小鼠跨胎盘RNA干扰EphB2基因在先天畸形中的实验研究

Experimental study of EphB2 gene in transplacental RNAi in congenital malformations in mice

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，48h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mRNATk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〈O．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。

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 real-time quantitative polymerase chain reaction （PCR） and Western blot were used for detecting the expression of EphB2. The anorectal development of newborn and l-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 Ringel＇s 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 hematoxylin ＆ eosin （HE） staining of sagittal section. No anoreetal malformation was found in SiEphB2 group. Conclusions Systemic delivery of SiEphB2 provides a valuable approach to gene silencing in embryos. words]