本文選題：腭裂 + 動物模型　； 參考：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：第一部分TCDD誘導(dǎo)C57BL/6J胎鼠腭裂的最適劑量再探討目的:觀察不同劑量環(huán)境污染物2,3,7,8-四氯二苯二VA英(2,3,7,8-tetrachlorodibenzo-p-dioxin,TCDD)誘導(dǎo)的C57BL/6J胎鼠腭部發(fā)育情況及死胎發(fā)生,探討腭裂發(fā)生率和死胎發(fā)生率與TCDD劑量的相關(guān)性。方法:于合籠交配后第10.5天(GD10.5),將判斷受孕并稱重的C57BL/6J孕鼠分為不同劑量給藥組給予0、8、18、28μg/kg的TCDD。觀察孕鼠活動情況至GD17.5,處死孕鼠,剖腹取胎,觀察死胎發(fā)生情況。于解剖顯微鏡下觀察存活胚胎腭部發(fā)育情況。結(jié)果:對照組與實(shí)驗(yàn)組在孕鼠體重增加、每窩胚胎數(shù)上無差異(P0.05),各劑量組產(chǎn)生的腭裂表型無差異。對照組無腭裂及死胎發(fā)生。各劑量組腭裂發(fā)生率均顯著高于對照組(P0.01);但TCDD18μg/kg和28μg/kg組間腭裂發(fā)生率無統(tǒng)計(jì)學(xué)差異(P0.05);僅TCDD28μg/kg組與更低劑量組及對照組的死胎發(fā)生率存在差異(P0.05)。結(jié)論:TCDD20μg/kg~30μg/kg劑量區(qū)間是誘導(dǎo)腭裂高發(fā)生率的平臺區(qū)間,即為誘導(dǎo)C57BL/6J胎鼠腭裂高發(fā)生率的最適劑量。第二部分腭發(fā)育過程中基因組DNA甲基化的初步研究目的:研究TCDD作用于C57BL/6J孕鼠后,胎鼠腭組織基因組DNA甲基化水平變化及正向調(diào)控甲基化水平的DNA甲基轉(zhuǎn)移酶(DNA methyltranferases,Dnmts)和負(fù)向調(diào)控甲基化水平的十-十一轉(zhuǎn)位蛋白(Ten-Eleven Translocations,TETs)m RNA的表達(dá)情況,明確基因組DNA甲基化在腭發(fā)育過程中的作用。方法:C57BL/6J孕鼠共40只,隨機(jī)分為TCDD組和對照組,每組20只。TCDD組于受孕后第10.5天(gestation day,GD10.5)以TCDD28μg/kg一次性灌胃;對照組以等量玉米油一次性灌胃;分別于GD13.5、GD14.5、GD15.5、GD16.5、GD17.5處死孕鼠,收集各時間點(diǎn)的胎鼠腭組織,分別提取基因組DNA、總RNA。應(yīng)用MethylampT.M.基因組DNA甲基化定量試劑盒檢測基因組DNA甲基化水平,實(shí)時熒光定量PCR檢測Dnmt1、Dnmt3a、Dnmt3b以及TET1、TET2、TET3 m RNA的表達(dá)量。結(jié)果:TCDD組標(biāo)準(zhǔn)化甲基化率在GD13.5顯著高于對照組組(P0.01);而在GD14.5、16.5均低于對照組(P0.05,P0.05)。在GD13.5、16.5,TCDD組Dnmt1 m RNA表達(dá)量高于對照組(P0.05,P0.01)。在GD13.5、16.5,TCDD組Dnmt3a m RNA表達(dá)量高于對照組(P0.05,P0.05)。在GD14.5,TCDD組Dnmt3b m RNA表達(dá)量顯著高于對照組(P0.01)。在GD13.5,TCDD組TET3 m RNA表達(dá)量顯著低于對照組(P0.01)。結(jié)論:胎鼠腭組織內(nèi)可能存在復(fù)雜的DNA甲基化調(diào)控機(jī)制。由Dnmt1、Dnmt3a表達(dá)升高以及TET3表達(dá)降低引起的腭組織GD13.5基因組DNA甲基化水平的顯著升高可能是TCDD致胎鼠腭發(fā)育障礙的表觀遺傳機(jī)制之一。第三部分TGF-β3、Dnmt1、Dnmt3a基因啟動子區(qū)Cp G島甲基化狀態(tài)的實(shí)驗(yàn)研究目的:探討TGF-β3、Dnmts基因啟動子Cp G島甲基化狀態(tài)與TCDD所致胎鼠腭發(fā)育障礙的相關(guān)性。方法:將已知孕期的C57BL/6J孕鼠共18只,隨機(jī)分為TCDD組和對照組。TCDD組于GD10.5給予28μg/kg的TCDD,對照組給予等體重體積的玉米油。分別于GD13.5、14.5、15.5處死孕鼠,收集各組胎鼠腭突組織。通過試劑盒提取組織DNA、亞硫酸氫鹽轉(zhuǎn)化、甲基化特異性PCR,將PCR產(chǎn)物進(jìn)行瓊脂糖凝膠電泳分析各組各時間點(diǎn)腭組織表達(dá)的TGF-β3、Dnmts啟動子Cp G島甲基化狀態(tài)。結(jié)果:各組各時間點(diǎn)TGF-β3啟動子區(qū)Cp G島均處于低甲基化水平,相同時間點(diǎn)兩組間無統(tǒng)計(jì)學(xué)差異(P0.05)。Dnmt1啟動子區(qū)Cp G島均處于高甲基化水平,相同時間點(diǎn)兩組間亦無統(tǒng)計(jì)學(xué)差異(P0.05)。TCDD組Dnmt3a啟動子區(qū)Cp G島1的甲基化水平在GD13.5和GD15.5高于對照組(P0.01,P0.01),在GD14.5低于對照組(P0.01)。對照組Dnmt3a啟動子區(qū)Cp G島2的甲基化水平在各時間點(diǎn)均高于TCDD組(P0.01,P0.01,P0.01)。結(jié)論:TCDD誘導(dǎo)的胎鼠腭發(fā)育期間Dnmt3a啟動子區(qū)接近第一外顯子處Cp G島低甲基化水平可能是Dnmt3a基因在GD13.5高表達(dá)的原因,并因此形成異常的基因組高甲基化狀態(tài),進(jìn)而誘發(fā)胎鼠腭裂。
