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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 silence the expression of Cbl-b in mouse T lymphocytes by using small interfering RNA (siRNA) specific for Cbl-b gene and to observe the expression of silencing Cbl-b Effect of posterior T lymphocyte on murine breast cancer TM40D cells in. Methods: The si RNA sequence of Cbl-b gene was silenced and transfected into T lymphocytes of BALB / c mice. After 72 hours of transfection, enzyme linked immunosorbent assay (ELISA) was used to detect T The secretion of T lymphocyte cytokines such as interleukin-2 (IL-2) and transforming growth factor-β (TNF-β) in the supernatant of lymphocytes; CCK-8 cell counting kit-8) method was used to detect the killing rate of Cbl-b gene silencing T lymphocytes on murine breast cancer TM40D cells. Results: The purity of T lymphocytes isolated from mouse spleen was 94.77%. The results of Western blotting showed that Cbl-b-siR NA-1 had the best ability of inhibiting the expression of Cbl-b protein. After transfection of T lymphocytes with Cbl-bsiR NA-1 for 72 h, the transfection group, negative control group and blank The levels of IL-2 secretion in the control group were (1 678.96 ± 126.91) pg / mL, (1 141.69 ± 83.67) pg / mL and (1 054.03 ± 85.14) pg / mL respectively The levels of IL-2 secreted by the two groups were significantly higher than those in the negative control group and the blank control group (P <0.001). The secretion levels of TNF-β were (769.33 ± 65.46) pg / mL, (573.33 ± 58.67) pg / mL and (581.72 ± 51.72) pg / mL respectively. The secretion of TNF-β in transfection group was significantly higher than that in negative control group and blank control group (P <0.05). In in vitro experiments, compared with the blank control group and the negative control group, the T lymphocytes of the transfected group could kill the TM40D cells more efficiently and the killing rate to the tumor cells was the highest (59.18 ± 3.82)%. CONCLUSIONS: The silencing of Cbl-b gene expression can promote the ability of T lymphocytes to secrete cytokines IL-2 and TNF-β and enhance the immunosuppressive effects of T lymphocytes on murine breast cancer TM40D cells.