T细胞免疫球蛋白黏蛋白分子3对黑素瘤TRP-2180-188肽疫苗刺激小鼠淋巴细胞的影响  

Effect of T-cell immunogiobulin and mucin domain-3 on TRP-21180-188 peptide-sthnulated murine spleen lymphocytes co-cultured with B16F10 murine melanoma cells

作　　者：吕雅琳[1] 周晓伟 胡彬[1] 吴琼[1] 曾学思[1] 刘毅[1] 孙建方[1] 

机构地区：[1]中国医学科学院北京协和医学院皮肤病研究所病理科,南京210042 [2]江苏省皮肤病与性病分子生物学重点实验室

出　　处：《中华皮肤科杂志》2016年第2期82-87,共6页Chinese Journal of Dermatology

基　　金：国家自然科学基金（81171513）;江苏省自然科学基金（BK2012506、BK20131063）;2012高等学校博士学科点专项科研基金（20121106110040）

摘　　要：目的探讨体外T细胞免疫球蛋白黏蛋白分子-3（TIM-3）对与小鼠黑素瘤B16F10细胞共培养的酪氨酸相关蛋白-2（TRP-2180-188）抗原肽刺激小鼠淋巴细胞的影响。方法构建TIM-3重组质粒pFUSE—TIM-3-mlgG2Aae1—Fc2，分别转染重组质粒及空质粒pFUSE—mIgG2Aae1—Fc2至人上皮293T细胞，继续培养48h，制备含TIM-3、免疫球蛋白（Ig—tail）的上清液。TRP-2180-188肽疫苗免疫C57BL／6小鼠，分离小鼠脾淋巴细胞，用TRP-2180-188抗原肽和白细胞介素（IL）-2刺激培养5d，以未刺激细胞作为对照组。构建B16F10细胞与上述TRP-2180-188抗原肽刺激淋巴细胞共培养体系，分为空白对照组（加293T细胞培养48h上清液）、TIM-3组（加含TIM-3上清液）、阴性对照组（加含Ig-tail上清液）。CCK-8法检测细胞增殖情况，酶联免疫吸附试验（ELISA）检测共培养体系中干扰素（IFN）-γ和肿瘤坏死因子（TNF）-α浓度，流式细胞仪检测共培养体系中CD8^＋淋巴细胞。结果酶切和测序鉴定证实目的基因正确插入真核表达载体中，检测到转染重组质粒pFUSE—TIM-3-mIgG2Aae1-Fc2的293T细胞上清液中有TIM-3表达，转染空质粒pFUSE-mIgG2Aae1-Fc2的293T细胞上清液中有Ig-tail的表达。CCK-8法检测显示24h时空白对照组、阴性对照组、TIM-3组淋巴细胞增殖活力分别为（100．00±10．42）％、（108．70±9．90）％、（78．06±6．37）％，48h时分别为（100．00±6．24）％、（168．00±2．98）％、（42．93±5．93）％；24h、48h时TIM-3组淋巴细胞活力低于空白对照组和阴性对照组（均P〈0．05）。TIM-3组48h相比24h淋巴细胞增殖倍数低于空白对照组和阴性对照组（均P〈0．05）。24h时，空白对照组、阴性对照组、TIM-3组IFN-γ浓度分别为（216．44±7．85）、（223．67±7．79）、（192．96±5．05）ng／L，48h时分别为（230．06±4．23）、（167．24±3．33）、（54．95±0．57）ng／L。2Objective To evaluate the effect ofT-cell immunoglobulin and mucin domain-3 （TIM-3） onTRP-2180-188 peptide-stimulated murine spleen lymphocytes co-cuhured with B16F10 murine melanoma cells. Methods A recombinant plasmid pFUSE-TIM-3-mIgG2Aae1-Fc2 encoding TIM-3 was constructed. Then, the recombinant plasmid and an empty plasmid pFUSE-mIgG2Aae1-Fc2 were transfected into human 293T epithelial cells followed by 48-hour culture for the preparation of supematants containing TIM-3 and Ig-tail respectively. C57BL/6 mice were immunized with the TRP-21180-188 peptide vaccine for 4 sessions. One week after the last vaccination, C57BL/6 mice were sacrificed, andspleen lymphocytes were collected and then cultured with the TRP-2180-188 peptide and interleukin-2 （IL-2） for 5 days, with lymphocytes untreated with the TRP-2180-188 peptide or IL-2 serving as the control group. Mitomycin-treated B16F10 murine melanoma cells and TRP-2180-188 peptide-stimulated lymphocytes were co-cultured with the presence of supernatants of 293T cells that had been cultured for 48 hours （blank control group ）, TIM-3-eontaining supematants （TIM-3 group ） and Ig-tail-containing supernatants （negative control group ） separately. After 24 and 48 hours of co-culture, cell counting kit-8 （CCK-8） assay was performed to estimate the proliferative activity of lymphocytes, enzyme-linked immunosorbent assay （ELISA） to determine the supernatant levels ofinterferon （INF）-γ and tumor neerosis factor （TNF）-α, flow cytometry to determine the percentage of CD8 ^± T cells in the co-culture system. Results Enzyme digestion and sequence analysis showed that the TIM-3 gene was successfully inserted into the eukaryotic expression plasmid. After 48-hour culture, TIM-3 and Ig-tail expressions were detected in the supernatants of 293T cells transfected with the recombinant plasmid and empty plasmid respectively. As CCK-8 assay showed, the proliferative activity of lymphocytes was significantly lower in the TIM-3 group than in the blan

关 键 词：黑色素瘤 实验性 疫苗 亚单位 T淋巴细胞 细胞毒性 干扰素Γ T细胞免疫球蛋白粘蛋白-3 

分 类 号：R739.5[医药卫生—肿瘤]