本文選題：結(jié)直腸癌 + DNMT3a��； 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:通過(guò)Real time-q PCR技術(shù)檢測(cè)DNA甲基化轉(zhuǎn)移酶3a(DNMT3a)mRNA在結(jié)直腸癌(colorenctal cancer,CRC)組織及相應(yīng)切緣正常粘膜組織中的表達(dá)水平,分析DNMT3a表達(dá)水平與結(jié)直腸癌患者病理和臨床特征之間的關(guān)系。同時(shí)進(jìn)一步應(yīng)用Cell Counting Kit-8(CCK-8)法、流式細(xì)胞技術(shù)(FCM)及Transwell遷移實(shí)驗(yàn)等方法檢測(cè)DNMT3a對(duì)結(jié)腸癌細(xì)胞株HCT116的增殖、凋亡以及遷移等生物學(xué)功能的影響。探討DNMT3a作為潛在腫瘤標(biāo)志物對(duì)結(jié)直腸癌早期診斷及預(yù)后評(píng)估的價(jià)值,進(jìn)一步探究DNMT3a在腫瘤發(fā)生發(fā)展過(guò)程中的作用機(jī)制,以便為結(jié)直腸癌的早期篩查、診斷、藥物治療提供新的理論和實(shí)驗(yàn)依據(jù)。方法:1收集2012年5月-2015年3月河北醫(yī)科大學(xué)第四醫(yī)院以及河北醫(yī)科大學(xué)第一醫(yī)院共計(jì)25例結(jié)直腸癌組織標(biāo)本,每例標(biāo)本均取自結(jié)直腸原發(fā)癌組織及其兩側(cè)切緣正常黏膜組織(術(shù)后病理證實(shí)切緣無(wú)癌細(xì)胞侵犯)。新鮮標(biāo)本離體后迅速置于液氮中,于-80℃冰箱保存?zhèn)溆谩?5例患者均為第一次手術(shù),且術(shù)前均未接受放療或化療。2應(yīng)用實(shí)時(shí)熒光定量PCR(RT-q-PCR)技術(shù)檢測(cè)DNA甲基化轉(zhuǎn)移酶3a(DNMT3a)mRNA在結(jié)直腸癌(colorenctal cancer,CRC)組織及相應(yīng)切緣正常粘膜組織中的表達(dá)水平,分析DNMT3a表達(dá)水平與結(jié)直腸癌患者病理和臨床特征之間的關(guān)系。選取結(jié)腸癌細(xì)胞株HCT116進(jìn)行培養(yǎng),應(yīng)用Lipofectamine 2000將DNMT3a小干擾RNA(siRNA)轉(zhuǎn)入HCT116細(xì)胞,設(shè)為實(shí)驗(yàn)組,同時(shí)設(shè)立對(duì)照組(轉(zhuǎn)染陰性對(duì)照siRNA),轉(zhuǎn)染后24小時(shí)檢測(cè)兩組細(xì)胞中DNMT3a mRNA的表達(dá)情況。3轉(zhuǎn)染DNMT3a siRNA后1d、2 d、3 d、4 d、5d分別應(yīng)用CCK-8法檢測(cè)5個(gè)時(shí)段的OD值,繪制細(xì)胞生長(zhǎng)曲線以觀察細(xì)胞增殖改變情況。4應(yīng)用流式細(xì)胞技術(shù)檢測(cè)轉(zhuǎn)染前后細(xì)胞凋亡的變化情況。5應(yīng)用Transwell技術(shù)檢測(cè)轉(zhuǎn)染前后腫瘤細(xì)胞遷移能力的變化情況。6應(yīng)用spss13.0軟件對(duì)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析,數(shù)據(jù)以中位數(shù)(四分位數(shù)間距)或均數(shù)±標(biāo)準(zhǔn)差表示,兩組間配對(duì)樣本采用Wilcoxon檢驗(yàn)或t檢驗(yàn),兩組間獨(dú)立樣本采用Mann-Whitney檢驗(yàn),多組間比較采用單因素方差分析,均以P0.05為差異有統(tǒng)計(jì)學(xué)意義。結(jié)果:1 DNMT3a mRNA在25例結(jié)直腸癌組織及其對(duì)應(yīng)切緣正常粘膜組織中的表達(dá)水平分別為2.332(1.3685,4.653)和0.9073(0.2708,1.5185),結(jié)直腸癌組織中的表達(dá)較正常黏膜組織顯著上調(diào)(P0.0001)。2 DNMT3a的表達(dá)水平與結(jié)直腸癌患者的部分臨床病理特征密切相關(guān),即T分期為T3、T4的患者明顯高于T1、T2期的患者(P=0.0239),腫瘤直徑5cm的患者明顯高于≤5cm的患者(P=0.0043),而與性別、年齡、病理類型、分化程度和有無(wú)淋巴結(jié)轉(zhuǎn)移無(wú)關(guān)(均P0.05)。3轉(zhuǎn)染24h后,實(shí)驗(yàn)組細(xì)胞中DNMT3a mRNA的表達(dá)水平明顯低于陰性對(duì)照組。4轉(zhuǎn)染DNMT3a siRNA實(shí)驗(yàn)組在轉(zhuǎn)染后1d、2d、3 d、4 d、5d,5個(gè)時(shí)段OD值均低于對(duì)陰性照組,差異具有統(tǒng)計(jì)學(xué)意義(均P0.05)。5 HCT116細(xì)胞中,實(shí)驗(yàn)組細(xì)胞凋亡率(3.35±0.451)%,相較于對(duì)照組(2.733±0.459)%呈升高趨勢(shì),但差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.4016)。6轉(zhuǎn)染24小時(shí)后對(duì)細(xì)胞進(jìn)行Transwell實(shí)驗(yàn),實(shí)驗(yàn)組細(xì)胞遷移數(shù)目為(140±12)個(gè),陰性對(duì)照組細(xì)胞遷移數(shù)目為(231±14)個(gè),實(shí)驗(yàn)組的遷移能力明顯弱于陰性對(duì)照組,(P=0.0395)。結(jié)論:1 DNMT3a可能作為一種促癌因子在結(jié)直腸癌發(fā)生過(guò)程中發(fā)揮作用;2 DNMT3a可能通過(guò)沉默多種抑癌基因的表達(dá)而促進(jìn)了直腸癌的發(fā)生及腫瘤惡性程度的進(jìn)展,并可以為腫瘤預(yù)后的判斷提供參考;3 DNMT3a可作為潛在的新型腫瘤標(biāo)記物和治療靶點(diǎn),為結(jié)直腸癌患者的診斷及腫瘤藥物的研發(fā)提供新的思路。
[Abstract]:Objective: to detect the expression level of DNA methyltransferase 3A (DNMT3a) mRNA in colorectal cancer (colorenctal cancer, CRC) tissue and the normal mucosal tissue by Real time-q PCR, and to analyze the relationship between DNMT3a expression level and the pathological and clinical characteristics of colorectal cancer patients. 8) method, flow cytometry (FCM) and Transwell migration test to detect the effects of DNMT3a on the proliferation, apoptosis and migration of colon cancer cell line HCT116, and explore the value of DNMT3a as a potential tumor marker for early diagnosis and prognosis of colorectal cancer, and further explore DNMT3a in the process of tumor development. The mechanism of action is provided to provide new theoretical and experimental basis for early screening, diagnosis and treatment of colorectal cancer. Methods: 1 a total of 25 specimens of colorectal cancer were collected from the fourth hospital of Hebei Medical University in March -2015 and the first hospital of Hebei Medical University in May 2012. Each sample was taken from the primary colorectal