Objective To investigate the effect of ultrasound-targeted microbubble destruction (UTMD) technology mediated silencing of Itch gene in T-lymphocytes on killing MFC gastric cancer cells. Methods T lymphocyte were isolated by magnetic beads. shRNA plasmid was constructed and transferred by UTMD technology to silence Itch gene in T lymphocytes. Twenty-four hours after transfection, the expression of CD69 was detected by flowcytometry; 48 hours after transfection, transfection efficiency and expression of Itch were identified by flowcytometry and Western blot, respectively; 72 hours after transfection, cytotoxicity activity against MFC gastric cancer cells were determined. Results Success rate of transfection by UTMD was 51.9％. Transfection of Itch increased expression of CD69 significantly when compared with remaining groups. Expression of Itch gene was sufficiently blocked by UTMD. Cytotoxicity of transfected T lymphocyte against MFC gastric cancer cells was dramatically upregulated when compared with negative control and blank group. Conclusion Silencing the expression of Itch gene by UTMD significantly promotes immune cytotoxicity of T lymphocyte against MFC gastric cancer cells in vitro.%目的 利用超声介导微泡破裂技术(UTMD)沉默T淋巴细胞Itch基因表达,观察转染T细胞对胃癌MFC细胞的免疫杀伤效率.方法 免疫磁珠分离T淋巴细胞,构建靶向Itch基因的shRNA表达质粒,超声微泡介导转染48 h后,流式细胞仪分析T细胞转染成功率,Western blot测定T细胞Itch蛋白表达.转染24 h后,流式细胞仪检测T淋巴细胞活化早期标志CD69表达.转染72 h后,观察对比单纯T淋巴细胞、阴性对照T淋巴细胞及转染T淋巴细胞与小鼠胃癌MFC细胞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51.9％, Itch蛋白表达能被有效抑制.转染24h后,转染组T淋巴细胞早期活化标志CD69表达较其他组更高.转染72 h后,与阴性对照组和空白组相比,在不同的效靶比水平(10: 1、 20: 1、40: 1), 转染T淋巴细胞杀瘤活性均增强.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淋巴细胞免疫活性,增强T淋巴细胞对胃癌MFC细胞的免疫杀伤效率.
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