超声微泡介导Itch基因沉默增强T细胞对肺腺癌细胞LA795免疫杀伤作用  

Silencing Itch gene expression by ultrasound-targeted microbubble destruction to enhance the immune killing efficiency of T lymphocyte against lung neoplasms cells LA795

作　　者：毛宁[1] 熊弢[1] MAO Ning;XIONG Tao(Dept.of Thoracic Surgery,the Affiliated Yongchuan Hospital,Chongqing Medical University,Chongqing 402160,China)

机构地区：[1]重庆医科大学附属永川医院心胸外科,重庆402160

出　　处：《基础医学与临床》2018年第4期516-521,共6页Basic and Clinical Medicine

基　　金：重庆医科大学附属永川医院院内课题(YJLCX201624)

摘　　要：目的利用超声介导微泡破裂技术(UTMD)沉默T细胞Itch基因表达,观察转染T细胞对肺腺癌细胞LA795的体外免疫杀伤效率。方法磁珠分选纯化T细胞,构建靶向Itch基因的shRNA表达质粒,超声微泡介导转染48 h后,荧光显微镜和流式细胞仪评估细胞转染效率,Western blot检测Itch蛋白表达。转染72 h后,酶联免疫吸附法(ELISA)检测细胞上清液中IL-2和IFN-γ分泌水平,观察对比单纯T细胞、阴性对照T细胞及转染T细胞与小鼠肺腺癌细胞LA795共培养时肿瘤杀伤率。结果利用UTMD技术介导shRNA转染效率达到52.3%±3.8%,Itch蛋白表达能够有效被抑制。转染72 h后,超声微泡介导沉默Itch基因显著增加T细胞因子IL-2和IFN-γ分泌水平(P<0.05);与空白组或阴性对照组T细胞相比,在不同的靶效比水平(10∶1、20∶1、40∶1),转染T细胞杀瘤活性均明显增高(P<0.05)。结论利用UTMD技术介导shRNA转染能有效沉默Itch基因表达,促进T细胞免疫活性,增强T细胞对小鼠肺腺癌细胞LA795的体外免疫杀伤效率。Objective To silence the expression of Itch gene of T-lymphocytes by ultrasound-targeted microbubble destruction(UTMD)and to investigate the cytotoxicity of transfected T lymphocytes against LA795 lung neoplasms cells in vitro.Methods T lymphocyte were isolated by magnetic bead,and an targeted shRNA to silence Itch gene of T lymphocytes was established.48 hours after transfection by UTMD,the transfection efficiency was detected and analyzed by fluorescence microscopy and flow cytometry;the expression of Itch protein was measured with Western blot;72 hours after transfection,the secretion of IL-2 and IFN-γin the cell supernatant was measured by enzyme-linked immunosorbent assay(ELISA).The cytotoxicity activity changes against LA795 lung neoplasms cells was compared between transfected T lymphocytes and negative control or simplex T lymphocytes in vitro.Results The transfection rate to silence Itch gene of T lymphocytes by UTMD was 52.3%±3.8%.At 48 h after transfection,the Itch gene expression can be effectively suppressed by UTMD.Seventy-two hours after transfection,the secretion level of cytokines including IL-2 and IFN-γ,was significantly increased in the group of targeted shRNA to silence Itch gene of T lymphocytes by UTMD(P<0.05)and transfected T lymphocyte also showed more efficient killing ability against LA795 lung neoplasms cells than negative control or blank group at E∶T of 10∶1,20∶1 and 40∶1(P<0.05).Conclusions Silencing the expression of Itch by UTMD can significantly promote immune activity of T lymphocyte,enhance the cytotoxicity activity of T lymphocyte against LA795 lung neoplasms cells in vitro.

关 键 词：ITCH 超声介导微泡破裂技术 基因沉默 肺肿瘤 免疫治疗 

分 类 号：R655[医药卫生—外科学]