中华口腔医学杂志
中華口腔醫學雜誌
중화구강의학잡지
Chinese Journal of Stomatology
2014年
12期
728-732
,共5页
张岱尊%许尧祥%肖文林%庄翠竹
張岱尊%許堯祥%肖文林%莊翠竹
장대존%허요상%초문림%장취죽
腭%腭裂%基因沉默%上颌面部发育
腭%腭裂%基因沉默%上頜麵部髮育
악%악렬%기인침묵%상합면부발육
Palate%Cleft palate%Gene silencing%Maxillofacial development
目的 研究沉默7-脱氢胆固醇还原酶(7-dehydrocholesterol reductase,Dhcr-7)表达对体外培养腭突器官中音猬基因(sonic hedgehog,Shh)-骨形成蛋白2(bone morphogenetic protein 2,BMP-2)信号通路的影响,探讨Dhcr-7参与腭突发育的信号通路.方法 取60只孕期(gestation day,GD) 13.5d小鼠胚胎根据简单随机抽样法平均分为3组:空白对照组(A组):不含胆固醇培养基培养腭突;Dhcr-7基因沉默组(B组):不含胆固醇培养基培养腭突+Dhcr-7-siRNA腺病毒;添加胆固醇组(C组);每组各20只.培养48 h后,A、B组更换不含胆固醇培养基,C组更换含有600 mg/L胆固醇培养基.继续培养72 h后,分别将腭突固定,组织染色和扫描电镜观察其形态变化;分别提取腭突RNA和蛋白质,应用反转录-聚合酶链反应(reverse transcriotion-polymerase chain reaction,RT-PCR)和蛋白质印迹法检测Dhcr-7、Shh和BMP-2表达量的变化.结果 组织染色和扫描电镜显示A组及C组腭突能完全融合,B组腭突未融合.Shh和BMP-2在B组的mRNA和蛋白质的表达量随Dhcr-7表达量降低而降低.B组mRNA和蛋白质的表达量Shh为0.063±0.018和0.092±0.065;BMP-2为0.054±0.018和0.049±0.021;A组mRNA和蛋白质的表达量Shh为0.667±0.093和0.639±0.078;BMP-2为0.591±0.043和0.569±0.081.A、B两组Shh和BMP-2的mRNA和蛋白质的表达量差异分别具有统计学意义(P＜0.05);C组Dhcr-7的mRNA表达量(0.074±0.034)和蛋白质表达量(0.075±0.028)基本无变化,与B组(Dhcr-7的mRNA表达量为0.083±0.045;蛋白质表达量为0.067±0.065)相比,差异无统计学意义(P＞0.05);RNA和蛋白质的表达量Shh(0.649±0.085和0.608±0.092)和BMP-2(0.578±0.062和0.548±0.065)均明显升高,与B组相比差异有统计学意义(P＜0.05).结论 Dhcr-7可影响Shh和BMP-2的表达,Dhcr-7通过Shh-BMP-2信号通路调控腭突发育.
目的 研究沉默7-脫氫膽固醇還原酶(7-dehydrocholesterol reductase,Dhcr-7)錶達對體外培養腭突器官中音猬基因(sonic hedgehog,Shh)-骨形成蛋白2(bone morphogenetic protein 2,BMP-2)信號通路的影響,探討Dhcr-7參與腭突髮育的信號通路.方法 取60隻孕期(gestation day,GD) 13.5d小鼠胚胎根據簡單隨機抽樣法平均分為3組:空白對照組(A組):不含膽固醇培養基培養腭突;Dhcr-7基因沉默組(B組):不含膽固醇培養基培養腭突+Dhcr-7-siRNA腺病毒;添加膽固醇組(C組);每組各20隻.培養48 h後,A、B組更換不含膽固醇培養基,C組更換含有600 mg/L膽固醇培養基.繼續培養72 h後,分彆將腭突固定,組織染色和掃描電鏡觀察其形態變化;分彆提取腭突RNA和蛋白質,應用反轉錄-聚閤酶鏈反應(reverse transcriotion-polymerase chain reaction,RT-PCR)和蛋白質印跡法檢測Dhcr-7、Shh和BMP-2錶達量的變化.結果 組織染色和掃描電鏡顯示A組及C組腭突能完全融閤,B組腭突未融閤.Shh和BMP-2在B組的mRNA和蛋白質的錶達量隨Dhcr-7錶達量降低而降低.B組mRNA和蛋白質的錶達量Shh為0.063±0.018和0.092±0.065;BMP-2為0.054±0.018和0.049±0.021;A組mRNA和蛋白質的錶達量Shh為0.667±0.093和0.639±0.078;BMP-2為0.591±0.043和0.569±0.081.A、B兩組Shh和BMP-2的mRNA和蛋白質的錶達量差異分彆具有統計學意義(P＜0.05);C組Dhcr-7的mRNA錶達量(0.074±0.034)和蛋白質錶達量(0.075±0.028)基本無變化,與B組(Dhcr-7的mRNA錶達量為0.083±0.045;蛋白質錶達量為0.067±0.065)相比,差異無統計學意義(P＞0.05);RNA和蛋白質的錶達量Shh(0.649±0.085和0.608±0.092)和BMP-2(0.578±0.062和0.548±0.065)均明顯升高,與B組相比差異有統計學意義(P＜0.05).結論 Dhcr-7可影響Shh和BMP-2的錶達,Dhcr-7通過Shh-BMP-2信號通路調控腭突髮育.
