中华小儿外科杂志
中華小兒外科雜誌
중화소인외과잡지
Chinese Journal of Pediatric Surgery
2015年
9期
656-660
,共5页
王大佳%唐爽%张志波%苏朋俊%白玉作%袁正伟%王维林
王大佳%唐爽%張誌波%囌朋俊%白玉作%袁正偉%王維林
왕대가%당상%장지파%소붕준%백옥작%원정위%왕유림
RNA干扰%基因,抑制%畸形
RNA榦擾%基因,抑製%畸形
RNA간우%기인,억제%기형
RNA Interference%Genes,suppressor%Abnormalities
目的 应用跨胎盘RNA干扰技术抑制胚胎鼠EphB2基因,探索研究易感基因在先天性畸形中的作用.方法 构建先天性肛门直肠畸形(anorectal malformation,ARM)相关易感基因EphB2下调质粒SiEphB2,设计3个不同位点SiEphB2,通过鼠尾静脉向孕鼠注射SiEphB2质粒,空载体及Ringer's液.孕鼠在孕9.5d注射,48 h后取出胚胎,应用实时定量PCR及Western blotting 检测EphB2基因在子代鼠的mRNA和蛋白质表达水平.并观察新生鼠的肛门直肠发育情况.结果 在mRNA水平,发现SiEphB2载体注射组(SiEphB2组)EphB2基因表达比较空载体注射组(Vector组)及释液注射组(Ringer's组)明显降低.Ringer's组均值为0.99±0.06,Vector组为0.87±0.07;SiEphB2组中,SiEphB2-1组均值为0.27±0.03,SiEphB2-2组为0.34±0.04,SiEphB2-3组为0.42±0.02.SiEphB2组胚胎EphB2基因较Ringer's组和Vector组分别下调(65.3±6.6)％和(60.6±8.6)％,差异有统计学意义(P＜0.05).在蛋白质水平,Ringer's组为1.00±0.05,Vector组为0.93±0.09;SiEphB2组中,SiEphB2-1组为0.20±0.03;SiEphB2-2组为0.33±0.02;SiEphB2-3组为0.39±0.02.SiEphB2组胚胎EphB2基因较Ringer's组和Vector组分别下调(69.3±7.8)％和(66.9±9.6)％,差异有统计学意义(P＜0.05).大体观察SiEphB2组胎鼠可见肛门及鼠尾发育,矢状位切片HE染色未见ARM发生.结论 胚胎鼠早期跨胎盘RNA干扰是研究基因功能现实可行的研究方法,跨胎盘SiEphB2可下调子代鼠基因.
目的 應用跨胎盤RNA榦擾技術抑製胚胎鼠EphB2基因,探索研究易感基因在先天性畸形中的作用.方法 構建先天性肛門直腸畸形(anorectal malformation,ARM)相關易感基因EphB2下調質粒SiEphB2,設計3箇不同位點SiEphB2,通過鼠尾靜脈嚮孕鼠註射SiEphB2質粒,空載體及Ringer's液.孕鼠在孕9.5d註射,48 h後取齣胚胎,應用實時定量PCR及Western blotting 檢測EphB2基因在子代鼠的mRNA和蛋白質錶達水平.併觀察新生鼠的肛門直腸髮育情況.結果 在mRNA水平,髮現SiEphB2載體註射組(SiEphB2組)EphB2基因錶達比較空載體註射組(Vector組)及釋液註射組(Ringer's組)明顯降低.Ringer's組均值為0.99±0.06,Vector組為0.87±0.07;SiEphB2組中,SiEphB2-1組均值為0.27±0.03,SiEphB2-2組為0.34±0.04,SiEphB2-3組為0.42±0.02.SiEphB2組胚胎EphB2基因較Ringer's組和Vector組分彆下調(65.3±6.6)％和(60.6±8.6)％,差異有統計學意義(P＜0.05).在蛋白質水平,Ringer's組為1.00±0.05,Vector組為0.93±0.09;SiEphB2組中,SiEphB2-1組為0.20±0.03;SiEphB2-2組為0.33±0.02;SiEphB2-3組為0.39±0.02.SiEphB2組胚胎EphB2基因較Ringer's組和Vector組分彆下調(69.3±7.8)％和(66.9±9.6)％,差異有統計學意義(P＜0.05).大體觀察SiEphB2組胎鼠可見肛門及鼠尾髮育,矢狀位切片HE染色未見ARM髮生.結論 胚胎鼠早期跨胎盤RNA榦擾是研究基因功能現實可行的研究方法,跨胎盤SiEphB2可下調子代鼠基因.
