In vitro effects of Cbl-b gene silencing on immunocompetence of primary murine lymphocytes / 中华皮肤科杂志

ABSTRACT

Objective To evaluate in vitro effects of specific small interfering RNA (siRNA)-silencing of the casitas B-lineage lymphoma b (Cbl-b)gene on immunocompetence of primary murine lymphocytes. Methods Spleens were resected from C57BL/6 mice, and splenic lymphocytes were sterily isolated and cultured in vitro. These lymphocytes were divided into 3 groups silence group transfected with a Cbl-b-specific siRNA using the EntransterTM-R 4000 reagent, negative control group transfected with a negative control siRNA using the EntransterTM-R4000 reagent, blank control group receiving no treatment. After additional culture for 72 hours, ELISA was performed to measure levels of interferon γ(IFN-γ)and tumor necrosis factor α (TNF-α)in culture supernatants of lymphocytes. In addition, the Cbl-b gene-silenced lymphocytes were co-cultured with B16F10 melanoma cells to evaluate their immunocytotoxic effects on melanoma cells. Results Splenic lymphocytes were successfully isolated from C57BL/6 mice and cultured in vitro, and the Cbl-b-specific siRNA was also successfully transfected into the primary murine lymphocytes and effectively down-regulated the expression of Cbl-b gene in them. Compared with the negative control group and blank control group, the silence group showed significantly increased supernatant levels of IFN-γ and TNF-α(all P < 0.05). The immunocytotoxic effect of lymphocytes on melanoma cells was significantly stronger in the silence group than in the negative control group. Conclusion Cbl-b gene silencing can promote secretion of IFN-γ and TNF-α by murine lymphocytes, and enhance their immunocytotoxic effects on B16F10 melanoma cells in vitro.