肠隐窝上皮细胞Myd88稳定沉默体外模型的建立及早期凋亡研究  

The Set-up of an in vitro Model for Stable Knockdown of MyD88 by Lentivirus-based RNAi in IEC-6 Cell Line and the Study on Its Early Apoptosis

作　　者：包平倩[1] 李杨[2] 陈珂玲[3] 周斌[3] 刘斌[1] 李园[1,3] 周总光[2,3,4] 

机构地区：[1]四川大学华西医院小儿外科,成都610041 [2]四川大学生物国家重点实验室,成都610041 [3]四川大学华西医院消化外科实验室,成都610041 [4]四川大学华西医院胃肠外科,成都610041

出　　处：《生物医学工程学杂志》2012年第6期1138-1143,1149,共7页Journal of Biomedical Engineering

基　　金：国家自然科学基金重点项目资助(30830100);国家自然科学基金面上项目资助(30972924);教育部留学回国人员启动基金资助项目(20101174-4-2)

摘　　要：肠道炎症性疾病临床常见,发病率逐年上升而病因不清。现已证实Toll样受体4(TLR4)信号通路与肠道炎性疾病密切相关。髓样分化蛋白88(Myd88)是TLR4炎症信号通路上游关键调控点,对肠道炎性疾病的研究具有重要价值,但目前尚缺乏Myd88稳定调控表达的体外模型。本实验利用慢病毒三质粒包装系统,构建特异靶向Myd88基因RNA干扰(RNAi)病毒载体。采用改良病毒离心法结合离心孵育法(MVC-S)行肠隐窝上皮细胞(IEC-6)转导,采用Real-time PCR及Western blot检测Myd88mRNA及蛋白水平,Annexin V染色、流式细胞术检测Myd88沉默后对IEC-6细胞早期凋亡影响。结果显示正确构建的慢病毒shRNA载体,可通过改良MVC-S法成功转导IEC-6细胞,转导后的IEC-6细胞Myd88表达明显下调,同时早期凋亡减少。本实验成功构建了慢病毒载体介导的RNAi的Myd88稳定沉默细胞系,并总结分享了实验中的关键技术条件及要点,同时描述了沉默Myd88后IEC-6细胞早期凋亡明显减少的特征,为肠道炎性疾病的发病机制及靶向治疗研究提供了一个新的、稳定的、可重复的细胞模型。Intestinal inflammatory disease is a kind of non-specific disease with morbidity increasing yearly. It has been proved that the Toll like receptor 4 （TLR4） signaling pathways are closely related to intestinal inflammatory diseases. Myeloid differentiation protein 88 （Myd88） is a critical adaptor protein of TLR4 signaling and critical for the study of intestinal inflammatory disease, but stable Myd88 knockdown in vitro models of cell line are still very few. In the present study, an HIV-l-based lentivirus three-plamid packaging system was used for the construction of a lentivirual vector mediating RNA interference （RNAi） against Myd88 in intestinal fossae epithelial cell line-6 （IEC- 6）. Real-time PCR and Western blot were used to detect Myd88 expression. Annexin V staining and flowcytometry （FLM） were applied to detect and evaluate the early apoptosis. The results showed that lentiviral vectors containing the shRNA expression cassette specifically targeting Myd88 were constructed and efficiently stably knocked down Myd88 expression in IEC-6 cell line. Early apoptosis was significantly decreased after Myd88 knockdown. This study successfully constructed a lentivirus-based RNAi for Myd88 and detailed the key technique combined with character- istics of the early apoptosis after the Myd88 knockdown, provided a novel, stable and repeatable in vitro model for the pathogenesis, targeting therapeutic study for the intestinal inflammatory diseases.

关 键 词：髓样分化蛋白88 RNA干扰 慢病毒表达载体 凋亡 肠隐窝上皮细胞 

分 类 号：R574[医药卫生—消化系统]