超声微泡介导沉默T 淋巴细胞Itch 基因对胃癌MFC 细胞免疫杀伤作用的实验研究

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24 h 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。

Effect of ultrasound-targeted microbubble destruction mediated silencing of Itch gene in T lymphocyte on cytotoxicity against MFC gastric cancer cells

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 Tlymphocyte were isolated by magnetic beads. shRNA plasmid was constructed and transferred by UTMD technologyto silence Itch gene in T lymphocytes. Twenty-four hours after transfection, the expression of CD69 was detectedby flowcytometry; 48 hours after transfection, transfection efficiency and expression of Itch were identified byflowcytometry and Western blot, respectively; 72 hours after transfection, cytotoxicity activity against MFCgastric cancer cells were determined. Results Success rate of transfection by UTMD was 51.9%. Transfection ofItch increased expression of CD69 significantly when compared with remaining groups. Expression of Itch genewas sufficiently blocked by UTMD. Cytotoxicity of transfected T lymphocyte against MFC gastric cancer cellswas dramatically upregulated when compared with negative control and blank group. Conclusion Silencing theexpression of Itch gene by UTMD significantly promotes immune cytotoxicity of T lymphocyte against MFC gastric cancer cells in vitro.