老年荷瘤小鼠髓源性抑制细胞亚群检测及功能研究  

Identification of the subpopulations of myeloid-derived suppressor cells and the function study in elderly tumor-bearing mice

作　　者：陈思文[1] 王翎[1] 苏楠[2] 张光波 毛燕青[1] 李洁[1] 潘旭东[1] 

机构地区：[1]苏州大学附属第一医院博习诊疗中心,215006 [2]苏州大学附属第一医院呼吸内科,215006 [3]江苏省临床免疫重点实验室,苏州215006

出　　处：《中华老年医学杂志》2016年第6期651-655,共5页Chinese Journal of Geriatrics

基　　金：江苏省自然科学基金（BK2012607）,苏州市科教兴卫青年科技项目（KJXW2011005）

摘　　要：目的 以健康老年小鼠为对照，检测老年荷瘤小鼠髓源性抑制细胞（MDSCs）亚群的水平，探讨MDSCs亚群免疫抑制功能的差异及机制。 方法 随机选取20只C57BL/6老年小鼠（18～20月龄）建立Lewis肺癌模型，采用流式细胞术检测健康老年小鼠和荷瘤老年小鼠单核系、粒系MDSCs的水平。免疫磁珠分选法分选荷瘤老年小鼠脾脏单核系、粒系MDSCs，经迈-格-吉染色后观察两亚群细胞形态。运用溴脱氧嘧啶核苷-酶联免疫吸附实验（BrdU-ELISA）法检测MDSCs亚群对T细胞增殖的影响，实时荧光定量PCR法检测MDSCs亚群抑制性免疫介质的表达。 结果 与健康老年小鼠相比，荷瘤老年小鼠脾脏中单核系MDSCs明显增加[（12.44±1.20）%比（38.42±3.66）%，t＝5.67，P〈0.001]，而粒系MDSCs水平无明显增加[（10.34±0.68）%比（12.18±1.27）%，t＝2.21，P＝0.09]；BrdU-ELISA检测结果显示：单核系MDSCs能显著抑制T细胞增殖[（0.30±0.18）比（3.38±0.96），t＝8.33，P〈0.001]，而粒系MDSCs不能明显抑制T细胞增殖[（2.69±0.45）比（3.38±0.96），t＝1.72，P＝0.11]。PCR结果表明，与粒系MDSCs相比，单核系MDSCs高表达精氨酸酶-1、诱导型一氧化氮合酶、白介素-10、干扰素-γ[t＝4.31、8.89、1.70、3.13，P〈0.01或P〈0.05]，而两者白介素-13、转化生长因子-β表达无明显差异（t＝4.94、2.75，P＝0.39、0.47）。 结论 老年Lewis肺癌小鼠脾脏内显著增加的是单核系MDSCs，该亚群可通过高表达精氨酸酶-1、诱导型一氧化氮合酶、白介素-10、干扰素-γ来抑制T细胞增殖，介导老年肺癌肿瘤免疫逃逸。Objective To investigate the level of subpopulations of myeloid-derived suppressor cells （MDSCs） in elderly tumor-bearing mice versus elderly tumor-free mice, and to study the difference in immune suppressive functions between different subpopulations and their mechanisms. Methods A total of 20 healthy C57BL/6 elderly mice（aged 18-20 months） were randomly chosen to establish Lewis lung cancer models. The amount of monocytic-MDSCs（MO-MDSCs） and polymorphonuclear granulocytic-MDSCs（PMN-MDSCs） in tumor-free and tumor-bearing elderly mice was evaluated by using flow cytometry. MO-MDSCs and PMN-MDSCs were separated with Magnetic-Activated Cell Sorting （MACS） MicroBeads and their morphological characteristics were observed after May- Grunwald-Giemsa staining. The effects of MO-MDSCs and PMN-MDSCs on the proliferation of T cells were determined by Brdu-enzyme-linked immunosorbent assay（ELISA）. And the immune suppressive mediators secreted by the subpopulations were detected by Real-time polymerase chain reaction（PCR）. Results Compared to the tumor-free group,the proportion of MO-MDSCs in the spleen of tumor- hearing group were increased [-（12.44±1.20） G vs. （38.42±3.66） G,t=5.67,P%0.001],while PMN-MDSCs were not [-（10.34±0.68） G vs. （12. 18±1.27） G,t=2.21,P=0.09]. The result of Brdu-ELISA showed that MO-MDSCs could suppress the proliferation of T cells [.（0. 30±0.18） vs. （3.38＋_0.96） ,t=8.33,P〈0. 001] ,while PMN-MDSCs could not [（2. 69±0.45）vs. （3. 38±0.96） ,t = 1.72,P= 0.11]. The result of PCR showed that as compared with PMN-MDSCs, Mo-MDSCs had the increased expression levels of arginase-1 （ARG-1）, inducible nitric oxide synthase （iNOS）, interleukin-10 （ IL-10 ）, interferon-7 （IFN-7） （ t = 4.31, 8.89, 1.70, 3.13, respectively, P 〈 0.01 or 0.05）, while the expression levels of interleukin-13 （IL-13）, transforming growth factor-β （TGF-β） had no differences（t = 4.94 and 2.75, P = 0.39 and0.47）. Conclusions MO-MDSCs

关 键 词：肺肿瘤 免疫 肿瘤逃逸 

分 类 号：R734.2[医药卫生—肿瘤]