2,3,7,8-四氯二苯二噁英诱导的胎鼠腭裂腭突组织细胞周期相关分子的表达变化  

Expression changes of cell cycle related molecules in palatal tissue of fetal mice with cleft palate induced by TCDD

作　　者：蒋亨 袁心刚[1] 魏光辉[2] 蒲玮 傅跃先[1] Jiang Hen;Yuan Xingang;Wei Guanghui;Pu Wei;Fu Yuexian(Department of Plastic Surgery,Children′s Hospital of Chongqing Medical University,Ministry of Education Key Laboratory of Child Development and Disorders,China International Science and Technology Cooperation Base of Child Development and Critical Disorders,Chongqing Key Laboratory of Pediatrics,Chongqing 400014,China;Chongqing Key Laboratory of Child Urogenital Development and Tissue Engineering,Chongqing 400014,China;Department of Biology,College of Medical Technology,Chengdu University of TCM,Chengdu 611137,China)

机构地区：[1]重庆医科大学附属儿童医院整形外科,儿童发育疾病研究教育部重点实验室,儿童发育重大疾病国家国际科技合作基地,儿科学重庆市重点实验室,400014 [2]重庆医科大学儿科研究所泌尿生殖发育与畸形研究室,400014 [3]成都中医药大学医学技术学院生物教研室,611137

出　　处：《中华整形外科杂志》2019年第7期686-694,共9页Chinese Journal of Plastic Surgery

基　　金：国家临床重点专科建设项目:小儿外科学(国卫办医函2013544);重庆市渝中区科技计划项目(20150112).

摘　　要：目的检测2,3,7,8-四氯二苯二噁英(2,3,7,8-tetrachlorodibenzo-p-dioxin,TCDD)诱导的胎鼠腭裂模型中腭突组织细胞周期相关分子的表达变化,初步探讨细胞周期相关分子在腭裂发生中的作用机制.方法使用随机数字表法将48只C57BL/6J孕鼠随机分为TCDD处理组与对照组,每组24只.TCDD处理组在胚胎日第10天(the embryonic day10.5,E10.5),按28μg/kg的量(含5μg/mlTCDD的玉米油)给予孕鼠一次性灌胃,对照组等量玉米油灌胃.分别在E13.5、E14.5及E15.5取出各组胎鼠腭突组织,提取总核糖核酸和总蛋白,分别用逆转录-聚合酶链式反应(RT-PCR)和蛋白质印迹法检测细胞周期相关分子的信使核糖核酸(mRNA)和蛋白表达水平.以不同浓度TCDD(0.01、0.1、0.5和1nmol/L)处理人肾胚293t细胞(HEK293t),并用噻唑蓝(MTT)检测其细胞增殖活力.以IBMSPSS24.0进行统计分析,两样本间均值比较均采用独立样本t检验,所有结果作K-S检验均符合正态分布,方差不齐者采用校正t检验,P<0.05表示差异有统计学意义.结果E13.5、E14.5和E15.5,干扰素调节因子6(Irf6)蛋白在对照组(1.26±0.13、1.67±0.14、1.42±0.15)较TCDD组(0.81±0.08、1.04±0.02、0.86±0.12)表达水平高,差异有统计学意义(t=2.836、3.662、2.867,P=0.0471、0.0146、0.0241);E14.5和E15.5时细胞周期蛋白依赖性激酶抑制因子1A(P21)蛋白在对照组(2.26±0.21、1.99±0.21)较TCDD组(1.43±0.12、0.93±0.22)表达水平高,差异有统计学意义(t值为3.398、3.378,P值为0.8726、0.0273).细胞周期相关分子的mRNA表达水平:各个时间点,细胞周期蛋白D1(cyclinD1)对照组(1.00±0.02、0.94±0.03、1.11±0.09)表达水平较TCDD组(0.28±0.01、0.33±0.06、0.88±0.01)高(t=22.53、22.35、14.27,均P<0.001);TCDD组cyclinE1、cyclinA2、cyclinB1、细胞周期蛋白依赖性激酶6(Cdk6)、Cdk2、Cdk1表达均较对照组高,差异有统计学意义(均P<0.05),其中E13.5cyclinB1和E15.5Cdk2在2组间差异无统计学意义(均P>0.05).0.1nmol/LTCDDObjective The purpose of this study is to investigate the expression change of cell cycle-related molecules in platal tissue of fetal mice with cleft palate, induced by 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin (TCDD), and to explore the mechanism of cell cycle-related molecules in cleft palate. Methods In vivo, 48 pregnant mice were randomly divided into TCDD treatment group and control group with Random number table, 24 mice in each group. On the embryonic day 10.5 (E10.5), pregnant mice were orally administrated with TCDD 28 μg/kg (containing 5 μg/ml TCDD of corn oil) in TCDD treatment group. The same volume of corn oil was given to the mice in control group. The pregnant mice in each group were sacrificed on E13.5, E14.5 and E15.5, to collect the fetal palates for analysis. Fetal palates were used to extract total RNA and total protein, so as to detect the expression levels of cell cycle-related molecules, using RT-PCR and western blotting respectively. In vitro, human kidney embryo 293t (HEK293t) cells were treated with different concentrations of TCDD (0.01, 0.1, 0.5 and 1 nmol/L), and cells proliferation activity was detected using MTT assay. Statistical analysis was performed with IBM SPSS 24.0. Kolmogorov-Smimov test was used for normal distribution check, and the distribution was normal. Independent t-test was carried out among two groups. P<0.05 was considered statistically significant. Results At E13.5, E14.5 and E15.5, the expression level of interferon regulatory factor 6 (Irf6) protein were higher in the control group (1.26 ± 0.13, 1.67 ± 0.14 and 1.42 ± 0.15, respectively) compared to that in the TCDD group (0.81 ± 0.08, 1.04± 0.02 and 0.86 ± 0.12, respectively), on each time point (t value were 2.836, 3.662 and 2.867, respectively;P values were 0.0471, 0.0146 and 0.0241, respectively). The expression level of cyclin-dependent kinase inhibitor 1A (P21) protein on E13.5 and E14.5 of the control group (2.26 ± 0.21, 1.99 ± 0.21)were higher than that in the TCDD group on each time point(1.43 ±

关 键 词：细胞周期 2 3 7 8-四氯二苯二噁英 腭裂 腭中缝上皮 

分 类 号：R782.2[医药卫生—口腔医学]