摘要
目的 观察跨膜型葡萄球菌肠毒素A(SEA TM)和糖基化磷脂酰肌醇锚定型mB7 1(mB7 1 GPI)二种免疫分子膜表面修饰瘤苗的抗肿瘤作用是否优于单种免疫分子膜表面修饰的瘤苗。方法 构建pcDNA3 1( + ) /mB7 1 GPI真核表达载体 ,转染中国仓鼠卵巢上皮细胞 (CHO)中 ,表达和纯化mB7 1 GPI。通过蛋白转染法将SEA TM、mB7 1 GPI单独或共同锚定到EL 4肿瘤细胞膜上 ,制成瘤苗 ,观察这些瘤苗刺激小鼠脾细胞的增殖和分泌白细胞介素 (IL) 2和γ干扰素 (IFN) γ的量及抗肿瘤作用。结果 mB7 1 GPI和SEA TM能单独或共同锚定在肿瘤细胞膜上 ,具有相当的稳定性 ;在体外有刺激小鼠脾细胞增殖和分泌IL 2、IFN γ的功能。mB 7 1 GPI、SEA TM、mB 7 1 GPI +SEA TM锚定肿瘤细胞 ,制成的瘤苗 ,能抑制荷瘤小鼠的肿瘤生长和延长荷瘤小鼠的存活时间。mB 7 1 GPI和SEA TM双锚定瘤苗 ,显示出比单一蛋白锚定瘤苗更强的抗肿瘤作用。结论 用蛋白转染法将SEA和mB7 1二种免疫分子同时锚定到肿瘤细胞膜上所制成的瘤苗 。
Objective To prepare the SEA TM and mB7 1 GPI dual anchored EL 4 cell vaccine and to investigate its antitumor effects Methods mB7 1 GPI anchored EL 4 cell vaccine, SEA TM anchored EL 4 cell vaccine, SEA TM and mB7 1 GPI dual anchored EL 4 cell vaccine were prepared In vitro the biological activities of these vaccines were measured using a lymphocyte proliferation assay and cytokine release assay on splenocytes derived from C57BL/6 mice The splenocytes were co cultured with EL 4 or EL 4/mB7 1 GPI or EL 4/SEA TM or EL 4/SEA TM+mB7 1 GPI (treated with Mitomycin C) Lymphocyte proliferation was determined with MTT assay, the concentrations of cytokines (IL 2 and IFN γ) were measured using a ELISA technique Forty C57BL/6 mice were inoculated with EL 4 cells, after 3 days the mice were randomly divided into 5 groups with 8 in each and were treated with PBS, EL 4 cell vaccine, EL 4/mB7 1 GPI cell vaccine, EL 4/SEA TM cell vaccine and EL 4/SEA TM+mB7 1 GPI cell vaccine respectively, vaccines were injected three time with two day interval Animals were observed daily, tumor sizes were measured every third day Twenty five days after tumor challenge, 3 mice in each group were sacrificed and splenic lymphocytes were isolated to examine the activity of natural killer cells (NK) and cytolytic T lymphocytes (CTL) The survival of the remaining 5 mice in each group was observed till the 90th day Results mB7 1 GPI or/and TM SEA fusion protein was stably anchored onto the surface of EL 4 tumor cells EL 4/mB7 1 GPI or EL 4/SEA TM had a stronger ability to stimulate lymphocyte proliferation and IL 2 and IFN γ production than EL 4 ( P <0 05); while EL 4/SEA TM+ mB7 1 GPI showed a further increased ability than EL 4/mB7 1 GPI and EL 4/SEA TM in stimulating lymphocyte proliferation and cytokine production in vitro ( P <0 05) Volume of tumor was smaller and survival time of mice was longer in EL 4/mB7 1 GPI vaccine group, EL 4/SEA TM vaccine group and EL 4/SEA TM+mB7 1 GPI vaccine group, comparing with PBS group and EL 4 cell vaccine group ( P <0 05) Tumor volume was much smaller and survival time of mice was much longer in EL 4/mB7 1 GPI+mB7 1 GPI vaccine group, comparing with EL 4/SEA TM vaccine group and EL 4/mB7 1 GPI vaccine group ( P <0 05) Lymphocytes derived from the mice treated with EL 4/SEA TM+ mB7 1 GPI showed much higher NK activity and CTL activity than those derived from EL 4/mB7 1 GPI vaccine group and EL 4/SEA TM vaccine group ( P <0 05), meanwhile the NK activity and CTL activity of EL 4/mB7 1 GPI vaccine group and EL 4/SEA TM vaccine group was higher than EL 4 vaccine group ( P <0 05) Conclusion mB7 1 GPI or/and SEA TM fusion protein was stably anchored onto the surface of EL 4 tumor cells The tumor cell vaccines prepared from these cells exhibited antitumor effect The mB7 1 GPI and SEA TM dual anchored tumor cell vaccine had much stronger antitumor effect than the single anchored tumor cell vaccine
出处
《中华医学杂志》
CAS
CSCD
北大核心
2004年第18期1567-1571,共5页
National Medical Journal of China
基金
国家自然科学基金资助项目 (3 9770 83 7)
浙江省自然科学基金资助项目(3 9913 1
3 0 15 80 )