목적 응용과태반RNA간우기술억제배태서EphB2기인,탐색연구역감기인재선천성기형중적작용.방법 구건선천성항문직장기형(anorectal malformation,ARM)상관역감기인EphB2하조질립SiEphB2,설계3개불동위점SiEphB2,통과서미정맥향잉서주사SiEphB2질립,공재체급Ringer's액.잉서재잉9.5d주사,48 h후취출배태,응용실시정량PCR급Western blotting 검측EphB2기인재자대서적mRNA화단백질표체수평.병관찰신생서적항문직장발육정황.결과 재mRNA수평,발현SiEphB2재체주사조(SiEphB2조)EphB2기인표체비교공재체주사조(Vector조)급석액주사조(Ringer's조)명현강저.Ringer's조균치위0.99±0.06,Vector조위0.87±0.07;SiEphB2조중,SiEphB2-1조균치위0.27±0.03,SiEphB2-2조위0.34±0.04,SiEphB2-3조위0.42±0.02.SiEphB2조배태EphB2기인교Ringer's조화Vector조분별하조(65.3±6.6)％화(60.6±8.6)％,차이유통계학의의(P＜0.05).재단백질수평,Ringer's조위1.00±0.05,Vector조위0.93±0.09;SiEphB2조중,SiEphB2-1조위0.20±0.03;SiEphB2-2조위0.33±0.02;SiEphB2-3조위0.39±0.02.SiEphB2조배태EphB2기인교Ringer's조화Vector조분별하조(69.3±7.8)％화(66.9±9.6)％,차이유통계학의의(P＜0.05).대체관찰SiEphB2조태서가견항문급서미발육,시상위절편HE염색미견ARM발생.결론 배태서조기과태반RNA간우시연구기인공능현실가행적연구방법,과태반SiEphB2가하조자대서기인.
Objective To explore a predisposing gene research platform for congenital malformations in mice caused by transplacental RNAi-EphB2.Methods The EphB2 down-regulatory plasmid SiEphB2 and vector in Ringer's solution were delivered into tail vein of mice 9.5 days of pregnancy.Plasmids for SiRNA contained three different sites of RNAi for silencing EphB2 gene.The embryos were removed 48 h after injection and reabtime quantitative polymerase chain reaction (PCR) and Western blot were used for detecting the expression of EphB2.The anorectal development of newborn and1-week-old mice was observed.Results The expressions of EphB2 mRNA in all three SiEphB2 groups (SiEphB2-1 : 0.27 ± 0.03;SiEphB2-2: 0.34 ± 0.04;SiEphB2-3: 0.42 ± 0.02) were significantly lower than those in Ringers group (0.99 ± 0.06) and in vector group (0.87 ± 0.07).The mRNA expression of EphB2 in SiEphB2 embryos was down-regulated by (65.3 ± 6.6) ％ versus Ringers group and by (60.6 ± 8.6) ％ versus vector group (P＜0.05).The expression of EphB2 protein in SiEphB2 groups (SiEphB2-1 : 0.20 ± 0.03;SiEphB2-2: 0.33 ± 0.02;SiEphB2-3: 0.39 ± 0.02) decreased markedly versus that in Ringer' s group (1.00 ± 0.05) and vector group (0.93 ± 0.09).The protein level of EphB2 decreased by (69.3± 7.8)％ in SiEphB2 embryo group versus Ringer's group and by (66.9 ± 9.6)％ versus vector group(P＜0.05).In SiEphB2 group, normal development of anus and tail was confirmed by gross observation and hernatoxylin & eosin (HE) staining of sagittal section.No anorectal malformation was found in SiEphB2 group.Conclusions Systemic delivery of SiEphB2 provides a valuable approach to gene silencing in embryos.
