沉默Cbl-b基因对增强T淋巴细胞杀伤小鼠乳腺癌细胞能力的影响

Cbl-b gene silencing enhances the ability of T lymphocytes to kill the mouse breast cancer cells

目的 :利用特异性针对Cbl-b(casitas B cell lymphoma-b)基因的小干扰RNA(small interfering RNA,si RNA)沉默小鼠T淋巴细胞中Cbl-b的表达,并观察沉默Cbl-b表达后T淋巴细胞对小鼠乳腺癌TM40D细胞体外免疫杀伤作用的影响。方法 :筛选高效特异性沉默Cbl-b基因的si RNA序列,转染BALB/c小鼠脾脏T淋巴细胞;转染72 h后,利用酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)检测T淋巴细胞上清液中白细胞介素-2(interleukin-2,IL-2)和转化生长因子-β(transforming growth factor-β,TNF-β)等T淋巴细胞因子分泌的情况;CCK-8(cell counting kit-8)法检测Cbl-b基因沉默的T淋巴细胞对小鼠乳腺癌TM40D细胞的杀伤率。结果 :从小鼠脾脏中分离获得的T淋巴细胞的纯度为94.77%。蛋白质印迹法检测结果显示,Cbl-b-siR NA-1抑制Cbl-b蛋白表达的能力最佳;Cbl-bsiR NA-1转染T淋巴细胞72 h后,转染组、阴性对照组和空白对照组T淋巴细胞培养上清液中IL-2的分泌水平分别为(1 678.96±126.91)pg/mL、(1 141.69±83.67)pg/mL和(1 054.03±85.14)pg/mL,转染组中IL-2的分泌水平明显高于阴性对照组和空白对照组(P值均<0.001);TNF-β的分泌水平分别为(769.33±65.46)pg/mL、(573.33±58.67)pg/mL和(581.72±51.72)pg/mL,转染组中TNF-β的分泌水平明显高于阴性对照组和空白对照组(P值均<0.05)。在体外实验中,与空白对照组及阴性对照组相比,转染组T淋巴细胞能更高效地杀伤TM40D细胞,对肿瘤细胞的杀伤率最高为(59.18±3.82)%。结论 :特异性沉默Cbl-b基因的表达能够促进T淋巴细胞分泌细胞因子IL-2和TNF-β的能力,并增强T淋巴细胞对小鼠乳腺癌TM40D细胞的免疫杀伤作用。

Objective： To use specific small interfering RNA （siRNA） targeting casitas B cell lymphoma-b （Cbl-b） gene to silence the expression of Cbl-b in T lymphocytes in mice, and investigate the immune killing activity of T lymphocytes against mouse breast cancer TM40D cells after Cbl-b gene silencing in vitro. Methods： The siRNAs with high ability of Cbl-b gene silencing were screened out and transfected into T lymphocytes obtained from spleen of BALB/c mice. The secretion levels of interleukin-2 （IL-2） and tumor necrosis factor-13 （TNF-β） in supernatant of T lymphocytes were measured by enzyme linked immunosorbent assay （ELISA） at 72 h after transfection. The cytotoxicity activity of T lymphocytes with Cbl-b gene silencing against mouse breast cancer TM40D cells was measured by cell counting kit-8 （CCK-8） method. Results： The purity of T lymphocytes isolated from mouse spleen was 94.77%. The result of Western blotting showed that Cbl-b-siRNA-1 had the best ability to inhibit the expression of Cbl-b protein. After transfection with Cbl-b-siRNA-1 for 72 h, the secretion levels of IL-2 in supernatant of T lymphocytes in transfection group, negative control group and the blank control group were 1 678.96 ± 126.91, 1 141.69 ± 83.67, and 1 054.03 ± 85.14 pg/ mL, respectively; the secretion level of IL-2 in transfection group was significantly higher than those of the negative control group and the blank control group （both P 〈 0.001）. The secretion levels of TNF-β in transfection group, negative control group and the blank control group were 769.33 ± 65.46, 573.33 ± 58.67, and 581.72 ± 51.72 pg/mL, respectively; the secretion level of TNF-β in transfection group was significantly higher than those of the negative control group and the blank control group （both P 〈 0.05）. T lymphocytes had a highly effective killing ability [the most high killing rate was （59.18±3.82） %] against TM40D cells in transfection group as compared with those in the negative control group and the blank control group, in vitro. Conclusion： Silencing Cbl-b gene can promote the secretion levels of IL-2 and TNF-β from mouse T lymphocytes, and enhance the immune killing activity of T lymphocytes against mouse breast cancer TM40D cells.