TCDD致胎鼠腭裂模型中miR-381-3p下调抑制腭间充质细胞成骨分化的实验研究  

Down-regulation of miR-381-3p inhibits osteogenic differentiation of mouse embryonic palatal mesenchymal cells in 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin-induced cleft palate of fetal mice

作　　者：蒋亨 袁心刚[1] 傅跃先[1] JIANG Heng;YUAN Xingang;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,P.R.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

出　　处：《中国修复重建外科杂志》2019年第9期1174-1180,共7页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家临床重点专科建设项目[国卫办医函(2013)544];重庆市渝中区科技计划项目(20150112)~~

摘　　要：目的探讨2, 3, 7, 8-四氯二苯二英(2,3,7,8-tetrachlorodibenzo-p-dioxin,TCDD)致胎鼠腭裂模型中miR-381-3p下调与胎鼠腭间充质(mouse embryonic palatal mesenchymal,MEPM)细胞成骨分化抑制的相关性。方法 32只6~8周龄SPF级建康C57BL/6J孕鼠随机分为TCDD组与对照组,每组16只。于胚胎日第10.5天(embryonic day 10.5,E10.5)时,TCDD组一次性灌胃TCDD(28μg/kg)、对照组给予等量玉米油。于E13.5和E14.5取出两组胎鼠大体观察腭突组织后,取E13.5和E14.5腭突组织行实时荧光定量PCR检测miR-381-3p表达;E14.5腭突组织Western blot检测成骨特异性转录因子RUNX2和骨桥蛋白(osteopontin,OPN)的表达。取对照组E14.5胎鼠腭突分离培养MEPM细胞,取第3代细胞以含10 nmol/L TCDD的完全培养基培养,于0、0.5、1、2、3 d检测miR-381-3p表达,0、1、2、3 d检测RUNX2和OPN蛋白表达。另取第3代MEPM细胞随机分为4组,沉默表达组及对照组分别转染miR-381-3p抑制物及抑制物对照,过表达组及对照组分别转染miR-381-3p模拟物及模拟物对照。转染48 h后,检测各组miR-381-3p及RUNX2和OPN蛋白表达。结果 E13.5和E14.5时对照组获得活胎鼠96只、TCDD组92只;其中,E14.5对照组活胎鼠可见双侧腭突接触,TCDD组双侧腭突中间有间隙。TCDD组胎鼠腭突组织miR-381-3p以及RUNX2、OPN蛋白相对表达量均明显低于对照组(P<0.05)。TCDD处理MEPM细胞0.5和1 d后miR-381-3p相对表达量较0 d时明显下降(P<0.05),2、3 d时表达量明显上升,与0 d时比较差异无统计学意义(P>0.05);1、2、3 d时RUNX2及OPN蛋白相对表达量均较0 d时显著降低(P<0.05)。MEPM细胞转染48 h后,沉默表达组miR-381-3p及RUNX2、OPN蛋白相对表达量均较其对照组下降,过表达组均较其对照组升高,差异有统计学意义(P<0.05)。结论在TCDD致胎鼠腭裂模型中,miR-381-3p表达下调可能抑制胎鼠MEPM细胞成骨分化。Objective To investigate the correlation between down-regulation of miR-381-3p and inhibition of osteogenic differentiation of mouse embryonic palatal mesenchymal (MEPM) cells in 2, 3, 7, 8-tetrachlorodibenzo-pdioxin (TCDD)-induced cleft palate of fetal mice. Methods Thirty-two pregnant mice were randomly divided into TCDD group and control group, 16 in each group. On embryonic day 10.5 (E10.5), the pregnant mice in TCDD group were orally administrated with TCDD at dosage of 28 μg/kg, while the pregnant mice in control group received equivalent corn oil. The pregnant mice in each group were sacrificed on E13.5 and E14.5, fetal palates were collected for analysis. The expression of miR-381-3p was detected by real-time fluorescent quantitative PCR and the protein expressions of runtrelated transcription factor 2 (RUNX2) and osteopontin (OPN) were detected by Western blot. MEPM cells were extracted from fetal palates on E14.5 in control group and passaged. The 3rd passage cells were cultured with TCDD at dosage of 10 nmol/L for 0, 0.5, 1, 2, and 3 days. The expression of miR-381-3p was detected after 0, 0.5, 1, 2, and 3 days and the protein expressions of RUNX2 and OPN were detected after 0, 1, 2, and 3 days. Then, the 3rd passage cells were divided into 4 groups. The MEPM cells were transfected with miR-381-3p inhibitor (inhibitor group), NC inhibitor (NC inhibitor group) and miR-381-3p mimics (mimics group), NC mimics (NC mimics group) for 48 hours, respectively. And the expressions of miR-381-3p and the protein expressions of RUNX2 and OPN were detected. Results On E13.5 and E14.5, 96 fetal mice in control group and 92 in TCDD group were obtained. The bilateral palates contacted in control group on E14.5, and a gap between the bilateral palates existed in TCDD group. On E13.5 and E14.5, the relative expressions of miR-381-3p and RUNX2 and OPN proteins were significant lower in TCDD group than in control group (P<0.05). The relative expression of miR-381-3p at 0.5 and 1 day after TCDD treatment of MEPM cells were

关 键 词：miR-381-3p 胎鼠腭间充质细胞 成骨分化 2 3 7 8-四氯二苯二英 腭裂 

分 类 号：R782.2[医药卫生—口腔医学]