摘要
目的 观察Toll样受体4（TLR4）在皮肤恶性黑色素瘤（CMM）中的表达及对叉状头/翅膀状螺旋转录因子（Foxp3）表达的影响,探讨其在肿瘤免疫逃逸中的作用.方法 采用免疫组织化学链霉菌抗生物素蛋白-过氧化物酶（SP）法检测TLR4和Foxp3在42例CMM、36例色素痣及30例正常皮肤的表达.采用反转录-聚合酶链反应（RT-PCR）法和Western blot法检测脂多糖（LPS）活化黑色素瘤A375细胞TLR4后Foxp3 mRNA和蛋白水平的表达.设计并构建TLR4小干扰RNA（siRNA）慢病毒表达载体转染A375细胞,检测TLR4基因沉默对Foxp3 mRNA和蛋白表达的影响.细胞计数试剂盒（CCK-8）实验检测CD4＋ CD25-T淋巴细胞增殖能力.结果 在CMM、色素痣及正常皮肤组织,TLR4、Foxp3的阳性表达率分别为66.7％（28/42）、41.7％ （15/36）、16.7％（5/30）和95.2％ （40/42）、11.1％ （4/36）、0％ （0/30）,TLR4、Foxp3在CMM组织的阳性表达率显著高于色素痣及正常皮肤组织,差异有统计学意义（P＜0.05）,且两者表达呈显著正相关（r =0.883,P＜0.05）.LPS活化A375细胞TLR4可显著上调Foxp3 mRNA和蛋白水平的表达,TLR4基因沉默后再用LPS刺激A375细胞,Foxp3表达较对照组明显降低,且肿瘤细胞对CD4＋ CD25-T淋巴细胞增殖的抑制作用明显减弱（P＜0.05）.结论 TLR4信号通路激活可能通过上调Foxp3表达参与恶性黑色素瘤的免疫逃逸.
Objective To explore the relationship between the expression of Toll-like receptor 4 （TLR4） and forkhead/winged helix transcription factor P3 （Foxp3） in cutaneous malignant melanoma （CMM） and the role of TLR4 in immune evasion.Methods Immunohistochemistry was used to detect the expression of TLR4 and Foxp3 in 42 cases of CMM,36 cases of pigmented nevus and 30 cases of normal skin specimens.Lipopolysaccharide （LPS） was employed as exogenous ligands to activate TLR4 in A375 cells.The mRNA and protein levels of Foxp3 were detected by reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blotting.Specific TLR4 small interfering RNA （siRNA） was synthesized and transfected into A375 cells by lentivirus.The expression of Foxp3 and the proliferation of CD4 ＋ CD25-T lymphocytes were examined.Results The positive expression rate of TLR4 and Foxp3 in CMM （66.7％ and 95.2％） was significantly higher than in the pigmented nevus （41.7％ and 11.1％） and normal skin （16.7％ and 0％） （P ＜0.05）,and they were positively correlated （r =0.883,P＜0.05）.The expression of Foxp3 at mRNA and protein levels was significantly increased by LPS stimulation.siRNA could suppress the expression level of Foxp3 and down-regulate the inhibitory ability of tumor cells on the proliferation of CD4 ＋ CD25-T lymphocytes （P ＜ 0.05）.Conclusion TLR4 signaling pathway may be involved in CMM immune evasion process by upregulating Foxp3 expression.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2015年第6期1387-1390,共4页
Chinese Journal of Experimental Surgery
关键词
恶性黑色素瘤
TOLL样受体4
叉头样转录因子
免疫逃逸
Malignant melanoma
Toll-like receptor 4
Forkhead/winged helix transcription factor P3
Immune evasion
