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

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

摘要:

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。

Abstract:

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.