FOLFOX4与1-D-甲基色氨酸联合治疗对荷胃癌615小鼠细胞毒性淋巴细胞的影响

The effect of combination FOLFOX4 and 1-D-methyl tryptophan on CTL cell activity of gastric cancer bearing 615 mice

目的研究FOLFOX4化疗方案与1-D-甲基色氨酸（1-methyl—D—tryptophan，1-D—MT）联合应用对荷胃癌小鼠的免疫耐受的影响。方法用脂质体转染法，将IDO—peDNA3．1质粒和pcDNA3．1（＋）质粒稳定转染MFC细胞，设定空白对照组；用RT—PCR和Westernblot法检测吲哚胺2，3-双加氧酶（indoleamine-2，3-dioxygenase，IDO）mRNA和蛋白表达；建立荷胃癌615小鼠模型，分别给予生理盐水、FOLFOX4、1．D．MT和FOLFOX4＋1-D—MT治疗。利用抑瘤率检测小鼠成瘤情况，流式细胞术检测各组小鼠脾脏中细胞毒性淋巴细胞（cytotoxicity Tlymphocyte，CTL）数量的变化，RT—PCR检测颗粒酶B（granzymeBGZMB）mRNA表达量的变化。结果（1）与MFC和空质粒生理盐水组相比，IDO生理盐水组肿瘤体积大，生长快，差异有统计学意义（P〈0．05）；与IDO生理盐水组比较，FOLFOX4组、1-D—MT组和联合组肿瘤体积均变小，重量减轻，其抑瘤率分别为83．5％±0．4％、81．8％±0．4％、95．7％±0．8％，差异有统计学意义（P〈0．05）。（2）给予生理盐水的IDO阴性对照组CTL及GZMBmRNA含量（22．1％±4．2％、0．370±0．019）较MFC组（46．9％±4．1％、0．450±0．017）、空质粒组（47．6％±4．8％、0．450±0．018）明显降低，差异有统计学意义（P〈0．05）；联合组（64．5％±2．5％、0．660±0．019）和1-D—MT组（55．3％±2．5％、0．560±0．015）与FOLFOX4组（34．2％±3．2％、0．330±0．012）相比，CTL及GZMBmRNA含量明显升高（P〈0．05），且联合治疗组较1—D—MT组升高更明显，差异均具有统计学意义（均P〈0．05）。结论FOLFOX4与1-D—MT联合应用提高CTL细胞对肿瘤细胞的杀伤活性，降低肿瘤的免疫逃逸能力。

Objective To investigate the effect of 1-methyl-D- tryptophan （1-D-MT） and FOLFOX4 on immune tolerance of gastric cancer bearing 615 mice. Methods By using the lipofectamineYM2000, the eukaryotic expression plasmid pcDNA3. 1-IDO and empty vector pcDNA3.1 （ ＋ ） were transfected in a MFC cell line. The expression of indoleamine-2,3-dioxygenase（IDO） was detected by reverse transcription polymerase chain reaction（RT-PCR） and Western blot. Animal model of gastric cancer bearing 615 mice were established to give normal saline, FOLFOX4, 1-D-MT and 1-D-MT ＋ FOLFOX4 therapy respectively. By using flow cytometry, to analysis cytotoxicity T lymphocyte （CTL） cell ratio change and by using RT-PCR, to analysis granzyme B （GZMB） mRNA expression change. Result （ 1 ） Compared with MFC and empty vector saline group, tumors in group IDO saline were much larger, grew faster （ P 〈 0. 05 ）. Compared with IDO normal saline group, tumors in FOLFOX4 group, 1-D-MT group and combination group were smaller, with the tumor inhibitory rates of 83.5%± 0.4% , 81.8% ± 0. 4% ,95.7% ±0. 8% respectively （P 〈0. 05）. （2）CTL cell ratio and GZMB mRNA expression in IDO negative control group treated by normal saline was（22. 1% ±4. 2% ,0. 370±0. 019） much lower than that in MFC group（46.9% ±4. 1% ,0.450 ± 0.017）, empty vector group（47.6%±4.8% ,0.450 ±0.018） （P 〈 0.05）. That in 1-D-MT ＋ FOLFOX4 group （64.5%±2.5%,0.660 ± 0.019）and 1-D-MT group （55.3% ±2.5% ,0. 560±0. 015） were higher than that of FOLFOX4 group （34. 2% ± 3.2% ,0. 330 ± 0. 012） （P 〈 0. 05）. Conclusions Combining 1-D-MT with FOLFOX4 enhances cytotoxicity of CTL cell against grafted cancer cells, by reducing the immune escape of cancer ceils.