【作者】 冯惠枝；

【导师】 王琦；

【作者基本信息】 山西医科大学 ， 消化内科学， 2011， 硕士

【摘要】 目的吲哚胺2,3-双加氧酶(indoleamine 2,3-dioxygenase , IDO)通过耗竭局部的色氨酸(L-tryptophan)或/和色氨酸的代谢产物抑制了T淋巴细胞的增殖,诱导了T淋巴细胞的凋亡,然而在T淋巴细胞凋亡/死亡之前是否还发生了功能上的改变,目前还缺乏这方面的研究。本研究就是为了探讨IDO对效应CD8+T细胞溶胞作用的免疫抑制作用,并初步探讨其机制,为IDO在T淋巴细胞免疫应答中的作用提供新的理论依据。方法通过lipofectamineTM2000转染试剂将pcDNA3.1-IDO转入肝癌细胞株SMMC-7721细胞,设置pcDNA3.1-IDO转染组(I组)、pcDNA3.1-IDO转染并加入1-甲基-D-色氨酸(1 -D-MT)组(I+1-D-MT组)、pcDNA3.1转染组(P组)和SMMC-7721细胞与CD8+T细胞共培养组(7721组),转染48h后应用RT-PCR和Western blot方法检测IDO基因在SMMC-7721细胞中的表达情况。从健康人外周血中分离出CD8+T细胞后与瞬时转染pcDNA3.1-IDO的SMMC-7721细胞(I组)、单纯转入pcDNA3.1的SMMC-7721细胞(P组)、SMMC-7721细胞(7721组)和用1 -D-MT干预的I组(I+1-D-MT组)混合培养4~6h后,用LDH检测试剂盒检测各组中效应CD8+T细胞对SMMC-7721细胞的溶胞作用;混合培养48h后用RT-PCR和Western blot检测各组中效应CD8+T细胞的颗粒蛋白酶B的表达情况。结果1、质粒鉴定质粒经测序后与基因库对比,结果与基因库完全一致。2、用RT-PCR与Western Blot检测瞬时转染的SMMC-7721细胞和与CD8+T细胞共培养的SMMC-7721细胞的IDO表达情况可知:I组表达IDO mRNA(0.95±0.021)及IDO蛋白(1.04±0.078)且高于I+1-D-MT组(0.58±0.032,0.87±0.051),两者比较差异有统计学意义(P<0.05)。P组和7721组则不表达IDO mRNA及IDO蛋白。3、用RT-PCR检测各组中效应CD8+T细胞的颗粒蛋白酶B mRNA的表达情况可知:各组的效应CD8+T细胞均表达颗粒蛋白酶B mRNA,且不受IDO的影响(I组、P组和7721组分别为1.38±0.017、1.21±0.021、1.32±0.027),前者与后两者比较差异无统计学意义(P>0.05);加入1 -D-MT(浓度为2.5 mmol/l)后也没有逆转的现象(1.39±0.016),与I组比较差异无统计学意义(P>0.05)。4、用Western blot检测各组中效应CD8+T细胞的颗粒蛋白酶B蛋白的表达情况可知:I组(0.52±0.017)效应CD8+T细胞的颗粒蛋白酶B蛋白的表达低于P组和7721组(分别为1.02±0.023、1.15±0.055),前者与后两者比较差异有统计学意义(P<0.05);I+1-D-MT组(浓度为2.5 mmol/l)效应CD8+T细胞的颗粒蛋白酶B蛋白的表达(1.01±0.025)高于I组(0.52±0.017),两者比较差异有统计学意义(P<0.05)。5、用LDH检测试剂盒检测各组中效应CD8+T细胞对SMMC-7721细胞的溶胞作用可知:I组(15.32±4.06%)效应CD8+T细胞的溶胞作用低于P组和7721组(分别为60.37±1.53%、60.88±1.49%),前者与后两者比较差异有统计学意义(P<0.05);I+1-D-MT组(浓度为2.5 mmol/l)效应CD8+T细胞的溶胞作用(60.34±1.23%)高于I组(15.32±4.06%),两者比较差异有统计学意义(P<0.05)。结结论瞬时转染IDO基因的SMMC-7721细胞表达IDO mRNA及IDO蛋白,且1-D-MT抑制了IDO mRNA及IDO蛋白的表达;SMMC-7721细胞表达的IDO通过降低效应CD8+T细胞的颗粒蛋白酶B蛋白的表达而非mRNA的表达,抑制了效应CD8+T细胞对SMMC-7721细胞的溶胞作用,而IDO的这种作用可以被1 -D-MT所逆转。更多还原

