利用经修饰的survivin短肽体外诱导对胃癌细胞系具有限制性杀伤作用的特异性细胞毒性T细胞

Efficient Induction of Specific Cytotoxic T Lymphocytes Specific to a Kind of Modified Survivin Peptide which Has Restricted Cytotoxicity Against Gastric Cancer Cell Lines in Vitro

采用一种简单有效的方法体外诱导经修饰的survivin短肽特异性细胞毒性T细胞,并观察其对胃癌细胞的杀伤作用。选取对HLA-A2分子亲和力更高的survivin短肽(96—104,LLLGEFLKL,HLA-A2限制性)以更有效地诱导特异性T细胞。选取正常志愿者外周血单核细胞,筛选其中HLA-A2+者,利用HLA-A2/survivin短肽有效诱导特异性细胞毒性T细胞。Pentamer实验检测特异性细胞毒性T细胞比率。Elispot实验检测细胞毒性T细胞IFN-γ分泌能力。选取胃癌细胞系MKN28、SGC7901及KATO Ⅲ,检测其HLA-A2和survivin表达情况。检测特异性细胞毒性T细胞对三种细胞系的杀伤效果。Pentamer实验结果显示,HLA-A2/survivin短肽刺激之前短肽/HLA Pentamer和CD8双阳性细胞仅为0.06%,接受刺激后双阳性细胞增加到27.5%。Elispot实验结果显示HLA-A2/survivin短肽刺激后IFN-γ分泌细胞比率明显强于阴性对照组。PCR结果显示,KATO Ⅲ为HLA-A2+,MKN28和SGC7901为HLA-A2-。Western Blotting结果显示KATO III和SGC7901为survivin阳性,MKN28为survivin阴性。特异性细胞毒性T细胞杀伤结果显示,细胞能有效杀伤KATO III细胞,但对其他两种细胞杀伤效果不明显。p<0.01。经修饰的survivin短肽能够有效诱导特异性细胞毒性T细胞,并对胃癌细胞系有HLA-A2/survivin限制性杀伤。

A simple and effective method was applied in the present study to induce specific cytotoxic T lym- phocytes via a modified survivin peptide, the cytotoxicity of which is observed against gastric cancer cells. HLA-- A2-restricted survivin peptide （96--104, LLLGEFLKL） used in this study is a modified analogue showing high af- finity to HLA--A2 to ensure the activating effect. Healthy donors with HLA--A2 phenotype are involved and PB- MCs of them are used to induce specific cytotoxic T lymphocytes via HLA--A2/survivin. Pentamer analysis is used to detect the ratio of specific cytotoxic T lymphocytes; Elispot assays to detect the capacity of cytotoxic T lympho- cytes to secrete IFN-~/. Gastric cancer cell lines MKN28, SGC7901, and KATO III are selected and HLA--A2 phenotype and expression of survivin of them are detected. Cytotoxicity of specific cytotoxic T lymphocytes against these gastric cancer cell lines is also detected. Results of Pentamer analysis showed that ratio of peptide/HLA Pen- tamer and CD8 double-positive lymphocytes increased （from 0.06% to 27.5% ） after stimulation. Results of Elis- pot assay showed the average spot number of responding lymphocytes induced by T2 pulsed with survivin/HLA-- A2 peptide is significantly higher than that of responding lymphocytes induced by T2 cells without peptide. Biomar- kers are also detected of KATO III （HLA--A2 ＋/survivin＋ ）, MKN28 （HLA--A2/surviving）, and SGC7901 （HLA--A2-/survivin ＋ ） via PCR and Western Blotting. Results of cytotoxicity show that the identified CTLs can effectively kill the double-positive cell line （KATO III） but not others（p 〈0. 01 ）. Specific CTLs can be induced efficiently via modified survivin peptide and kill gastric cancer ceils with special biomarkers.