùn)用Meta分析對(duì)中藥治療PLGC的綜合療效和胃黏膜病理變化情況進(jìn)行系統(tǒng)評(píng)價(jià),從而為中藥療法干預(yù)PLGC提供較高質(zhì)量的臨床醫(yī)學(xué)證據(jù),并為實(shí)驗(yàn)研究提供理論基礎(chǔ)；實(shí)驗(yàn)研究是在前期臨床觀察和理論研究的基礎(chǔ)上進(jìn)一步驗(yàn)證以益氣化瘀解毒法為組方的消痞顆粒對(duì)慢性萎縮性胃炎(CAG)伴Dys大鼠的一般情況、宏觀表征及胃黏膜病理改變的干預(yù)情況,并充分利用分子生物學(xué)等現(xiàn)代科學(xué)技術(shù)方法,探討其逆轉(zhuǎn)Dys表觀遺傳學(xué)作用機(jī)制。 第一部分文獻(xiàn)研究 目的：系統(tǒng)評(píng)價(jià)中藥療法與單純西藥療法治療胃癌前病變的綜合療效和胃黏膜病理變化情況。 方法：通過制定檢索策略和對(duì)國內(nèi)外數(shù)據(jù)庫檢索及手工查閱(不考慮盲法,語言為中文和英文),兩名評(píng)價(jià)人員對(duì)符合納入標(biāo)準(zhǔn)和排除標(biāo)準(zhǔn)的隨機(jī)對(duì)照試驗(yàn)進(jìn)行獨(dú)立篩選,對(duì)所納入研究的文獻(xiàn)進(jìn)行有效的數(shù)據(jù)提取和統(tǒng)計(jì)合并,并對(duì)文獻(xiàn)質(zhì)量進(jìn)行科學(xué)評(píng)價(jià),根據(jù)異質(zhì)性分析選用固定效應(yīng)模式或隨機(jī)效應(yīng)模式進(jìn)行系統(tǒng)分析。 結(jié)果：共有4篇隨機(jī)對(duì)照試驗(yàn)納入研究,中藥療法的臨床綜合療效為[RR=1.52,95%CI(1.27,1.82)]；中藥療法對(duì)中、重度異型增生的改善情況分別為[RR1.58,95%CI(1.09,2.29)]和[RR=2.14,95%CI(0.84,5.41)]；中藥療法對(duì)胃癌前病變的改善情況為[RR =1.85,95%CI (1.35,2.54)]. 結(jié)論：系統(tǒng)評(píng)價(jià)分析結(jié)果顯示中藥療法在胃癌前病變的綜合療效、改善中度異型增生和改善癌前病變方面優(yōu)于單純西藥治療,但中藥療法在改善重度異型增生上尚不能認(rèn)為優(yōu)于單純西藥療法。由于納入研究的文獻(xiàn)較少且部分文獻(xiàn)為低質(zhì)量偏倚風(fēng)險(xiǎn)不確定的隨機(jī)對(duì)照研究,尚需高質(zhì)量和低偏倚風(fēng)險(xiǎn)的研究進(jìn)一步證實(shí)。 第二部分實(shí)驗(yàn)研究 目的：建立CAG伴胃黏膜Dys大鼠實(shí)驗(yàn)?zāi)Ｐ?探討消痞顆粒對(duì)CAG伴Dys大鼠一般情況、宏觀表征及胃黏膜病理變化的干預(yù)情況,進(jìn)一步探討消痞顆粒對(duì)該模型大鼠p16和Pten抑癌基因啟動(dòng)子區(qū)域CpG島(CpG island)甲基化干預(yù)機(jī)制的研究。 方法：60只無特定病源體(Specified Pathogen Free, SPF)雄性Wistar大鼠按照體重隨機(jī)分為空白組10只,造模組50只�？