[Abstract]:The optimal dosage of the first part of the TCDD induced C57BL/6J rat palate of objective: To observe the different dosage of environmental pollutants 2,3,7,8- four chloro two benzene two VA (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) development and stillbirth induced C57BL/6J rat palate, to explore the correlation between the rate and the incidence of TCDD and dose of cleft palate. Methods: in mated 10.5 day (GD10.5), will determine the pregnancy and pregnant C57BL/6J rats weighing different dosage group were given 0,8,18,28 g/kg TCDD. to observe the activity of pregnant rats to GD17.5, pregnant mice were killed, caesarean section, to investigate the incidence of stillbirth. In under a dissecting microscope to observe the survival of embryonic palatal development. Results: control group and experimental group increased in pregnant rats body weight, the number of embryos per litter no difference (P0.05), no difference in all dose groups. The control group without cleft palate cleft palate phenotype and stillbirth. The incidence of cleft palate group were significantly higher than those in control group (P0.01); but TCDD18 g/kg and 28 g/kg group there was no significant difference between cleft palate (P0.05); only TCDD28 g/kg group and lower dose group and control group, the incidence of differences (P0.05). Conclusion: TCDD20 g/kg~30 g/kg dose interval is the high incidence of cleft palate induced by the platform interval, the optimal dosage of C57BL/6J fetal rats induced high incidence of cleft palate development. The second part objective to investigate the genomic DNA methylation process: To study the effect of TCDD on C57BL/6J rats after methyl palatal tissues of fetal rats by DNA methylation level and positive regulation of the methylation level of DNA transferase (DNA methyltranferases, Dnmts) and negatively regulates the methylation level of ten - eleven protein translocation (Ten-Eleven Translocations, TETs) expression of M RNA, clear the genomic DNA methylation process in the development of palate The role of C57BL/6J in pregnant rats. Methods: a total of 40 rats were randomly divided into TCDD group and control group, 20 rats in each group.TCDD after conception 10.5 days (gestation day, GD10.5) with TCDD28 g/kg administered once; the control group was injected with the same amount of disposable corn oil gavage; respectively in GD13.5, GD14.5, GD15.5 GD16.5, GD17.5, pregnant rats were killed at each time point, collecting the palatal tissues of fetal rats, respectively from the genomic DNA of total RNA. using MethylampT.M. genomic DNA methylation assay kit to detect the methylation level of genomic DNA, real-time fluorescence quantitative PCR detection of Dnmt1, Dnmt3a, Dnmt3b, TET1, TET2, m RNA expression of TET3. Results group TCDD: Standard methylation rate in GD13.5 group was significantly higher than that of control group (P0.01); while the GD14.5,16.5 was lower than the control group (P0.05, P0.05). In GD13.5,16.5, TCDD Dnmt1 