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目的:研究人工合成人端粒酶逆转录酶(hTERT)多表位混合肽经髓样树突状细胞(mDC)提呈后,诱导特异性细胞毒性T淋巴细胞(CTL)对HLA-A24+肿瘤细胞的免疫杀伤效应。方法:人工合成四分支的树状串联hTERT表位肽(MAPs)及其各单表位多肽。取HLA-A24+健康志愿者的外周血,用免疫磁珠分选并培养mDC。用尼龙毛柱纯化T淋巴细胞并培养。以各表位肽致敏mDC后,诱导特异性CTL增殖,并以表达hTERT且以HLA-A24+肿瘤细胞株SMMC-7721及HLA-A24-肿瘤细胞株SKOV3为靶细胞行杀伤实验。用ELISA法检测不同时间点培养基上清液中IL-12、TNF-α的分泌量;用流式细胞术检测CTL对肿瘤细胞的杀伤效应。结果:以源于hTERT的T淋巴细胞表位I540(ILAKFLHWL)、V461(VYGFVRACL)及L766(LTDLQPYMRQFVAHL)合成4分支MAPs多肽及各单表位混合多肽。以人工合成的hTERT多表位混合肽致敏mDC后,能刺激CTL增殖,并可诱导对HLA-A24+肿瘤细胞株SMMC-7721的特异性杀伤作用,且MAPs多肽较单表位混合多肽的致敏效果更具显著性(P<0.05)。结论:以人工合成的hTERT多表位混合肽致敏mDC能激活同源淋巴细胞(CTL),可特异性杀伤HLA-A24+的肿瘤细胞,在肿瘤免疫治疗中具有重要的意义。
AIM: To investigate the effect of CTL on HLA-A24 + tumor cells induced by mesenchyme-derived dendritic cells (mDC) presenting human telomerase reverse transcriptase (hTERT) Immune killing effect of cells. Methods: Four-branched dendritic hTERT epitope peptides (MAPs) and their single epitope peptides were synthesized. Peripheral blood from healthy volunteers was collected from HLA-A24 + cells, and then the immunomagnetic beads were used to sort and culture mDC. T lymphocytes were purified by nylon column and cultured. After sensitized with each epitope peptide, specific CTLs were induced to proliferate and expressed in hTERT. The killing experiments were performed using HLA-A24 + tumor cell line SMMC-7721 and HLA-A24-tumor cell line SKOV3 as target cells. The secretion of IL-12 and TNF-α in supernatant of medium at different time points was detected by ELISA. The killing effect of CTL on tumor cells was detected by flow cytometry. Results: The four branched MAPs polypeptides and the single epitope peptides were synthesized by hTERT - derived T lymphocyte epitopes I540 (ILAKFLHWL), V461 (VYGFVRACL) and L766 (LTDLQPYMRQFVAHL). After sensitized mDC with synthetic hTERT multi-epitope peptide, it can stimulate the proliferation of CTL and induce specific killing of HLA-A24 + tumor cell line SMMC-7721, and MAPs polypeptide is more specific than the single epitope mixed peptide Sensitivity effect was more significant (P <0.05). CONCLUSION: MdCs sensitized with synthetic hTERT multi-epitope peptide can activate lymphocyte (CTL) and specifically kill tumor cells of HLA-A24 +, which is of great significance in tumor immunotherapy.