瞻捉M予屏障環(huán)境下標(biāo)準(zhǔn)飲食飲水,造模組予120gg/ml N-甲基-N'-硝基-N-亞硝基胍(N-methyl-N'-nitro-N-nitrosoguanidine, MNNG)溶液(5ml/kg/24h)灌胃為主要負(fù)荷因素配合自由飲用0.05%氨水溶液和進(jìn)食含0.03%鹽酸雷尼替丁清潔級(jí)飼料,建立CAG伴Dys實(shí)驗(yàn)大鼠模型。32周末造模成功后將造模組剩余的33只大鼠按照體重隨機(jī)分為自然恢復(fù)組、維酶素組、消痞顆粒組,各組分別為11只,均給予屏障環(huán)境下標(biāo)準(zhǔn)飲食飲水飼養(yǎng)。自然恢復(fù)組予生理鹽水3mL/kg/24h灌胃；維酶素組予維酶素混懸液2ml/kg/24h(含維酶素0.3g/kg)灌胃；消痞顆粒組予消痞顆�；鞈乙�3ml/kg/24h(含生藥9g/kg)灌胃,持續(xù)12周。動(dòng)物實(shí)驗(yàn)第44周末大鼠全部處死。造模階段和干預(yù)階段均觀察大鼠一般情況(體重、飲食及飲水)、宏觀表征情況。干預(yù)階段結(jié)束后采用普通HE染色觀察各組大鼠胃黏膜病理情況,同時(shí)選用甲基化特異性PCR法(Methylation-specific PCR, MSP)、免疫組化、光鏡等手段和技術(shù)方法檢測各組大鼠p16和Pten抑癌基因啟動(dòng)子區(qū)域CpG島甲基化狀態(tài)及蛋白表達(dá)情況。 結(jié)果： 1.在造模階段,模型組大鼠一般情況(體重、飲食及飲水量)及宏觀表征(皮毛色澤、反應(yīng)靈敏程度,大便及舌象等)均明顯差于空白組。在干預(yù)階段,消痞顆粒組與維酶素組大鼠的一般情況及宏觀表征均優(yōu)于自然恢復(fù)組,其中尤以消痞顆粒組明顯,接近空白組。自然恢復(fù)組一般情況及宏觀表征未見明顯恢復(fù)。 2.在造模階段,模型組大鼠于實(shí)驗(yàn)第26周出現(xiàn)胃黏膜萎縮,于實(shí)驗(yàn)第28周萎縮進(jìn)一步加重明顯,在此基礎(chǔ)上出現(xiàn)腸上皮化生,于實(shí)驗(yàn)第30、32周萎縮繼續(xù)加重,IM減少但是程度加重,伴有程度不同的Dys,未見明顯恢復(fù)現(xiàn)象。在干預(yù)階段,自然恢復(fù)組大鼠胃黏膜持續(xù)萎縮,伴有IM及Dys,未見明顯組織修復(fù)現(xiàn)象,消痞顆粒組大鼠的病理變化情況優(yōu)于自然恢復(fù)組(P0.05),維酶素組大鼠的病理變化情況不優(yōu)于自然恢復(fù)組(P0.05)。 3.干預(yù)階段結(jié)束后,經(jīng)兩變量相關(guān)分析大鼠體重與病理變化情況關(guān)系：Spearman相關(guān)系數(shù)=-0.155,P=0.3520.05,不能認(rèn)為體重與大鼠胃黏膜病理變化情況有關(guān)。 4.經(jīng)MSP結(jié)果顯示空白組、自然恢復(fù)組、維酶素組及消痞顆粒組均可檢測到大鼠p16抑癌基因啟動(dòng)子區(qū)域CpG島甲基化陽性條帶,甲基化陽性表達(dá)率分別為16.7%、85.7%、50.0%、40.0%。經(jīng)目的條帶圖像半定量分析計(jì)算得出,消痞顆粒組與自然恢復(fù)組甲基化水平比較均有顯著差異(P0.01),維酶素組p16抑癌基因CpG島甲基化水平與自然恢復(fù)組比較差異,有統(tǒng)計(jì)學(xué)意義(P0.05),消痞顆粒組p16抑癌基因啟動(dòng)子區(qū)域CpG島甲基化水平優(yōu)于維酶素組(P0.05)。 