m RNA group was higher than that of control group (P0.05, P0.01). In GD13.5,16.5 group the expression of TCDD Dnmt3a m RNA The amount is higher than the control group (P0.05, P0.05). In GD14.5, Dnmt3b m RNA expression in TCDD group was significantly higher than that of the control group (P0.01). In GD13.5, TET3 m RNA expression in TCDD group was significantly lower than the control group (P0.01). Conclusion: there may be DNA methylation regulation of complex palate tissues of fetal rats. By Dnmt1, the expression of Dnmt3a increased and decreased expression of TET3 caused by the palatal tissue GD13.5 DNA methylation significantly increased may be one of the mechanisms of epigenetic TCDD induced mouse embryonic palatal developmental disorder. The third part of TGF- beta 3, Dnmt1, Dnmt3a gene objective to study the promoter region of Cp G island methylation status: To investigate TGF- beta 3, Dnmts gene relationship between promoter Cp methylation of the G and TCDD induced mouse embryonic palatal developmental disorder. Methods: C57BL/6J rats were only 18 known during pregnancy, were randomly divided into TCDD group and control group in.TCDD group GD10.5 given 28 g/kg TCDD, the control group was given The volume weight of corn oil. Pregnant mice were killed in GD13.5,14.5,15.5 respectively, were collected to mouse embryonic palatal tissue. Through tissue DNA extraction kit, bisulfite conversion, methylation specific PCR, PCR agarose gel electrophoresis analysis of the expression of each group at different time points of palate tissue TGF- beta 3, Dnmts promoter Cp the G Island methylation status. Results: each group at different time points of TGF- beta 3 promoter region Cp island of G are in the low level of methylation at the same time, no significant difference between the two groups (P0.05.Dnmt1) Cp in the promoter region G island methylation were at the high level, at the same time there was no statistical difference between the two groups (P0.05).TCDD group Dnmt3a launched 1 Cp promoter G island methylation level was higher than the control group in GD13.5 and GD15.5 (P0.01, P0.01), in GD14.5 was lower than the control group (P0.01). The control group, the methylation level of Dnmt3a promoter Cp island of G 2 were higher than that of group TCDD at each time point (P0.01 , P0.01, P0.01). Conclusion: TCDD induced mouse embryonic palatal development during the Dnmt3a promoter region close to the first exon of Cp G island methylation level may be the reason for the high expression of Dnmt3a gene in GD13.5 genome, and thus the formation of high methylation abnormalities, and induce fetal cleft palate.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R782.22

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本文編號：1739684