运用纳米板转导siRNA抑制HPV基因表达的量效学探究  

Dose-effect Research Using Nanopatch to Deliver siRNA in the Inhibition of HPV Gene Expression

作　　者：熊倬[1] 董晓静[1] 孙培文[1] 张莹[1] 

机构地区：[1]重庆医科大学附属第二医院妇产科,重庆400010

出　　处：《生物医学工程学杂志》2013年第6期1283-1289,共7页Journal of Biomedical Engineering

基　　金：重庆市卫生局资助项目(2010-2-162);重庆市教委资助项目(KJ110310);重庆市科学技术委员会(重庆市自然科学基金)资助项目(CSTC;2009BB5071)

摘　　要：Hela细胞是人宫颈腺癌细胞株,人乳头状瘤病毒(HPV)18表达阳性。本实验用纳米板转导靶向于HPV18E7mRNA的siRNA进入Hela细胞裸鼠皮下移植瘤(宫颈癌动物模型),分析siRNA对HPV基因表达的抑制作用,进而探讨纳米板在动物水平上转导siRNA的优越性及转导的最佳作用时间和浓度。合成针对HPV18E7mRNA的siRNA(简称siE7),细胞实验检测siE7转录后的沉默效果。采用纳米板转导siE7与GenEscort TMⅢ(转染试剂)复合物进入Hela细胞裸鼠皮下移植瘤,逆转录PCR(RT-PCR)、Western blot分别检测作用0、24、48、72h后瘤体组织HPV18E7mRNA及蛋白的表达变化,分析纳米板作用的最佳时间。不同浓度siE7与GenEscort TMⅢ体外孵育复合物,分别用纳米板及腹腔注射转导进入Hela细胞裸鼠皮下移植瘤,RT-PCR、Western blot分别检测72h后瘤体组织HPV18E7mRNA及蛋白的表达变化,选择纳米板最佳作用浓度,对照腹腔注射组分析纳米板转导siRNA在动物水平上的优越性。体外实验结果证明siE7能有效沉默HPV18E7mRNA表达并促进Hela细胞凋亡。动物实验结果证明,纳米板转导的siE7能有效沉默HPV18E7mRNA,并抑制HPV18E7蛋白表达,转导siE7的最佳作用时间及浓度分别是72h、2μmol/L。与腹腔注射组比较,纳米板转导siRNA在动物水平上有明显的优越性。因此,纳米板能有效转导siRNA进入裸鼠皮下移植瘤组织,并且转导进入的siRNA可有效抑制HPV基因表达。Hela is the cell line of adenocarcinoma of the uterine cervix, and human papillomavirus （HPV） 18 shows positive. We delivered siRNA with target specifically to HPV18 E7 mRNA into nude mice Hela tumor xenografts by nanopatch to inhibit the HPV gene expression, and further to study the superiority, the best action time and concen- tration of siRNA of using nanopatch to transfer siRNA in vivo. We designed siRNA that target specifically to HPV18 E7 mRNA （siET）and checked the effect of siE7 in vitro. Tumor xenografts were transfected with siE7 and GenEscortTM Iti by nanopatch. Expression of HPV18 E7 mRNA and protein were detected 0 hours, 24 hours, 48 hours,72 hours after transfection with PT-PCR and Western blot, and the best action time was analyzed using nano patch to thansfect siRNA in vivo. We transfected GenEscortTM l]I and siE7 of Different concentration into tumor xen ografts respectively by nanopatch and intraperitoneal injection. Expression of HPV18 E7 mRNA and protein was de tected 72 hours after transfeetion by PT-PCR and Western blot, to analyze the best action concentration of siRNA and the superiority of using nanopatch to thansfect siRNA in vivo. The results proved that SiE7was efficient to inhibit expression of HPV18 E7 mRNA and to advance Hela apoptosis in vitro. SiE7 transfected by nanopatch into xeno grafts could inhibit effectively expression of HPV18 E7 mRNA and protein. The best action time and concen- tration of siRNA of using nanopateh to thansfect siRNA in vivo are 72 hour post-transfection and 2μmol/I. siE7. To compare intraperitoneal injection in delivering siRNA in vivo, the effect of nanopatch is very predominant. It can be well concluded that Nanopatch can effectively transfer siRNA in vivo, which can effectively inhibit the HPV gene ex-

关 键 词：宫颈癌 HELA细胞 纳米板 RNA干扰 人乳头状瘤病毒 

分 类 号：R737.33[医药卫生—肿瘤]