摘要
目的:探讨Toll样受体-4(TLR-4)表达对树突状细胞(DC)表型及功能的影响及其机制。方法:以构建的含有TLR-4全基因序列的pc DNA3.1质粒载体为模板,通过m MESSAGE m MACHINE T7 Kit体外转录获得TLR-4 m RNA,并采用脂质体法将其转染健康人外周血PBMC来源的DC,用流式细胞术检测鉴定转染后DC表面功能性分子的表达及DC体外诱导细胞毒性T淋巴细胞(CTL)分泌细胞因子的能力。结果:成功构建人TLR-4-pc DNA3.1质粒载体;体外扩增成功人TLR-4和EGFP m RNA片段。TLR-4 m RNA转染后的DC表面趋化因子CCR7及功能性分子HLA-DR的表达显著高于control m RNA转染DC、转染前成熟DC[CCR-7:(42.4±4.93)%vs(20.1±3.09)%、(17.1±4.33)%,P<0.05;HLA-DR:(62.1±7.23)%vs(17.7±6.01)%、(25.8±4.16)%,P<0.05],同时转染后的DC体外诱导CTL分泌IFN-γ细胞因子的能力强于control m RNA-DC、空载转染DC诱导的CTL及加入DC前CTL[(66.5±3.58)%vs(41.1±4.27)%、(37.9±2.96)%、(3.2±2.03)%,P<0.05]。结论:TLR-4 m RNA转染DC可显著增强DC的功能,提高了DC抗原提呈及诱导CTL的能力,为增强DC疫苗抗肿瘤效应提供实验依据。
Objective：To investigate whether the expression of Toll like receptor-4（TLR-4） has an effect on the phenotypes and function of dendritic cells（DCs） and the possible mechanism.Methods：pc DNA3.1 plasmid encoding full TLR-4 gene sequence was constructed as a template to obtain TLR-4 m RNA through in vitro transcription using m MESSAGE m MACHINE T7 Kit;DCs from healthy human peripheral blood were transfected with TLR-4m RNA by liposomal transfection.The functional molecule expression on DC surface and the ability of DC inducing cytotoxic T lymphocytes（CTLs） to secrete cytokines in vitro were detected by Flow cytometry after transfection.Results：Human TLR-4-pc DNA3.1 plasmid was successfully constructed;human TLR-4 and EGFP m RNA fragments were successfully amplified.The Flow cytometry results showed that the expressions of chemokine CCR7 and functional molecule HLA-DR on dendritic cell surface were significantly increased after transfection with TLR-4 m RNA compared with control m RNA transfection or pre-transfection（CCR7：[42.4 ± 4.93]% vs [20.1 ± 3.09]%,[17.1±4.33]%,P〈0.05;HLA-DR：[62.1±7.23]% vs [17.7±6.01]%,[25.8±4.16]%,P〈0.05）;and the ability of secreting IFN-γ from CTLs induced by TLR-4 m RNA-DCS was significantly enhanced in vitro compared with control m RNA-DC,empty-DC and PBMC（[66.5±3.58]% vs [41.1±4.27]%,[37.9±2.96]%,[3.2±2.03]%,P〈0.05）.Conclusion：The function of dendritic cells could be significantly enhanced by TLR-4 m RNA transfection.The ability of antigen presentation and inducing CTLs of dendritic cell were improved,which would provide an experimental basis for enhancing the anti-tumor effect of DC vaccines.
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
北大核心
2017年第9期950-954,共5页
Chinese Journal of Cancer Biotherapy
基金
国家临床重点专科建设项目资助
福建省科技计划项目资助(No.2017Y0022)
福建省自然基金项目资助(No.2016J01514)~~