목적 연구침묵7-탈경담고순환원매(7-dehydrocholesterol reductase,Dhcr-7)표체대체외배양악돌기관중음위기인(sonic hedgehog,Shh)-골형성단백2(bone morphogenetic protein 2,BMP-2)신호통로적영향,탐토Dhcr-7삼여악돌발육적신호통로.방법 취60지잉기(gestation day,GD) 13.5d소서배태근거간단수궤추양법평균분위3조:공백대조조(A조):불함담고순배양기배양악돌;Dhcr-7기인침묵조(B조):불함담고순배양기배양악돌+Dhcr-7-siRNA선병독;첨가담고순조(C조);매조각20지.배양48 h후,A、B조경환불함담고순배양기,C조경환함유600 mg/L담고순배양기.계속배양72 h후,분별장악돌고정,조직염색화소묘전경관찰기형태변화;분별제취악돌RNA화단백질,응용반전록-취합매련반응(reverse transcriotion-polymerase chain reaction,RT-PCR)화단백질인적법검측Dhcr-7、Shh화BMP-2표체량적변화.결과 조직염색화소묘전경현시A조급C조악돌능완전융합,B조악돌미융합.Shh화BMP-2재B조적mRNA화단백질적표체량수Dhcr-7표체량강저이강저.B조mRNA화단백질적표체량Shh위0.063±0.018화0.092±0.065;BMP-2위0.054±0.018화0.049±0.021;A조mRNA화단백질적표체량Shh위0.667±0.093화0.639±0.078;BMP-2위0.591±0.043화0.569±0.081.A、B량조Shh화BMP-2적mRNA화단백질적표체량차이분별구유통계학의의(P＜0.05);C조Dhcr-7적mRNA표체량(0.074±0.034)화단백질표체량(0.075±0.028)기본무변화,여B조(Dhcr-7적mRNA표체량위0.083±0.045;단백질표체량위0.067±0.065)상비,차이무통계학의의(P＞0.05);RNA화단백질적표체량Shh(0.649±0.085화0.608±0.092)화BMP-2(0.578±0.062화0.548±0.065)균명현승고,여B조상비차이유통계학의의(P＜0.05).결론 Dhcr-7가영향Shh화BMP-2적표체,Dhcr-7통과Shh-BMP-2신호통로조공악돌발육.
Objective To investigate the effect of 7-dehydrocholesterol reductas(Dhcr-7) gene silencing on the palatal development by sonic hedgehog(Shh)-bone morphogenetic protein2(BMP-2) signal pathway in vitro.Methods A total of 60 pairs of palatal shelves fromgestation day(GD) 13.5 mouse embryos were divided into three groups(A,B,C) of 20 randomly.In group A(control),palatal shelves were cultured with medium containing no cholesterol.In group B(Dhcr-7-siRNA),palatal shelves were cultured without cholesterol medium but containing Dhcr-7 siRNA adenovirus.After 48h,the culture medium of groups A and B were changed with medium without cholesterol.In group C(cholesterol),palatal shelves were cultured without cholesterol medium but containing Dhcr-7 siRNA adenovirus.After 48h,the culture medium of group C was changed with medium containing 600 mg/L cholesterol.After 72h again,tissues dyeing and scanning electron microscope(SEM) technique were used to observe morphological changes of palates.Both RT-PCR and Western blottingtechniques were used to measure mRNA and protein expressions for Dhcr-7,Shh,and BMP-2,respectively.Results The tissues dyeing and SEM showedthat the palates fusedin groups A and Candthe palates did not fuse in group B eventually.The expression of both mRNA and proteins for Shh and BMP-2 in group B wasdecreased with the Dhcr-7 reduction.In group B,the mRNA and protein expression of Shh was separately 0.063±0.018 and 0.092±0.065;the mRNA and protein expression quantity of BMP-2 was separately 0.054±0.018 and 0.049±0.021.In group A,the mRNA and protein expression of Shh was separately 0.667±0.093 and 0.639±0.078;the mRNA and protein expression of BMP-2 was separately 0.591 ±0.043 and 0.569± 0.081.The difference of Shh and BMP-2 mRNA and protein expression between A and B group were statistically significant separately(P＜0.05).The expression of both mRNA and protein for Dhcr-7(0.074±0.034 and 0.075±0.028) did not changebasicallyin group C,compared with the Dhcr-7expression of mRNA and protein(0.083 ± 0.045;0.067± 0.065) in group B,the difference wasnot statistically significant(P＞0.05).In group C,the mRNA and protein expressionof Shh(0.649±0.085 and 0.608±0.092) and BMP-2(0.578±0.062 and 0.548±0.065) were significantly increased.The difference of Shh and BMP-2 mRNA and protein expression between B and C group were statistically significant separately(P＜ 0.05).Conclusions Dhcr-7 could influence the expression of Shh and BMP-2.Dhcr-7 reductase regulated the palatal development by the Shh-BMP-2 signal pathway.