【Abstract】 Objects Indoleamine 2,3-dioxygenase(IDO)suppress T lymphocyte proliferation and induce T lymphocyte apoptosis by depleting local L-tryptophan or/and tryptophan metabolites, but the research of the possibility of IDO-associated functional alteration before T lymphocyte apoptosis/death is lacked. Now, we reported that inhibition of effector CD8+ T cell mediated cytolytic function is an important mechanism behind IDO’s immune suppressing property.Methods The SMMC-7721 cell was cultured in vitro and transfected with the gene of IDO (IDO group) or with pcDNA3.1 (P group) by lipofectamineTM2000 reagent. There are four groups: the SMMC-7721 cell trancfected with pcDNA3.1-IDO (I group), the I group added 1-methyl-D-tryptophan (1-D-MT) (I+1-D-MT group), the SMMC-7721 cell trancfected with pcDNA3.1 (P group) and the SMMC-7721 cell cocultured with CD8+ T lymphocyte(7721 group). The IDO expression of SMMC-7721 cell was detected by RT-PCR and Western blot after transfected 48h. When CD8+ T lymphocytes were freshly isolated from healthy volunteers’peripheral blood, they were then cocultured with the four groups of cells. The cytolytic activity of effector CD8+ T lymphocyte was detected by LDH assay kit after cocultured 4~6h. The expression of granzyme B of effector CD8+ T lymphocyte was detected by RT-PCR and Western blot after cocultured 48h.Results1. Plasmid identified shows: The sequence of the plasmid pcDNA3.1-IDO is completely concordance with the Gene banks.2.The IDO expression of SMMC-7721 cell transiently transfected and SMMC-7721 cell cocultured with CD8+ T lymphocyte were detected by RT-PCR and Western blot: There was a significant increase of IDO mRNA (0.95±0.021)and protein (1.04±0.078) in SMMC-7721 cell transfected with recombinant plasmid pcDNA3.1-IDO(I group) over I group added 1-D-MT(I+1-D-MT group)(0.58±0.032,0.87±0.051), it was considered statistically significant comparing the two group (P<0.05); but the P group and the 7721 group didn’t express IDO mRNA and protein.3. The granzyme B mRNA expression of effector CD8+ T lymphocyte in each group was detected by RT-PCR: The granzyme B mRNA of effector CD8+ T lymphocyte was expressed in each group, but not subject to the IDO(the I group, the P group and the 7721 group was respectively 1.38±0.017、1.21±0.021、1.32±0.027), it was considered no statistically significant comparing the former with the latter two (P>0.05); adding 1-D-MT (concentration of 2.5mmol/l ) to the I group(I+1-D-MT group) (1.39±0.016)did not reverse the granzyme B mRNA expression of effector CD8+ T lymphocyte, it was considered no statistically significant comparing the I group with the I+1-D-MT group(P>0.05).4. The granzyme B protein expression of effector CD8+ T lymphocyte in each group was detected by Western blot: The granzyme B protein expression of effector CD8+ T lymphocyte in the I group(0.52±0.017) was significant lower than in the P group and the 7721 group(1.02±0.023、1.15±0.055 respectively), it was considered statistically significant comparing the former with the latter two (P<0.05); after 1-D-MT (concentration of 2.5mmol/l ) was added to the I group(I+1-D-MT group), the granzyme B protein expression of effector CD8+ T lymphocyte(1.01±0.025) was higher than the I group(0.52±0.017), it was considered statistically significant comparing the I group with the I+1-D-MT group(P<0.05).5. The cytolytic activity of effector CD8+ T lymphocyte against SMMC-7721 cell was detected by LDH assay kit: A significant decrease of cytolytic activity was observed in the I group (15.32±4.06%) over the P group and the 7721 group(60.37±1.53%、60.88±1.49%), it was considered statistically significant comparing the former with the latter two (P<0.05); after 1-D-MT (concentration of 2.5mmol/l ) was added to the I group(I+1-D-MT group), the cytolytic activity of effector CD8+ T lymphocyte (60.34±1.23%)was higher than the I group(15.32±4.06%), it was considered statistically significant comparing the I group with the I+1-D-MT group(P<0.05).Conclusions The IDO mRNA and IDO protein was expressed by the SMMC-7721 cell transiently transfected with recombinant plasmid pcDNA3.1-IDO, and the 1-D-MT inhibit the expression of the IDO mRNA and IDO protein. The IDO expressed by SMMC-7721 cell suppress the cytolytic activity of effector CD8+ T lymphocyte by reducing the granzyme B protein expression rather than the mRNA expression, but the IDO inhibitor 1-D-MT could reverse the immune inhibitory effect of IDO.更多还原