TLR配体对小鼠模型中HPV11E7CTL表位肽特异性免疫应答的调节作用

发布时间：2018-05-28 18:45

本文选题：TLR配体 + 树突状细胞　；参考：《浙江大学》2010年硕士论文

【摘要】： 目的 1.研究尖锐湿疣皮损中Toll样受体(Toll-like receptor,TLR)各亚型的表达,以明确皮肤人乳头瘤病毒(HPV)感染高相关性TLR亚型。 2.探讨TLR配体——咪喹莫特、PIC(polyinosinicacid:polycytidylic acid)以及CpG oligonucleotide对人乳头瘤病毒抗原肽-HPV11E7CTL表位肽(后简称“肽”)在小鼠模型中免疫应答的调节作用。方法 1.将尖锐湿疣病人的病损组织标本冰冻切片经免疫组化染色检测10种TLR亚型的表达情况,以正常皮肤组织作对照,比较两者TLRs表达谱的差异。 2.提取小鼠骨髓树突状细胞(BM-DC),体外培养后先与HPV11E7CTL表位肽共孵育,再分别与咪喹莫特或PIC或CpG共孵育,以促进DC成熟,流式细胞仪检测孵育后DC表面标志改变；体内实验：将刺激成熟的DC分别注射于各组小鼠腹腔,以免疫小鼠,然后在小鼠皮下接种已转染HPV11E7基因的B16肿瘤细胞；体外实验：将HPV11E7CTL表位肽和TLR配体处理后的成熟DC与未经任何处理的小鼠脾细胞混合培养；ELISA法检测小鼠脾脏T淋巴细胞培养液中细胞因子TNF-α和IFN-γ的浓度；乳酸脱氢酶(LDH)释放法测定细胞毒性T淋巴细胞(CTL)活性；监测小鼠皮下肿瘤大小以观察DC免疫后对肿瘤的预防作用。结果 1.正常表皮角质层和颗粒层TLR6、9呈强阳性表达,TLRR1、3、10呈中等度表达,TLR2、4、5、7、8弱表达,棘层和基底层细胞不表达或弱表达TLR；尖锐湿疣皮损表皮中除基底层弱表达或不表达TLR外,其余细胞层TLR1、3、6、9强阳性表达,TLR4、8、10中等度表达,TLR2、5、7弱表达。两组的真皮层均未见明显TLR阳性细胞。 2.BM-DC经与HPV11E7CTL表位肽及TLR配体共孵育后,反映其成熟度的表面标志CD40、CD80、CD86及MHC II (I-Ak)表型均明显提高；肽加CpG处理组小鼠脾细胞分泌细胞因子TNF-α和IFN-γ的水平均为最高,且与对照组有明显差异(P0.05)；各处理组小鼠肿瘤生长速度及脾脏T淋巴细胞的细胞毒性T淋巴细胞(CTL)活性与对照组未见明显差异。结论 1.尖锐湿疣皮损增生的角质形成细胞中TLR表达较正常皮肤明显增高,尤以TLR1、3、6、9亚型较显著,可能与HPV感染启动机体抗病毒免疫有关。 2.TLR激动剂尤其是CpG对小鼠模型中HPV11E7CTL表位肽所致的特异性免疫应答具有上调作用。
[Abstract]:objective
1. the expression of the Toll like receptor (Toll-like receptor, TLR) subtypes in the skin lesions of condyloma acuminata was studied to identify the high correlation TLR subtype of human papillomavirus (HPV) infection.
2. the effects of TLR ligand, imiquimod, PIC (polyinosinicacid:polycytidylic acid) and CpG oligonucleotide on the immune response of human papillomavirus antigenic peptide -HPV11E7CTL epitopes in mouse models, are investigated.
Method
1. the expression of 10 TLR subtypes was detected by immunohistochemical staining in the frozen section of the patients with condyloma acuminata, and the difference of TLRs expression profiles was compared between the normal skin tissues and the normal skin tissues.
2. the mouse bone marrow dendritic cells (BM-DC) were extracted and incubated with HPV11E7CTL epitope, then incubated with imiquimod or PIC or CpG respectively to promote the maturation of DC. The flow cytometry was used to detect the changes of the DC surface markers after incubation. In vivo experiment, the mature DC was injected into the abdominal cavity of mice in each group to immunize mice. B16 tumor cells transfected with HPV11E7 gene were inoculated subcutaneously in mice. In vitro, the mature DC treated with HPV11E7CTL epitope peptide and TLR ligand was mixed with the untreated mouse splenocytes; the concentration of cytokines TNF- A and IFN- in the mouse spleen T lymphocyte culture solution was detected by ELISA; lactate dehydrogenase (LDH) was detected. Release method was used to detect cytotoxic T lymphocyte (CTL) activity, and the size of subcutaneous tumor was monitored to observe the preventive effect of DC on tumor.
Result
1. the normal epidermal cuticle and granular layer TLR6,9 showed strong positive expression, TLRR1,3,10 was moderately expressed, TLR2,4,5,7,8 was weakly expressed, the spinous and basal layer cells did not express or weakly express TLR, and the TLR1,3,6,9 in the epidermis of condyloma acuminata was strongly expressed except for the weak expression of the basal layer or the non expression of TLR, and the TLR4,8,10 medium expression, TLR2. 5,7 was weakly expressed. No obvious TLR positive cells were found in the two groups of dermis.
After incubation with HPV11E7CTL epitopes and TLR ligands, 2.BM-DC reflected the surface marker of its maturity, CD40, CD80, CD86 and MHC II (I-Ak), and the level of TNF- alpha and IFN- gamma secreted by the peptiderma and CpG treatment group was the highest, and was significantly different from that in the irradiated group. The activity of cytotoxic T lymphocyte (CTL) in long - term and splenic T lymphocytes was not significantly different from that in the control group.
conclusion
1. the expression of TLR in keratinocytes of condyloma acuminate lesions was significantly higher than that of normal skin, especially TLR1,3,6,9 subtype, which may be related to the antiviral immunity of HPV infected body.
2.TLR agonist, especially CpG, has an up-regulated effect on the specific immune response induced by HPV11E7CTL epitope peptide in mouse models.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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