5.空白組、自然恢復(fù)組、維酶素組及消痞顆粒均可檢測到大鼠p16蛋白表達(dá),陽性表達(dá)率分別為100%、25%、55.6%和72.7%。經(jīng)免疫組化圖像半定量分析計(jì)算得出,消痞顆粒組p16陽性蛋白表達(dá)情況優(yōu)于維酶素組(P0.05)和自然恢復(fù)組(P0.01),但是與空白組p16蛋白表達(dá)比較仍然有顯著差異(P0.01)。 6.14例大鼠p16抑癌基因啟動(dòng)子區(qū)域CpG島甲基化大鼠中有2例p16蛋白表達(dá)陽性；而16例大鼠p16抑癌基因啟動(dòng)子區(qū)域CpG島非甲基化大鼠中僅有1例蛋白表達(dá)陰性表達(dá)。經(jīng)兩變量相關(guān)分析檢驗(yàn),Spearman系數(shù)-0.800,P0.01,可以認(rèn)為p16抑癌基因CpG島甲基化與p16蛋白陰性表達(dá)有關(guān)。 7.進(jìn)行二分類Logistic回歸分析,可以認(rèn)為p16抑癌基因啟動(dòng)子區(qū)域CpG島高甲基化(P0.01)及p16蛋白陰性表達(dá)(P0.05)與大鼠胃黏膜病理情況有關(guān)。 8.經(jīng)MSP結(jié)果顯示,各組均未出現(xiàn)大鼠Pten抑癌基因啟動(dòng)子區(qū)域CpG島甲基化現(xiàn)象,但是陽性標(biāo)準(zhǔn)品可擴(kuò)增出目的條帶�？瞻捉M、自然恢復(fù)組、維酶素組及消痞顆粒均可檢測到Pten蛋白表達(dá),陽性表達(dá)率分別為100%、50%、66.7%和81.8%。經(jīng)免疫組化圖像半定量分析計(jì)算得出,消痞顆粒組大鼠Pten蛋白表達(dá)情況明顯優(yōu)于維酶素組(P0.01)和自然恢復(fù)組(P0.01),同時(shí)與空白組大鼠Pten蛋白表達(dá)情況沒有差異,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。 9.經(jīng)兩變量相關(guān)分析大鼠病理變化與Pten抑癌基因蛋白表達(dá)關(guān)系：Spearman系數(shù)-0.619,P0.01,可以認(rèn)為大鼠胃黏膜病理變化情況與Pten抑癌基因蛋白表達(dá)有關(guān)。 結(jié)論： 1.建立以MNNG溶液灌胃為主要負(fù)荷因素的CAG伴Dys大鼠模型； 2.以益氣化瘀解毒法為組方的消痞顆�？捎行Ц纳坡晕s性胃炎伴異型增生大鼠的一般情況、宏觀表征,逆轉(zhuǎn)大鼠胃黏膜Dys的病理變化,防治胃癌的發(fā)生發(fā)展； 3.以益氣化瘀解毒法為組方的消痞顆粒通過降低大鼠p16抑癌基因啟動(dòng)子區(qū)域CpG島甲基化水平及其上調(diào)p16蛋白表達(dá),逆轉(zhuǎn)異型增生,從而達(dá)到阻斷胃癌發(fā)生發(fā)展； 4.各組大鼠Pten抑癌基因啟動(dòng)子區(qū)域CpG島未出現(xiàn)甲基化產(chǎn)物,不能認(rèn)為大鼠Pten抑癌基因啟動(dòng)子區(qū)域CpG島高甲基化與胃癌發(fā)生發(fā)展有關(guān)； 5.以益氣化瘀解毒法為組方的消痞顆�？赏ㄟ^提高Pten蛋白表達(dá)來逆轉(zhuǎn)異型增生,從而阻斷胃癌發(fā)生發(fā)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錛，

本文編號(hào)：1801188