TGF-β3、BMP4基因及组蛋白H3乙酰化在TCDD致胎鼠腭裂发生的作用
本文选题：转化生长因子-β3 + 骨形成蛋白4 ； 参考：《重庆医科大学》2014年硕士论文
【摘要】：第一部分TGF-β3、BMP4mRNA在TCDD致胎鼠腭裂中的表达 目的：研究C57BL/6J孕鼠在2,3,7,8-四氯二苯二VA英(2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD)致胎鼠腭裂的最适诱导剂量下，腭突组织中转化生长因子-β3（transforming growth factor-β3, TGF-β3）和骨形成蛋白4（bone morphogenetic protein4, BMP4）mRNA的表达情况，并探讨其中可能的发生机制。 方法：取10只C57BL/6J孕鼠，，随机分为TCDD组（5只）和对照组（5只），TCDD组于GDl0以TCDD28μg／kg灌胃1次，对照组于GDl0以等量玉米油灌胃，在GD16.5行大体解剖学形态观察；同时，使用4%的多聚甲醛固定胎头，HE染色行组织学观察。另取36只孕鼠随机分为TCDD组（18只）和对照组（18只），在GD13.5、GD14.5和GD15.5（处理方法同前），分别取各组胎鼠腭突组织提取总RNA，利用Q-PCR技术检测各组胎鼠TGF-β3、BMP4mRNA表达水平。 结果：在GD16.5，TCDD组胎鼠出现腭裂，对照组完全正常。在GD13.5、GD14.5和GD15.5，对照组TGF-β3mRNA相对表达量分别为0.7720±0.1548、0.8373±0.2458和0.9475±0.1737，TCDD组分别为1.0948±0.1733、1.1624±0.1892和1.0482±0.3438，TCDD组TGF-β3在GD13.5、GD14.5高于对照组，差异有统计学意义（p＜0.01, p＜0.05）；在GD15.5，TGF-β3亦高于对照组，差异无统计学意义（p＞0.05）。在GD13.5、GD14.5和GD15.5，对照组BMP4mRNA相对表达量分别为0.8470±0.2168、1.0531±0.0178和0.6865±0.1436，TCDD组分别为1.1114±0.0587、0.8617±0.0780和0.5825±0.1614，TCDD组在GD13.5、GD14.5差异有统计学意义（p＜0.05, p＜0.01），在GD15.5差异无统计学意义（p＞0.05）。 结论：TCDD可稳定地致胎鼠腭裂，并可使基因TGF-β3、BMP4mRNA表达水平发生显著性改变。 第二部分组蛋白H3乙酰化在TCDD致胎鼠腭裂发生中的作用 目的：探讨组蛋白H3乙酰化在2,3,7,8-四氯二苯二VA英所致的C57BL/6J胎鼠腭裂畸形发生中可能的作用，及其可能的作用机制。 方法：将18只C57BL/6J孕鼠随机分为TCDD组和对照组，每组9只，TCDD组于受孕后第10天(GDl0)以TCDD28μg／kg灌胃1次；对照组于GDl0以等量玉米油灌胃；分别于GD13.5、GD14.5及GD15.5处死孕鼠，收集胎鼠的上腭组织提取核蛋白，用比色法测定组蛋白乙酰化酶(histone acetyltransferases, HATs)活性。另选取18只孕鼠随机分为TCDD组和对照组（分组及处理方法同前），提取上述时间点各组组织核蛋白，用Western-blot方法检测乙酰化的组蛋白H3(Acetylatedhistone H3, Ac-H3)在上腭组织的表达情况。 结果：在GD13.5、GD14.5和GD15.5所测HATs相对活性OD值在对照组分别是0.4097±0.0147、0.5223±0.01709和0.6435±0.0139,TCDD组分别是0.8650±0.0129、0.7191±0.0178和0.5512±0.0168;在GD13.5, GD14.5TCDD组的HATs活性分别高于对照组，差异有统计学意义(P㩳0.01);而在GD15.5TCDD组HATs活性低于对照组，两组比较差异有统计学意义(P0.01)。在GD13.5、GD14.5和GD15.5Ac-H3相对表达量对照组分别是0.7450±0.1135、1.0559±0.2494和1.7955±0.0819, TCDD组分别是1.4490±0.1460、1.6418±0.0997和1.5121±0.1502; TCDD组的Ac-H3表达在GD13.5、GD14.5分别高于对照组GD13.5、GD14.5,差异均有统计学意义(P0.01, P0.05)；而在GD15.5，TCDD组的Ac-H3表达低于对照组，两组比较差异有统计学意义(P0.05)。 结论：组蛋白H3乙酰化参与了TCDD所致的C57BL/6J胎鼠腭裂的发生，可能是TCDD诱导腭裂发生的机制之一。
[Abstract]:Part one: expression of TGF- beta 3 and BMP4mRNA in TCDD induced cleft palate of fetal rats
Objective: To study the expression of transforming growth factor - beta 3 (transforming growth factor- beta 3, TGF- beta 3) and bone morphogenetic protein 4 (bone morphogenetic protein4) in C57BL/6J gestation rats with 2,3,7,8- four chloro two benzene two VA English (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) induced cleft palate. Discuss the possible mechanism in it.
