本文選題：氯乙烯 + 肝細(xì)胞��； 參考：《山西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:檢測(cè)氯乙烯對(duì)大鼠DNA損傷作用,測(cè)定大鼠的全基因組DNA甲基化水平,檢測(cè)癌基因KRAS,損傷修復(fù)基因CDKN2A、RASSF1A,DNA烷化損傷修復(fù)基因MGMT,抑癌基因SYK基因啟動(dòng)子區(qū)甲基化水平及mRNA的表達(dá)量改變,探討氯乙烯致癌在遺傳機(jī)制和表觀遺傳機(jī)制的關(guān)系。方法:選取96只健康大鼠,按體重隨機(jī)分成4組,每組24只,分別為陰性對(duì)照組和低劑量(5mg/kg)、中劑量(25mg/kg)、高劑量(125mg/kg)三個(gè)氯乙烯染毒劑量組;腹腔注射,隔日染毒,每周三次。每組大鼠分別于6、8、12周隨機(jī)處死8只,取其肝臟,應(yīng)用彗星實(shí)驗(yàn)評(píng)價(jià)肝細(xì)胞DNA損傷水平,DNA甲基化定量檢測(cè)試劑盒(比色法)檢測(cè)大鼠肝細(xì)胞全基因組甲基化水平,選用甲基化特異性PCR(QMSP)和實(shí)時(shí)熒光定量PCR(QPCR)分別檢測(cè)上述基因啟動(dòng)子區(qū)甲基化水平及mRNA的表達(dá)量。采用SPSS22.0對(duì)實(shí)驗(yàn)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析,本次實(shí)驗(yàn)數(shù)據(jù)符合正態(tài)或近似正態(tài)分布,采用單因素方差分析ANOVA進(jìn)行組間比較,兩兩比較采用LSD法;相關(guān)性分析采用雙變量相關(guān)分析,選用Pearson相關(guān)系數(shù),顯著性水平為α=0.05。結(jié)果:1、氯乙烯可誘發(fā)大鼠肝臟組織肝細(xì)胞壞死,肝脂肪變性,肝硬化等組織病理變化,大鼠肝細(xì)胞DNA損傷水平隨著染毒劑量的增加和染毒時(shí)間的延長(zhǎng)而升高,肝細(xì)胞全基因組甲基化水平在染毒組中明顯均高于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。2、大鼠肝細(xì)胞5種基因啟動(dòng)子區(qū)甲基化水平的改變:2.1各組別之間比較:氯乙烯染毒6周時(shí),各染毒組大鼠肝細(xì)胞MGMT基因啟動(dòng)子區(qū)甲基化水平均高于對(duì)照組(P0.05),mRNA的表達(dá)量隨著劑量的增加而升高;染毒8周時(shí),染毒組KRAS、SYK甲基化水平隨著染毒劑量的增加而下降,SYK mRNA的表達(dá)量隨著染毒劑量的增加而升高;染毒12周時(shí),RASSF1A、MGMT甲基化水平隨著染毒劑量的增加而明顯升高,RASSF1A mRNA表達(dá)量隨著染毒劑量的增加而下降。2.2各染毒時(shí)間之間比較:對(duì)照組中,KRAS、CDKN2A、RASSF1A、MGMT、SYK基因啟動(dòng)子區(qū)甲基化水平及mRNA表達(dá)量在各染毒時(shí)間差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);低劑量組中,MGMT、SYK甲基化水平隨著染毒時(shí)間的延長(zhǎng)而下降,MGMT mRNA表達(dá)量隨著染毒時(shí)間的延長(zhǎng)而升高;中劑量組中,KRAS、MGMT、SYK甲基化水平在8周和12周時(shí)低于6周(P0.05),mRNA表達(dá)量隨著染毒時(shí)間的延長(zhǎng)而升高;高劑量組中,CDKN2A、RASSF1A基因甲基化水平在6周和8周時(shí)低于12周(P0.05),RASSF1A mRNA表達(dá)量在6周和8周明顯高于12周(P0.05)。3、大鼠肝細(xì)胞DNA損傷和相關(guān)基因甲基化相關(guān)性分析:大鼠肝細(xì)胞DNA損傷與RASSF1A、MGMT基因啟動(dòng)子區(qū)甲基化水平呈正相關(guān)關(guān)系(P0.05)。結(jié)論:1、氯乙烯可引起大鼠肝細(xì)胞DNA損傷增加,全基因組甲基化水平升高。2、氯乙烯可引起KRAS和SYK基因啟動(dòng)子區(qū)甲基化水平的下降,CDKN2A、MGMT甲基化水平的升高。在短期、低劑量下,氯乙烯可引起RASSF1A啟動(dòng)子區(qū)甲基化水平下降,mRNA表達(dá)量增加;但當(dāng)染毒時(shí)間延長(zhǎng),染毒劑量增加時(shí),RASS1A啟動(dòng)子區(qū)甲基化水平升高,mRNA表達(dá)量下降。3、氯乙烯引起RASSF1A、MGMT基因啟動(dòng)子區(qū)甲基化水平的升高可能影響大鼠肝細(xì)胞DNA損傷的修復(fù),使大鼠肝細(xì)胞DNA損傷增加。
[Abstract]:Aim: to detect the effect of vinyl chloride on DNA injury in rats and to determine the level of DNA methylation in the whole genome of rats, detect the gene KRAS, damage repair gene CDKN2A, RASSF1A, DNA alkylation repair gene MGMT, the level of methylation and mRNA in the promoter region of the tumor suppressor gene SYK gene, and explore the genetic mechanism and apparent mechanism of the carcinogenesis of vinyl chloride. Methods: the relationship of genetic mechanism. Method: 96 healthy rats were randomly divided into 4 groups according to body weight, 24 rats in each group, which were negative control group and low dose (5mg/kg), middle dose (25mg/kg), high dose (125mg/kg) three chloroethylene exposure dose group; intraperitoneal injection, infected every week, three times per week. Rats in each group were killed at random for 8 rats in 6,8,12 weeks respectively. Liver cell DNA damage level was evaluated by comet assay. DNA methylation quantitative detection kit (colorimetric assay) was used to detect the whole genome methylation level of rat liver cells. Methylation specific PCR (QMSP) and real-time fluorescent quantitative PCR (QPCR) were used to detect the methylation level of the promoter region and the expression of mRNA respectively. SPSS22.0 The experimental data were statistically analyzed. The experimental data conformed