CAJ | 학술논문

目的分离培养内毒素耐受状态小鼠脾脏CD11clowCD45 RBhigh DC并探讨其生物学特性.方法 12只体质量为20～25 g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0.2 mL;内毒素耐受组小鼠以0.1 μg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CD11clowCD45 RBhigh DC,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Dunnett T3检验.结果正常对照组小鼠CD11clowCD45RBhigh DC浓度为30％,细胞计数为(5.30±0.12)×105个;内毒素耐受组CD11clowCD45 RBhigh DC浓度为80％,细胞计数为(1.20±0.13)×106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CD11clowCD45RBhigh DC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1∶10、1∶50、1∶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CD 11clowCD45 RBhigh DC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.
목적 분리배양내독소내수상태소서비장CD11clowCD45 RBhigh DC병탐토기생물학특성.방법 12지체질량위20～25 g적건강BALB/c소서,채용수궤수자표법분성량조,매조6지.정상대조조소서복강주사0.9％록화납용액0.2 mL;내독소내수조소서이0.1 μg지다당복강주사,매일1차,련속주사5d건립내독소내수소서모형.면역자주분선법분선정상대조조화내독소내수소서비장CD11clowCD45 RBhigh DC,류식세포술검측세포표형,사갑기우담서염(MTT)법검측T림파세포증식억제솔,ELISA법검측상청액중IL-10화IL-12표체량.양본균수비교채용단인소방차분석,Levene법검험방차제성,방차제시채용LSD-t검험,방차불제시채용Dunnett T3검험.결과 정상대조조소서CD11clowCD45RBhigh DC농도위30％,세포계수위(5.30±0.12)×105개;내독소내수조CD11clowCD45 RBhigh DC농도위80％,세포계수위(1.20±0.13)×106개,량조차이유통계학의의(t=3.23,P＜0.01).량조세포형태상미견명현차별.내독소내수소서비장CD11clowCD45RBhigh DC표면표지주요조직상용성항원(MHC)-Ⅱ、CD40、CD80적표체균교정상대조조명현강저.내독소내수조재세포농도위1∶10、1∶50、1∶100시세포증식솔균명현저우정상대조조,차이균유통계학의의(t치분별위1.36、2.49화1.88,균P＜0.01).내독소내수조각농도세포여정상대조조비교,IL-10분비량현저증가,차이유통계학의의(t치분별위13.63、13.45화9.31,균P＜0.01);IL-12분비량명현하강,차이유통계학의의(t치분별위2.62、2.74화2.99,균P＜0.05).결론 내독소내수소서비장CD 11clowCD45 RBhigh DC구유교약적항원제정능력급자격이기인림파세포증식능력.
Objective To isolate and culture splenic CD11clow CD45RBhigh 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 lipopolysaccharide (LPS) for several days (pretreated with LPS 0.1 μg/mouse for 5 d).Mice in control group were given the same volume of normal saline (NS).CD11clowCD45RBhighDC were isolated from spleen by magnetic activated cell sorting (MACS).The immunological phenotypes were detected by flow cytometry.The suppressive capacity of CD11clow CD45RBhigh 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)-10 and IL-12 produced by CD11clow CD45RBhigh DC 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 CD1 1 clow CD45RBhigh DC in control group was 30 ％,reaching the amount of (5.30±0.12) × 105/mouse ;In ET group,the percentage of CD11clow CD45RBhighDC 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-Ⅱ,CD40 and CD80) in normal mice,the cell surface expression levels of CD11clowCD45RBhigh DC in ET mice were much lower.The difference in two groups was statistically significant.Splenic CD11clowCD45RBhighDC derived from ET mice with cell concentration of 1∶ 10,1∶50and 1∶100 had more obvious prohibitory effects on allogenic T cells (t1∶0 =1.36,P1∶10 ＜0.01,t1∶50 =2.49,P1∶50 ＜0.01,1∶100 =1.88,Pm00 ＜0.01).Secretion of IL-10 produced by CD11clowCD45RBhighDC of ET mice was significantly increased (t1∶0=13.63,P1∶10 ＜0.01,t1∶50 =13.45,P1∶50 ＜0.01,t1∶00 =9.31,P1∶00 ＜0.01),but the expression of IL-12 was lower (t1∶0 =2.62,P1∶0 =0.02,1∶∶50 =2.74,P1∶0=0.02,t1∶100 =2.99,P1∶100 =0.01).Conclusion Splenic CD11clow CD45RBhigh DC from ET mice have weaker ability of antigen presenting and allogeneic lymphocytes proliferation stimulating than those from normal mice.