lncRNA AC119762.8-201在乙烯硫脲诱导大鼠胚胎肛门直肠畸形中表达的初步研究  

Expression of lncRNA AC119762.8-201 in rat embryonic anorectal malformation induced by ethylenethiourea

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作  者:戈舞 刘丹[1] 曹正浓 廉鹏 沈苗苗 贾慧敏[1] Ge Wu;Liu Dan;Cao Zhengnong;Lian Peng;Shen Miaomiao;Jia Huimin(Department of Pediatric Surgery,Affiliated Shengjing Hospital,China Medical University,Shenyang 110004,China)

机构地区:[1]中国医科大学附属盛京医院小儿外科,沈阳110004

出  处:《中华小儿外科杂志》2021年第7期650-658,共9页Chinese Journal of Pediatric Surgery

基  金:国家自然科学基金(82070531);国家自然科学基金(818671503);盛京医院345人才计划项目基金(40A);辽宁省兴辽英才计划项目资助(XLYC1908008)。

摘  要:目的验证lncRNA AC119762.8-201在乙烯硫脲(ethylene thiourea,ETU)诱导的大鼠胚胎肛门直肠畸形(anorectal malformation,ARM)中的差异表达;研究干扰及过表达lncRNA AC119762.8-201后对大鼠小肠隐窝上皮细胞株(intestinal epithelial cell line,IEC-6)的细胞增殖、迁移(上皮-间质转换)及Wnt5a蛋白表达水平的影响。方法选用体重250~300 g的健康成年未育的Wistar大鼠30只,其中雌鼠24只,雄鼠6只;雌雄大鼠按4∶1的比例于晚上合笼过夜,次日晨雌鼠做阴道涂片,在光镜下见到精子或阴道栓即确认已交配,当日为孕0 d(E0),选取明确受孕的18只雌鼠按随机数字表法随机分为大鼠ETU-ARM致畸组(9只)和大鼠生理盐水对照组(9只)。孕10 d(E10)时,将大鼠ETU-ARM致畸组一次性灌胃注入1%ETU(125 mg/kg),大鼠生理盐水对照组灌入等量生理盐水。在孕16 d(E16)、17 d(E17)和19 d(E19),对两组孕鼠剖宫并取出胎鼠。无菌操作下,从大鼠ETU-ARM致畸组中共取得162只胎鼠,根据肉眼及解剖显微镜下观察,若胎鼠出现无尾或短尾畸形,且肛门与外界不相通,直肠末端与尿道之间有瘘管连通,则纳入胎鼠ARM组(简称ARM组),ARM组共122只致畸胎鼠,致畸率为75.31%(122/162);从大鼠生理盐水对照组取得141只胎鼠,胎鼠尾部均正常,无肛门直肠畸形,肛门与外界相通且直肠末端与尿道之间无瘘管连通,为胎鼠正常组(简称正常组)。解剖显微镜下,在无菌条件下取出ARM组和正常组胎鼠的直肠末端组织置于-80℃冰箱保存备用。采用实时定量聚合酶链反应(quantificational real-time polymerase chain reaction,qRT-PCR)检测lncRNA AC119762.8-201的表达;提取IEC-6细胞质和细胞核RNA,通过qRT-PCR检测lncRNA AC119762.8-201在细胞中的定位;采用siRNA序列或过表达质粒转染的方法下调或上调IEC-6细胞中lncRNA AC119762.8-201的表达,通过细胞计数试剂盒8(cell counting kit-8,CCK-8)法检测细胞增殖,蛋白质印迹法检测细胞上皮-间�Objective To verify the differential expression of lncRNA AC119762.8-201 in ethylenethiourea(ETU)-induced rat embryonic anorectal malformation(ARM)and explore the effects of interference and over-expression of lncRNA AC119762.8-201 on cell proliferation,migration(epithelial-mesenchymal transition)and Wnt5a protein expression levels in rat small intestinal crypt epithelial cell line(IEC-6).Methods Thirty healthy adult juvenile Wistar rats weighing 250 to 300 grams were used,including 24 female and 6 male rats;male and female rats were cohabited overnight at night in a ratio of 4:1 and vaginal smears were performed in female rats the next morning to confirm that they had mated by visualizing sperm or vaginal emboli under light microscope.At 0 day of gestation(EO)on the same day,18 clearly conceiving female rats were selected and randomly divided into rat ETU-ARM teratogenic group(n=9)and rat saline control group(n=9)according to the digital randomization method.At E10,ARM group received a single intragastric injection of 1%ETU(125 mg/kg)and normal group the same amount of normal saline.At E16/17/19,two groups of pregnant rats were dissected and fetuses removed.Under aseptic handling,a total of 162 fetuses were obtained from rat ETU-ARM teratogenic group.Based upon the observations under naked eye and dissecting microscope,if fetuses had anuran or short tail deformity,anus had non-communication with the outside and a fistula connection between rectal end and urethra,they were included into fetal rat ARM group.There were 122 teratogenic rats in ARM group and 75.31%(122/162)were teratogenic;141 fetuses were obtained from saline control group,fetal tails were normal,there was no anorectal malformation,anus communicating with the outside and no fistula connection between rectal end and urethra(normal group).Under dissecting microscope,terminal rectal tissues of ARM normal fetuses were removed under aseptic conditions and stored in a-80℃freezer for later use.The expression of lncRNAAC119762.8-201 was detected by real-t

关 键 词:大鼠 肛门直肠畸形 长链非编码RNA 细胞增殖 细胞迁移 

分 类 号:R725.7[医药卫生—儿科]

 

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