CAJ | 학술논문

目的探讨转染人吲哚胺2,3双加氧酶(IDO)基因的胃癌细胞系的IDO表达与免疫逃逸的关系.方法根据IDO基因编码序列,构建真核表达载体pIRES_2-EGFP-IDO,电转染胃癌BGC-823细胞,用G418筛选稳定表达IDO细胞株.用RT-PCR和Western blot检测其IDO mRNA和IDO蛋白表达,测定培养上清中色氨酸和犬尿氨酸的水平.分离胃癌患者外周血T细胞,与转染细胞及加入1甲基色氨酸(1-MT)的细胞共孵育,检测对细胞毒性T淋巴细胞(CTL)和T细胞增殖的影响.结果 pIRES_2-EGFP-IDO真核载体经酶切验证并测序后证实连接成功.稳定表达IDO的BGC-823细胞IDO mRNA明显高于未转染组和对照组,并有IDO蛋白表达.转染组和未转染组色氨酸水平分别为(3.23±0.53)mg/L、(6.03±0.51)mg/L(t=13.32,P=0.000),犬尿氨酸浓度分别为(4.84±0.11)mg/L、(1.83±0.10)mg/L(t=42.916,P=0.000).转染IDO组、1-MT逆转组、转染空质粒组、BGC-823组之间对T淋巴细胞增殖抑制相比差异均有统计学意义(F=114.817,P=0.000),转染IDO组和1-MT逆转组抑制率明显高于转染空质粒组及BGC-823组,同时逆转组抑制率低于转染IDO组(P<0.05).4组CTL杀伤活性之间相比差异均有统计学意义(F=397.449,P=0.000).转染IDO组和1-MT逆转组杀伤活性明显低于转染空质粒组及BGC-823组,同时逆转组CTL杀伤活性也明显高于转染IDO组(P<0.05).结论 IDO转染胃癌细胞后显示IDO抑制了T细胞的增殖和CTL活性,参与胃癌的免疫逃逸作用.
목적 탐토전염인신타알2,3쌍가양매(IDO)기인적위암세포계적IDO표체여면역도일적관계.방법 근거IDO기인편마서렬,구건진핵표체재체pIRES_2-EGFP-IDO,전전염위암BGC-823세포,용G418사선은정표체IDO세포주.용RT-PCR화Western blot검측기IDO mRNA화IDO단백표체,측정배양상청중색안산화견뇨안산적수평.분리위암환자외주혈T세포,여전염세포급가입1갑기색안산(1-MT)적세포공부육,검측대세포독성T림파세포(CTL)화T세포증식적영향.결과 pIRES_2-EGFP-IDO진핵재체경매절험증병측서후증실련접성공.은정표체IDO적BGC-823세포IDO mRNA명현고우미전염조화대조조,병유IDO단백표체.전염조화미전염조색안산수평분별위(3.23±0.53)mg/L、(6.03±0.51)mg/L(t=13.32,P=0.000),견뇨안산농도분별위(4.84±0.11)mg/L、(1.83±0.10)mg/L(t=42.916,P=0.000).전염IDO조、1-MT역전조、전염공질립조、BGC-823조지간대T림파세포증식억제상비차이균유통계학의의(F=114.817,P=0.000),전염IDO조화1-MT역전조억제솔명현고우전염공질립조급BGC-823조,동시역전조억제솔저우전염IDO조(P<0.05).4조CTL살상활성지간상비차이균유통계학의의(F=397.449,P=0.000).전염IDO조화1-MT역전조살상활성명현저우전염공질립조급BGC-823조,동시역전조CTL살상활성야명현고우전염IDO조(P<0.05).결론 IDO전염위암세포후현시IDO억제료T세포적증식화CTL활성,삼여위암적면역도일작용.
Objective To study tumor immune escape in a gastric carcinoma cell line expressing human indoleamine 2,3-dioxygenase(IDO).Methods Human IDO gene was cloned by RT-PCR and the vector for pIRES_2-EGFP-IDO was constructed.BGC-823 cells were transfected with the plasmid using eleetroporation.The integrated INDO genes were detected by RT-PCR and Western blot.The enzyme activity of IDO were measured.T cells from gastric cancer patients were cecuhured with BGC-823 transfected with IDO or added with 1-MT circumstance,T cell-mediated cytotoxicity and proliferation were detected.Results Higher level expression of IDO mRNA and IDO protein Was detected in tumor cells transfected with IDO gene.The level of kynurenic acid was higher in transfected cells compared with no-transfeeted group (4.84±0.11)mg/L vs.(1.83±0.10)mg/L,P=0.000.The cytotoxicity ratio of the IDO transfected group and transfected group with 1-MT circumstance (1-MT group) was lower than control group (P<0.05).The inhibition rate of transfected group with 1-MT group Was higher than control group(P<0.05).Conclusion Gastric cancer cell lines encoded with IDO inhibits T cell-mediated cytotoxicity and proliferation.