carcinoma and two The normal mucosa tissue of the lateral incisor (confirmed by postoperative pathology confirmed that there was no cancer cell invasion in the cutting edge). Fresh specimens were quickly placed in liquid nitrogen in vitro, and.25 patients were first operated at -80 C fridge for the first time, and no radiotherapy or chemotherapy.2 was used to detect 3A (DNMT3a) mRNA DNA methyltransferase 3A (DNMT3a) mRNA before operation. The expression level in the colorenctal cancer (CRC) tissue and the normal mucosal tissue of the corresponding cutting edge was analyzed. The relationship between the level of DNMT3a expression and the pathological and clinical features of colorectal cancer patients was analyzed. The colon cancer cell line HCT116 was cultured and Lipofectamine 2000 was used to transfer DNMT3a small interference RNA (siRNA) into HCT116 cells. In the experimental group, the control group was set up (transfected negative control siRNA), and the expression of DNMT3a mRNA in the two groups of cells was detected 24 hours after transfection..3 transfected to DNMT3a siRNA 1D, 2 D, 3 D, 4 D, 5D applied CCK-8 method to detect the 5 time periods respectively, and plotted the cell growth curve to observe the cell proliferation changes. Changes of cell apoptosis before and after dyeing.5 Transwell technique was used to detect the change of migration ability of tumor cells before and after transfection.6 applied SPSS13.0 software to analyze the data, the data were expressed with median (four quantile spacing) or mean standard deviation, and two pairs of paired samples were examined by Wilcoxon test or t test, and the two groups were independent. The samples were compared with single factor analysis of variance with Mann-Whitney test. The results showed that the expression level of 1 DNMT3a mRNA was 2.332 (1.3685,4.653) and 0.9073 (0.2708,1.5185) in the normal mucosa of 25 cases of colorectal cancer and its corresponding cutting edge, and the expression in colorectal cancer tissues was compared. The expression level of normal mucosal tissue (P0.0001).2 DNMT3a is closely related to some clinicopathological features of colorectal cancer patients, that is, T staging is T3, T4 patients are significantly higher than T1, T2 stage patients (P=0.0239), tumor diameter 5cm patients are significantly higher than those of 5cm (P=0.0043), and sex, age, pathological type, differentiation degree. After transfection of 24h with or without lymph node metastasis (P0.05), the expression level of DNMT3a mRNA in the experimental group was significantly lower than that of the negative control group.4 transfected DNMT3a siRNA experimental group, 1D, 2D, 3 D, 4 D, and the 5 periods were lower than the negative group, the difference was statistically significant. The rate of death (3.35 + 0.451)%, compared with the control group (2.733 + 0.459)%, was higher, but the difference was not statistically significant (P=0.4016).6 transfection for 24 hours after the Transwell experiment, the number of cell migration in the experimental group was (140 + 12), and the number of cell migration numbers in the negative control group was (231 + 14). The migration ability of the experimental group was significantly weaker than the negative control. Group, (P=0.0395). Conclusion: 1 DNMT3a may play a role in the carcinogenesis of colorectal cancer; 2 DNMT3a may promote the progression of rectal cancer and the malignancy of cancer by silencing the expression of a variety of tumor suppressor genes, and can provide reference for the prognosis of cancer; 3 DNMT3a may be a potential new type of swelling. Tumor markers and therapeutic targets provide new ideas for the diagnosis of colorectal cancer and the development of tumor drugs.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.34

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相關(guān)期刊論文 前2條

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本文編號(hào)：2046834