中华小儿外科杂志
中華小兒外科雜誌
중화소인외과잡지
CHINESE JOURNAL OF PEDIATRIC SURGERY
2013年
10期
766-771
,共6页
金鑫%郭振华%向丽%丁雄辉%孙艳辉%赵占波%刘宏%刘靓%陈中美
金鑫%郭振華%嚮麗%丁雄輝%孫豔輝%趙佔波%劉宏%劉靚%陳中美
금흠%곽진화%향려%정웅휘%손염휘%조점파%류굉%류정%진중미
RNA干扰%骨形态发生蛋白4%小鼠
RNA榦擾%骨形態髮生蛋白4%小鼠
RNA간우%골형태발생단백4%소서
RNA Interference%Bone morphogenetic protein 4%Mice
目的 应用跨胎盘RNA干扰技术抑制胚胎鼠骨形态发生蛋白4(BMP4)基因,为获取基因下调小鼠探索经济高效技术.方法 构建发育相关基因BMP4下调质粒后通过尾静脉向孕鼠注射Ringer's液、pSES质粒、pSES-SiBMP4质粒.部分孕鼠注射48 h后取出胚胎观测胚胎发育变化,并应用RT-PCR及Western blotting检测BMP4基因mRNA和蛋白质；部分孕鼠自然分娩后研究子代鼠生长发育变化.结果 在mRNA水平,pSES-SiBMP4组胚胎BMP4基因较Ringer's组、pSES组分别下调(29.4±5.0)％,(42.2±9.2)％(P＜0.05)；在蛋白质水平,pSES-SiBMP4组胚胎BMP4基因较Ringer's组、pSES组分别下调(27.0±6.4)％、(31.1±5.8)％(P＜0.05).pSES-SiBMP4组胚胎鼠头部、胃肠道及脊柱发育异常.新生子代鼠中pSES-SiBMP4组出现癫痫,肠息肉,先天性巨结肠.结论 胚胎鼠早期跨胎盘RNA干扰现实可行,跨胎盘SiBMP4可下调子代鼠基因.该方法可用来研究基因功能,构建先天性巨结肠,诱导肠息肉形成.
目的 應用跨胎盤RNA榦擾技術抑製胚胎鼠骨形態髮生蛋白4(BMP4)基因,為穫取基因下調小鼠探索經濟高效技術.方法 構建髮育相關基因BMP4下調質粒後通過尾靜脈嚮孕鼠註射Ringer's液、pSES質粒、pSES-SiBMP4質粒.部分孕鼠註射48 h後取齣胚胎觀測胚胎髮育變化,併應用RT-PCR及Western blotting檢測BMP4基因mRNA和蛋白質；部分孕鼠自然分娩後研究子代鼠生長髮育變化.結果 在mRNA水平,pSES-SiBMP4組胚胎BMP4基因較Ringer's組、pSES組分彆下調(29.4±5.0)％,(42.2±9.2)％(P＜0.05)；在蛋白質水平,pSES-SiBMP4組胚胎BMP4基因較Ringer's組、pSES組分彆下調(27.0±6.4)％、(31.1±5.8)％(P＜0.05).pSES-SiBMP4組胚胎鼠頭部、胃腸道及脊柱髮育異常.新生子代鼠中pSES-SiBMP4組齣現癲癇,腸息肉,先天性巨結腸.結論 胚胎鼠早期跨胎盤RNA榦擾現實可行,跨胎盤SiBMP4可下調子代鼠基因.該方法可用來研究基因功能,構建先天性巨結腸,誘導腸息肉形成.
목적 응용과태반RNA간우기술억제배태서골형태발생단백4(BMP4)기인,위획취기인하조소서탐색경제고효기술.방법 구건발육상관기인BMP4하조질립후통과미정맥향잉서주사Ringer's액、pSES질립、pSES-SiBMP4질립.부분잉서주사48 h후취출배태관측배태발육변화,병응용RT-PCR급Western blotting검측BMP4기인mRNA화단백질；부분잉서자연분면후연구자대서생장발육변화.결과 재mRNA수평,pSES-SiBMP4조배태BMP4기인교Ringer's조、pSES조분별하조(29.4±5.0)％,(42.2±9.2)％(P＜0.05)；재단백질수평,pSES-SiBMP4조배태BMP4기인교Ringer's조、pSES조분별하조(27.0±6.4)％、(31.1±5.8)％(P＜0.05).pSES-SiBMP4조배태서두부、위장도급척주발육이상.신생자대서중pSES-SiBMP4조출현전간,장식육,선천성거결장.결론 배태서조기과태반RNA간우현실가행,과태반SiBMP4가하조자대서기인.해방법가용래연구기인공능,구건선천성거결장,유도장식육형성.
Objective To assess an economical and efficient technology to establish knock-down mouse by trans-placental RNAi-BMP4 (Bone morphogenetic protein) to mouse embryos.Methods After established BMP4 down-regulatory plasmid,the Ringer's,pSES and pSES-SiBMP4 were delivered to pregnant mouse via tail vein.Some embryos were taken out 48h after injection,then RTPCR and Western blotting were used to analyze the expression of BMP4 in embryos.The growth and development of newborn mouse were observed.Results The mRNA expression of BMP4 in pSES-SiBMP4 embryo group was downregulated by (29.4± 5.0)％,compared with Ringer's group and by (42.2 ±9.2)％ compared with pSES group (P＜0.05).The protein level of BMP4 in pSES-SiBMP4 embryo group was reduced by (27.0 ± 6.4)％ compared with Ringer's group and by (31.1 ± 5.8)％ compared with pSES group (P＜0.05).There was no significant malformation detected in Ringer's group or control group.In mouse embryos of pSES-SiBMP4 group,abnormalities in head,gastrointestinal tract or backbone were detected,and epilepsy,intestinal polyps and Hirschsprung were found in offsprings.Conclusions It is feasible to deliver plasmid to early staged mouse embryos,pSES-SiBMP4 can knock down BMP4 gene expression.This technique can potentially be used to evaluate gene function or to induce Hirschsprung's disease or intestinal polyps mouse model.
