Hepal-6细胞热休克后裂解蛋白致敏的树突状细胞瘤苗对肝细胞癌瘤内CD25^+Foxp3^+Treg细胞的影响  

Effect of administration of BMDC vaccine sensitized by heat shocked hepal-6 cell proteins on intratumoral CD25^+Foxp3^+ Tregs in mouse hepatocellular carcinoma

作　　者：李日伦[1] 周爽[2] 秦杰[2] 梁春敏[2] 罗国容[1] 

机构地区：[1]广西医科大学组织胚胎学教研室,广西壮族自治区南宁市530021 [2]复旦大学上海医学院解剖与组织胚胎学系,上海市200032

出　　处：《世界华人消化杂志》2014年第15期2081-2090,共10页World Chinese Journal of Digestology

基　　金：国家自然科学基金青年基金资助项目;No.30500280;国家自然科学基金面上基金资助项目;No.30871312~~

摘　　要：目的:观察注射Hepal-6细胞热休克后裂解蛋白致敏的骨髓来源树突状细胞(bone marrowderived dendritic cells,BMDCs)瘤苗对小鼠肝细胞癌(hepatocellular carcinoma,HCC)瘤内CD25+叉头盒转录因子P3(forkhead box p3,Foxp3)+调节T淋巴细胞(regulatory T cells,Tregs)浸润的影响.方法:在粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony stimulatingfactor,GM-CSF)和白介素-4(interleukin-4,IL-4)诱导下体外扩增BMDCs,使用Hepal-6细胞热休克后裂解蛋白体外致敏BMDCs制备瘤苗,荧光免疫化学染色和FACS检测致敏前后BMDCs CD11c、CCR7、CD80和CD86的表达变化.使用Hepal-6细胞皮下注射的方法制备小鼠(C57BL/6J)HCC模型,成瘤小鼠分组注射Hepal-6细胞热休克后裂解蛋白致敏的BMDCs瘤苗(足垫部和瘤内,每7 d注射1次,共2次),并另设对照(空白对照组、BMDCs组和Hepal-6细胞裂解蛋白组).在治疗结束后9 d获取组织标本,免疫荧光组织化学染色和FACS检测瘤苗注射后肿瘤内CD8+T细胞和CD25+Foxp3+Tregs细胞的浸润情况.结果:光镜和扫描电镜显示:GM-CSF和IL-4在体外诱导扩增的BMDCs具有树突状细胞特征性的形态特征,且免疫细胞化学染色显示:该细胞表达CD11c,CCR7,CD80和CD86.使用Hepal-6细胞热休克后裂解蛋白致敏的BMDCs组,与对照组(BMDCs组和Hepal-6细胞裂解蛋白组)相比该组细胞CD11c(67.2±4.49 vs 52.4±5.20,58.4±4.43,P<0.01),CCR7(65.4±5.34 vs 45.9±5.04,57.0±3.46,P<0.01),CD80(62.9±4.69 vs 46.9±4.75,54.4±3.47,P<0.01)和CD86(73.3±3.58 vs 60.1±2.98,63.7±3.10,P<0.01)的表达均明显增高.使用Hepal-6细胞热休克后裂解蛋白致敏的BMDCs瘤苗为HCC荷瘤小鼠进行注射治疗,治疗后的检测结果显示:该组小鼠瘤内CD8+T细胞的浸润明显高于对照组(空白对照组、BMDCs组和Hepal-6细胞裂解蛋白组)(55.0±4.11 vs 38.2±3.34,44.6±4.29,45.6±4.92,P<0.01),而同时瘤内CD25+Foxp3+Tregs细胞的浸润则明显低于相应对照组(0.37±0.028 vs 1.31±0.020,0.77±0.0AIM： To determine whether the bone marrow derived dendritic cell （BMDC） vaccine sensitized by heat shocked hepal-6 cell proteins affects the infiltration of intratumoral CD25＋Foxp3＋ Tregs in a mouse hepatocellular carcinoma （HCC） model.METHODS： In the presence of GM-CSF and IL-4, BMDCs were induced in vitro. BMDCs were sensitized by heat shocked hepal-6 cell pro- teins to generate a vaccine for HCC. The expression of CD11c, CCR7, CD80 and CD86 on these sensitized BMDCs were analyzed by FACS. The anti-tumor effect of this vaccine was evaluated using a mouse HCC model established by sub- cutaneous injection of Hepal-6 cells. Eight days later, the tumor-bearing mice were divided into four groups, which underwent intratumoral injection of BMDCs sensitized by heat shocked hepal-6 cell proteins, serum-free culture me- dium, BMDCs without sensitization and BMDCs sensitized by unheated hepal-6 cell proteins （once every 7 d, 2 times altogether）, respectively. Nine days after final administration, the mice were sacrificed and the tumor samples were taken for immunofluorescence staining for CD8＋ cells and intratumoral CD25＋Foxp3＋ Tregs. RESULTS： Light microscopy and scanning electron microscopy showed that BMDCs propagated in the presence of GM-CSF and IL-4 displayed the typical morphological characteristics of dendritic ceils. Immunocytochemical staining showed that they expressed the dendritic cell marks including CD11c, CCR7, CD80 and CD86. Compared with the controls （BMDCs without sensitization or sensitized by unheated hepal-6 cells proteins）, the BMDCs sensitized by heat shocked hepal-6 cells proteins showed increased expression of CD11c （67.2 ± 4.49 vs 52.4 ± 5.20, 58.4 ± 4.43）, CCR7 （65.4 ± 5.34 vs 45.9 ± 5.04, 57.0 ± 3.46）, CD80 （62.9 ± 4.69 vs 46.9 ± 4.75, 54.4 ± 3.47） and CD86 （73.3 ± 3.58 vs 60.1 ± 2.98, 63.7 ± 3.10） （P 〈 0.01 for all）. Compared with the controls, the mice administrated with the BMDC vaccine sensitized by heat shocked Hepal-6 cell

关 键 词：Hepal-6细胞热休克后裂解蛋白 骨髓来源树突状细胞 CD8+T细胞 CD25+Foxp3+Treg细胞 肝细胞癌 

分 类 号：R735.7[医药卫生—肿瘤]