to normal or approximately normal distribution. The single factor variance analysis ANOVA was used to compare group comparison, and 22 was compared with LSD; correlation analysis adopted bivariate correlation analysis and Pearson correlation coefficient was selected. The significant water level was alpha =0.05. results: 1, vinyl chloride could induce rat liver group. Hepatocyte necrosis, hepatic steatosis, liver cirrhosis and other histopathological changes, the level of DNA damage in hepatocytes of rats increased with the increase of dose and prolongation of time. The total genome methylation level of hepatocytes in the infected group was significantly higher than that in the control group. The difference has the significance of P0.05 (.2) and the initiation of 5 genes in rat liver cells. The changes in the level of methylation in the subregion: 2.1 the comparison between each group: the methylation water of the MGMT gene promoter region of the rat hepatocytes was higher than that of the control group (P0.05) at 6 weeks, and the expression of mRNA increased with the increase of the dose. At 8 weeks, the level of KRAS and SYK methylation decreased with the increase of the dose. The expression of SYK mRNA increased with the increase of the dose, and at 12 weeks, the level of RASSF1A and MGMT methylation increased obviously with the increase of the dose, and the RASSF1A mRNA expression decreased with the increase of the dose of the infected.2.2, and the comparison between the duration of the.2.2 exposure time: KRAS, CDKN2A, RASSF1A, MGMT, and the methylation of the SYK gene promoter region. There was no significant difference between the level and the expression of mRNA in the time of exposure. In the low dose group, the level of MGMT, SYK methylation decreased with the prolongation of the exposure time, and the expression of MGMT mRNA increased with the prolongation of the exposure time; in the middle dose group, the level of KRAS, MGMT and SYK methylation was lower than 6 weeks (P0.05) at 8 and 12 weeks, and the mRNA expression was associated with the low dose group. In the high dose group, the methylation level of CDKN2A and RASSF1A genes was lower than 12 weeks (P0.05) at 6 and 8 weeks in the high dose group. The RASSF1A mRNA expression was significantly higher than 12 weeks (P0.05).3 in the 6 and 8 weeks. The correlation of DNA damage and related gene methylation in rat hepatocytes was analyzed: DNA injury in rat liver cells and RASSF1A, MGMT gene initiation The methylation level in the subregion is positive correlation (P0.05). Conclusion: 1, ethylene chloride can cause the increase of DNA damage in rat hepatocytes and the increase of.2 in the whole genome methylation level. Chloroethylene can cause the decrease of methylation level in the promoter region of KRAS and SYK genes, the increase of CDKN2A, MGMT methylation level. In the short term and low dose, vinyl chloride can cause RASSF1A initiation. The methylation level of the promoter region decreased and the expression of mRNA increased, but when the time was prolonged and the dose increased, the level of methylation in the RASS1A promoter region increased, the expression of mRNA decreased by.3. The elevated methylation level of RASSF1A in the promoter region of the MGMT gene may affect the repair of DNA damage in the rat liver cells and the DNA loss of rat liver cells. The injury increased.

【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R114

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