小鼠肺部TMEM16A基因表达沉默模型的建立  

Establishment mouse model of lung TMEM16A gene knockout

作　　者：李宏林[1] 阎锡新[1] 张会然[1] 郭丽娜[2] 

机构地区：[1]河北医科大学第二医院呼吸一科,石家庄050000 [2]河北医科大学第二医院中心实验室,石家庄050000

出　　处：《国际呼吸杂志》2016年第10期757-762,共6页International Journal of Respiration

摘　　要：目的 构建小鼠TMEM16A-shRNA慢病毒载体,通过气管内注射慢病毒载体,局部部分沉默小鼠肺部TMEM16A基因的表达,为后续在体研究TMEM16A在肺部作用提供基础.方法 ①构建小鼠TMEM16A-shRNA慢病毒载体;②免疫荧光法验证LA795小鼠肺腺癌细胞膜上TMEM16A的表达,体外行慢病毒感染实验,确定感染的最佳条件,最后体外实验确定慢病毒的干扰效率;③链霉菌抗生素蛋白-过氧化物酶连接法（streptavidin-perosidase,SP）法验证肺组织中TMEM16A的表达;④气管内注射慢病毒2周后行支气管肺泡灌洗,BAC法检测灌洗液中蛋白及酶联免疫吸附法测定（ELISA）法检测炎症因子肿瘤坏死因子α（tumornecrosisfactor-a,TNF-α）、IL-1β及IL-10的浓度;⑤气管内注射慢病毒2周后取材肺组织提取蛋白用蛋白印迹法（Western blot）验证在体TMEM16A-shRNA慢病毒载体的沉默效率.结果 ①LA795细胞膜上存在TMEM16A的表达,LA795细胞在5 mg/L的Eni.S＋ polybrene条件下容易被感染,病毒感染复数（MOI）为20;②Western blot进一步显示体外细胞慢病毒shRNA的沉降效率约81.35％（P＜0.05）;③气管内注入Lenti-Neg-shRNA后BALF的蛋白含量无增加,细胞因子TNF-α、IL-1β及IL-10的浓度无明显变化（P＞0.05）;光镜下肺组织结构正常,未造成病理损伤;④气管内注入Lenti-TMEM16A-shRNA后,Western-blot结果显示TMEM16A表达水平降低（P＜0.05）,达到在肺部部分沉默TMEM16A的目的.结论 本研究成功构建小鼠TMEM16A-shRNA慢病毒载体,体内实验证实慢病毒气管内注射是比较安全,未增加肺组织的蛋白渗漏及病理损伤,并能有效转染气道及肺泡上皮细胞并在局部表达,成功沉默肺部TMEM16A的表达.Objective To construct the mouse TMEM16A-shRNA lentiviral vector and TMEM16A gene expression in lung was silenced partly by endotracheal lentivirus injection in vivo.Methods ①The mouse TMEM16A-shRNA lentiviral vector was constructed.②The location of TMEM16A expression was detected in LA795 cell by immunofluorescence.We got optimum multiplicity of infection （MOI） values by analysis the transfection efficiency in conditions of different transfection reagents.③The location expression of TMEM16A was observed in lung tissue by streptavidin-perosidase.④Concentrated Lenti-Neg-shRNA was delivered into male wild-type C57BL/6 mice.14 days after lentiviral supernatant delivery,the lung tissue was harvested to be observed the pathological variation.At the same time,bronchoalveolar lavage （BAL） was performed to detected the epithelial permeability by BACand the concentrations of tumornecrosisfactor-α （TNF-α）、interleukine-1 beta （IL-1β） 及 interleukine-10 （IL-10） by enzyme linked immunosorbent assay （ELISA）.⑤After 14 days of lentiviral supernatant delivery,TMEM16A was drawed to be verified the knock-out effect by Western-blot.Results ①The expression of TMEM16A located in cell membranes in LA795 and optimum MOI values was 20 in the condition of 5 mg/L Eni.S＋polybrene.②The effect of knockdown of lenti-shRNA in protein level was about 81.35% by Western blot in vitro （P 〈0.05）.③The verification of the silent effect of transfection of the lenti-NC into lung tissue：The protein concentrations showed no significant difference between control group and Lenti-NC group in BALF （P 〉 0.05）.At the same time,there was no significant difference in the concentrations of TNF-α,IL-1β and IL-10 in groups （P 〉0.05）.④Western blot analysis revealed that the level of TMEM16A protein in lenti-shRNA group was reduced about 50% compared with that of Lenti-NC group （P 〈0.05）.Conclusions Lentivial vector expressing TMEM16A-shRNA for mouse has been constructed successfully.Intrat

关 键 词：TMEM16A RNA干扰 慢病毒 LA795 

分 类 号：R734.2[医药卫生—肿瘤] R-332[医药卫生—临床医学]