冬凌草甲素增强对小鼠H22肝癌细胞免疫杀伤作用的实验性研究  

Experimental study on the immunological mechanisms of oridonin's cytotoxicity to mouse H22 cell line

作　　者：沈青[1,2] 季萍[1] 杨忠英[2] 樊洪中[2] 王树军[1] 王颖[1] 

机构地区：[1]上海市免疫学研究所上海交通大学医学院,上海200025 [2]上海交通大学医学院附属新华医院崇明分院,上海202150

出　　处：《现代免疫学》2016年第2期123-129,共7页Current Immunology

基　　金：上海市教委项目(上海高校实验技术队伍建设计划);上海市自然科学基金(14ZR1436300)

摘　　要：为阐明冬凌草甲素(ORI)抗小鼠H22肝癌中的T细胞免疫应答机制,本研究利用C57/BL6小鼠H22肝癌皮下移植瘤模型,随机分为荷瘤对照组(PBS组)、ORI高、中、低剂量处理组(75、50、25mg·kg^(-1)·d^(-1))、阳性对照组(5-Fu组)和空白组(NC组),连续腹腔注射给药12d后处死。以瘤重、抑瘤率观察ORI对移植瘤生长的影响;用乳酸脱氢酶释放法检测对小鼠H22细胞杀伤活性;流式细胞术检测小鼠脾脏细胞中CD4^+T细胞分泌IL-17、IL-2、TNF-α、IFN-γ等细胞因子能力,并检测CD8^+T和CD4^+T细胞表面PD-1的表达。结果显示:低、中、高剂量ORI处理组和荷瘤对照组比较均有显著性缩小(P<0.05);ORI能明显提高荷瘤小鼠脾脏细胞对H22细胞的杀伤活性(P<0.05);抑制CD4^+T细胞中细胞因子的分泌,尤其对IL-17和IL-2的抑制最为明显,对TNF-α和IFN-γ抑制在ORI高浓度情况下发挥作用;同时,ORI处理组中CD8^+T细胞的PD-1表达降低,而CD4^+T细胞表面PD-1的表达呈现一定程度的上调。上述结果表明ORI对小鼠H22肿瘤具有明显的体内抑瘤作用,除了直接的杀伤作用外,ORI还可以通过提高CD8^+T的杀伤作用,降低CD4^+T细胞的炎症特性发挥抗肿瘤免疫应答功效,其中的可能机制是影响两种细胞表面PD-1的表达。因此,ORI还可通过协同T细胞抗肿瘤免疫应答机制促进对肿瘤的抑制作用。Mouse graft hepatoma model was established by using H22 cell line to determine the immunological anti-tumor mech- anisms mediated by oridonin.. C57/BL6 mice bearing H22 graft tumors were treated intra-peritoneally with oridonin at high, medium and low concentrations （75, 50 and 25mg · kg-1 · day-1） respectively while PBS and 5-Fu treatment served （were considered） as negative and positive controls, respectively. Mice were sacrificed after 12 days injection intra-peritoneally. The inhibitory rate of graft tumors was evaluated based on the tumor weight and volume. Cytotoxicity of splenocytes against H22 in vitro was determined by LDH releasing assay. CD4＋ T cells secreting IL-17 ,IL-2 ,TNF-α and IFN-γ were detected by using in- tracellular cytokine staining. PD-1 expressions on CD8＋ T and CD4＋ T ceils were also analyzed. The results showed that orido- nin suppressed graft tumor growth significantly in mice in a dose-dependent manner （with inhibitory rates of 32.51%, 40. 02 %, 52.51% at low, medium and high concentrations, respectively, P〈0.05） when compared to PBS treatment. Spleno- cytes from ORI treated mice exhibited higher cytotoxicity against H22 cells in vitro （P〈0. 05）. The percentages of CD4＋ T cells secreting IL-17 and IL-2 dramatically decreased in ORI treated tumor-bearing mice at all three different concentrations while treatment with high concentration of ORI also exerted the decrease of TNF-α and IFN-γ producing CD4＋ T cells. More interestingly, ORI treatment was associated with the decrease of PD-1 expression on CD8＋ T cells whereas upregulation of PD- 1 on CD4＋ T cells in the spleens of tumor bearing mice. We conclude that ORI exhibits anti-tumor effects in vivo. Besides its direct killing on tumor ceils reported previously, ORI also display immunological anti-tumor effects orchestrating the enhanced cytotoxicity against tumor cells and anti-inflammtoryinflammatory effects on CD4＋T cells, which are associated with modula- tion on PD-1 expression on T cells.

关 键 词：冬凌草甲素 小鼠H22肝癌细胞 T细胞 炎症因子 PD-1 

分 类 号：R392.5[医药卫生—免疫学]