Methods: 10 C57BL/6J pregnant rats were randomly divided into TCDD group (5 rats) and control group (5 rats), group TCDD was given 1 times with TCDD28 muon g / kg, and the control group was observed on GDl0 with equal amount of corn oil and observed in GD16.5 on the gross anatomy form. At the same time, 4% polyformaldehyde was used to fix fetal head and HE staining for histological observation. And 36 pregnant rats were randomly divided into two groups. In group TCDD (18) and control group (18 rats), the total RNA was extracted from the palatine process of each group of fetal rats, and the expression level of TGF- beta 3 and BMP4mRNA was detected by Q-PCR technique in GD13.5, GD14.5 and GD15.5.
Results: in GD16.5, TCDD group had cleft palate, and the control group was completely normal. In GD13.5, GD14.5 and GD15.5, the relative expression of TGF- beta 3mRNA in the control group was 0.7720 + 0.1548,0.8373 + 0.2458 and 0.9475 + 0.1737 respectively. The TCDD group was 1.0948 + 0.1733,1.1624 + 0.1892 and 1.0482 + 0.3438 respectively, TCDD group TGF- beta 3 was higher than the control group. There were statistical significance (P < 0.01, P < 0.05), and in GD15.5, TGF- beta 3 was also higher than that in the control group (P > 0.05). In GD13.5, GD14.5 and GD15.5, the relative expression of BMP4mRNA in the control group was 0.8470 + 0.2168,1.0531 + 0.0178 and 0.6865 + 0.1436 respectively. The TCDD group was 1.1114 + + 0.0780 and 0.5825 + 0.1614, respectively. The difference between GD13.5 and GD14.5 was statistically significant (P < 0.05, P < 0.01), and there was no significant difference in GD15.5 (P > 0.05).
Conclusion: TCDD can cause fetal cleft palate in a stable manner, and can significantly change the expression level of TGF- beta 3 and BMP4mRNA.
The role of histone H3 acetylation in the development of cleft palate induced by TCDD in second parts
Objective: To investigate the possible role of histone H3 acetylation in the occurrence of cleft palate in C57BL/6J fetal rats induced by 2,3,7,8- four chloro two benzene two VA and its possible mechanism.
Methods: 18 C57BL/6J pregnant rats were randomly divided into TCDD group and control group, 9 rats in each group. Group TCDD was given 1 times after tenth days of pregnancy (GDl0) with TCDD28 mu g / kg, and the control group was fed with equal amount of corn oil in GDl0. The pregnant rats were killed in GD13.5, GD14.5 and GD15.5, and the upper palate tissues of fetal mice were collected and the protein B was determined by colorimetric method. The activity of histone acetyltransferases (HATs) was selected, and 18 pregnant mice were randomly divided into TCDD and control groups (group and treatment group). The nucleoprotein of each group was extracted at the time point, and the expression of the histone H3 (Acetylatedhistone H3, Ac-H3) in the upper palate tissues was detected by Western-blot.
Results: the relative active OD values of HATs in GD13.5, GD14.5 and GD15.5 were 0.4097 + 0.0147,0.5223 + 0.01709 and 0.6435 + 0.0139 in the control group, and TCDD group was 0.8650 + 0.0129,0.7191 + 0.0178 and 0.5512 + 0.0168 respectively. In GD13.5, the HATs activity of GD14.5TCDD group was higher than that of the control group, and the difference was statistically significant (P? 0.01). The activity of HATs in group DD was lower than that of the control group. The difference between the two groups was statistically significant (P0.01). In GD13.5, the relative expression of GD14.5 and GD15.5Ac-H3 in the control group was 0.7450 + 0.1135,1.0559 + 0.2494 and 1.7955 + 0.0819 respectively. The TCDD group was 1.4490 + 0.1460,1.6418 + 0.0997 and 1.5121 + 0.1502, respectively. The Ac-H3 expression in the TCDD group was higher than that of GD13.5. The control group GD13.5, GD14.5, the difference was statistically significant (P0.01, P0.05), but in GD15.5, TCDD group Ac-H3 expression was lower than the control group, the two groups were statistically significant (P0.05).
Conclusion: histone H3 acetylation is involved in TCDD induced cleft palate in C57BL/6J fetal rats, and may be one of the mechanisms of TCDD induced cleft palate.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714
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