Chen Dian-Jun, Li Xiao-Song, Zhao Hui, Shi Xiu-Li, Zhang Huan-Huan, Fan Zhong-Yi, Yao Yong-Ming, DU Nan
The Second Department of Oncology, The First Affiliated Hospital of Chinese PLA General Hospital, Beijing 100048, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2012 Apr;28(4):337-9.
To explore the feasibility of RNA interference in the treatment of melanoma by inhibiting the Foxp3 gene expression in mouse B16 melanoma cells using RNA interference (RNAi) in vitro.
Small interfering RNA (siRNA) was designed according to Foxp3 gene. A short hairpin RNA (shRNA) lentivirus expression vector was constructed and transfected into mouse B16 cells, and RNA interference was induced in vitro. Western blot and real-time RT-PCR were performed to detect the expression of Foxp3 gene. ELISA was applied to detect the changes of TGF-β(1);, TGF-β(2);, IL-10 and other cytokines. The B16 cells after interference were co-cultured with CD4(+);CD25(-);T lymphocytes. CCK8 assay was used to monitor the proliferation of CD4(+);CD25(-);T lymphocytes.
shRNA could suppress the expression level of Foxp3, down-regulate the inhibitory ability of tumor cells on the proliferation of CD4(+);CD25(-);T lymphocytes, and reduce the secretion of TGF-β(1);, TGF-β(2);, IL-10 and other cytokines, in particular the expression of TGF-β(2);.
RNA interference can inhibit the expression of target gene Foxp3 in mice melanoma cells and the proliferation of tumor cells. It can also reduce the inhibition on the proliferation of CD4(+);CD25(-);T lymphocytes, and the secretion of inhibitory cytokines.
在体外利用RNA干扰（RNAi）抑制小鼠B16黑色素瘤细胞中Foxp3基因表达，探讨RNA干扰在黑色素瘤治疗中的可行性。
根据Foxp3基因设计小干扰RNA（siRNA）。构建短发夹RNA（shRNA）慢病毒表达载体并转染至小鼠B16细胞，在体外诱导RNA干扰。采用蛋白质免疫印迹法和实时逆转录-聚合酶链反应检测Foxp3基因表达。应用酶联免疫吸附测定法检测转化生长因子-β（1）、转化生长因子-β（2）、白细胞介素-10及其他细胞因子的变化。将干扰后的B16细胞与CD4⁺CD25⁻T淋巴细胞共培养。采用CCK8法监测CD4⁺CD25⁻T淋巴细胞的增殖情况。
shRNA可抑制Foxp3表达水平，下调肿瘤细胞对CD4⁺CD25⁻T淋巴细胞增殖的抑制能力，减少转化生长因子-β（1）、转化生长因子-β（2）、白细胞介素-10及其他细胞因子的分泌，尤其是转化生长因子-β（2）的表达。
RNA干扰可抑制小鼠黑色素瘤细胞中靶基因Foxp3的表达及肿瘤细胞的增殖。还可减轻对CD4⁺CD25⁻T淋巴细胞增殖的抑制及抑制性细胞因子的分泌。
