内毒素耐受小鼠脾脏CD11clowCD45RBKgh树突状细胞培养与生物学特性鉴定  

Cultivation and biological characterization of splenic CDllclow CD45RBhigh dendritic cells derived from endotoxin tolerance mice

作　　者：施春玮[1] 董进中[1] 张赛男[1] 董培红[1] 许烂漫[1] 卢明芹[1] 陈永平[1] 

机构地区：[1]温州医科大学附属第一医院感染科,325000

出　　处：《中华传染病杂志》2014年第12期710-714,共5页Chinese Journal of Infectious Diseases

基　　金：浙江省自然科学基金资助项目（LY12H03002）

摘　　要：目的分离培养内毒素耐受状态小鼠脾脏CDIiclowCD45RBhlghDC并探讨其生物学特性。方法12只体质量为20~25g的健康BALB／c小鼠，采用随机数字表法分成两组，每组6只。正常对照组小鼠腹腔注射0．9％氯化钠溶液0．2mL；内毒素耐受组小鼠以0．1弘g脂多糖腹腔注射，每日1次，连续注射5d建立内毒素耐受小鼠模型。免疫磁珠分选法分选正常对照组和内毒素耐受小鼠脾脏CDIIclowCD45RBhigchDC，流式细胞术检测细胞表型，四甲基偶氮唑盐（MTT）法检测T淋巴细胞增殖抑制率，ELISA法检测上清液中IL-10和IL-12表达量。样本均数比较采用单因素方差分析，Levene法检验方差齐性，方差齐时采用LSD-t检验，方差不齐时采用DunnettT3检验。结果正常对照组小鼠CDIIclowCD45RBhighDC浓度为30％，细胞计数为（5．30±0．12）×10。个；内毒素耐受组CDlllowCD45RBhightDC浓度为80％，细胞计数为（1．20±0．13）×i0。个，两组差异有统计学意义（t=3．23，P〈0．01）。两组细胞形态上未见明显差别。内毒素耐受小鼠脾脏CDIIc“”CD45RB“ghDC表面标志主要组织相容性抗原（MHC）-II、CD40、CD80的表达均较正常对照组明显降低。内毒素耐受组在细胞浓度为i：10、1：50、I：100时细胞增殖率均明显低于正常对照组，差异均有统计学意义（t值分别为1．36、2．49和1．88，均P〈0．01）。内毒素耐受组各浓度细胞与正常对照组比较，IL-10分泌量显著增加，差异有统计学意义（t值分别为13．63、13．45和9．31，均P〈0．01）；IL-12分泌量明显下降，差异有统计学意义（t值分别为2．62、2．74和2．99，均P〈0．05）。结论内毒素耐受小鼠脾脏CDllclowCD45RBhighDC具有较弱的抗原提呈能力及刺激异基因淋巴细胞增殖能力。Objective To isolate and culture splenic CDllcl~~ CD45RBhlgh dendritic cells （DC） derived from endotoxin tolerance （ET） mice and investigate its biological characterization. Methods Mice weighed 20 to 25 gram were completely randomized into two groups including ET group and control group with 6 each. ET mice were modeled by intraperitoneal injection of low-dose lipopolysaecharide （LPS） for several days （pretreated with LPS 0. 1 /,g/mouse for 5 d）. Mice in control group were given the same volume of normal saline （NS）. CDllcI^wCD45RBh^ghDC were isolated from spleen by magnetic activated cell sorting （MACS）. The immunological phenotypes were detected by flow cytometry. The suppressive capacity of CDllcI^w CD45RBhi^h DC was determined by 3-（4, 5-dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide （MTT） colorimetric assay in allogenic mixed T cells reaction. The expressions of interleukin （IL）-I0 and IL-12 produced by CDllclow CD45RBhighDC were measured by enzyme-linked immunosorbent assay （ELISA）. Statistical significance was analyzed through one-way analysis of variance （ANOVA）.The homogeneity of variances was detected by Levene test. If variances were homogeneous, the least significant difference （LSD） test was used. If not, Dunnett T3 test was applied. Results The consistence of CD11clowCD45RBhighDC in control group was 30%, reaching the amount of （5.30＋0.12） X 105/mouse; In ET group, the percentage of CDllclow CD45RBhhigh DC achieved 80 % and the production was （1. 20± 0. 13） ~ 106/mouse the difference was statistically significant （t= 3.23, P〈0.01）. The cellar morphology in two groups showed no obvious difference. Compared to expression levels of all cell phenotypes （histocompatibility complex-l] , CD40 and CD80） in normal mice, the cell surface expression levels of CDllclowCD45RBhigh DC in ET mice were much lower. The difference in two groups was statistically significant. Splenic CDllclowCD45RBhigh DC derived from ET mice with cell concen

关 键 词：内毒素类 树突细胞 白细胞介素10 白细胞介素12 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]