沉默T细胞Cbl-b基因对4T1细胞免疫杀伤作用影响

Silencing Cbl-b gene expression enhances the immune killing efficiency of T lymphocyte against 4T1 breast cancer cells

目的乳腺癌发生发展与机体免疫水平有密切关系,而Cbl-b基因可作为抗肿瘤治疗的免疫靶点。利用超声介导微泡破裂技术(ultrasound-targeted microbubble destruction,UTMD)沉默T细胞Cbl-b基因表达,观察转染T细胞对4T1乳腺癌细胞的体外免疫杀伤效率。方法磁珠分选纯化T细胞,构建靶向Cbl-b基因的短发夹RNA(short-hairpin RNA,shRNA)表达质粒,超声微泡介导转染48h后,荧光显微镜和流式细胞仪评估细胞转染效率,蛋白质印迹法检测Cbl-b蛋白表达情况。转染72h后,利用酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)比较各组细胞上清液中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)分泌水平。转染72h后,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4T1乳腺癌细胞共培养时肿瘤杀伤效率。结果原代培养T细胞纯度为93.7%。利用UTMD技术介导shRNA转染效率达到61.3%。qPCR和蛋白质印迹法检测结果显示,Cbl-b基因和蛋白水平表达均能被有效抑制。与空白组(1.007±0.022)相比,实验组Cbl-b mRNA表达量(0.333±0.046)明显降低,P<0.001;实验组、对照组和空白组Cbl-b蛋白相对表达量分别为0.301±0.080、0.773±0.101和0.719±0.090,检验统计量组间总变异,F=38.751,P<0.001;实验组Cbl-b蛋白表达显著降低。与空白组[(74±6)pg/mL]相比,实验组T细胞因子TNF-α分泌[(157±26)pg/mL]水平明显升高,P=0.001。转染72h后,与空白组及阴性对照组T细胞相比,在15∶1(P=0.046)、30∶1(P=0.028)和60∶1(P=0.003)的效靶比水平,转染T细胞杀瘤活性均明显增高。结论利用UTMD技术介导shRNA转染能有效沉默Cbl-b基因表达,促进T细胞免疫活性,增强T细胞对小鼠4T1乳腺癌细胞的体外免疫杀伤效率。

OBJECTIVE The development of breast cancer treatment is closely related to the physical immunity,and the Cbl-b gene can be used as the immune target for antitumor therapy.In this study,ultrasound-targeted microbubble destruction（UTMD）technology was performed to silence the expression of Cbl-b gene of T-Lymphocytes and the cytotoxicity activity of transfected T lymphocytes against 4 T1 breast cancer cells in vitro was investigated.METHODS T lymphocyte were separated by magnetic bead,and an targeted shRNA was established to silence Cbl-b gene expression of T lymphocytes.Forty-eight hours after transfection by UTMD,transfection efficiency was detected and analyzed by fluorescence microscope and flowcytometry;48 hours after transfection by UTMD,the expression of Cbl-b protein was measured with Western Blot;Seventy-two hours after transfection,the secretion of TNF-αin the cell supernatant were measured by enzyme-linked immunosorbent assay（ELISA）.Seventy-two hours after transfection,we compared the cytotoxicity activity changes against 4 T1 breast cancer cells between transfected T lymphocytes,negative control and simplex T lymphocytes in vitro.RESULTS The purity of the primarily cultured T lymphocytes was 93.7%.The transfection rate to silence Cbl-b gene expression of T lymphocytes by UTMD was 61.3%.At 48 hafter transfection,qPCR and Western blot analysis showed the Cbl-b gene and protein expression can be effectively suppressed by UTMD.Compared with the blank group（1.007±0.022）,the expression of cbl-b mRNA was significantly decreased in the experimental group（0.333±0.046）,P〈0.001.The relative expressions of Cbl-b protein in the experimental group,control group and blank group were0.301±0.080,0.773±0.101 and 0.719±0.090,respectively,the total variation of the test statistics group was F=38.751,P〈0.001,and the expression of Cbl-b protein was significantly decreased in the experimental group.Seventy-two hours after transfection,compared with the blank group[（74±6）pg/mL],the secretion level of TNF-αwas significantly increased in the experimental group[（157±26）pg/mL],P=0.001.Seventy-two hours after transfection,transfected T lymphocyte also showed more efficient killing ability against 4 T1 breast cancer cells than that of negative control and blank group at each effect to target ratio in vitro,P=0.046（15∶1）,P=0.028（30∶1）,P=0.003（60∶1）.CONCLUSION Silencing the expression of Cbl-b by UTMD can significantly promote immune activity of T lymphocyte,and enhance the cytotoxicity activity of T lymphocyte against 4 T1 breast cancer cells in vitro.