分离培养人外周血CD4^+CD25^-T细胞及其生物学特性研究  

Biological characteristics of human peripheric CD4^+CD25^-T cells isolated and cultured in vitro

作　　者：苏苗赏[1] 徐漫欢[2] 陈福将[1] 李昌崇[1] 张维溪[1] 陈小芳[3] 陈慧[4] 

机构地区：[1]温州医学院附属第二医院儿科,浙江温州325027 [2]温州医学院附属第二医院神经内科,浙江温州325027 [3]温州医学院附属第二医院科研中心,浙江温州325027 [4]温州医学院附属第二医院检验科,浙江温州325027

出　　处：《细胞与分子免疫学杂志》2007年第8期711-714,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30571981);浙江省自然科学基金项目(Y205426)

摘　　要：目的:分离培养人外周血CD4+CD25-T细胞,并鉴定其生物学特性。方法:设对照组(A组)、LPS组(B组)、LPS+抗TGF-β1mAb组(D),用Percoll不连续密度梯度离心与免疫磁珠法,分离培养健康人外周血CD4+CD25-T细胞。用光镜及电镜观察其形态特征,台盼蓝试验检测其活力,流式细胞术(FCM)鉴定其纯度。体外培养4h、3d及5d后,用FCM检测CD4+CD25+T细胞的阳性率,ELISA法检测培养上清中TGF-β1的浓度,RT-PCR法检测细胞中叉状头/翼状螺旋转录因子(FOXP3)mRNA的表达。结果:(1)光镜下观察分离的CD4+CD25-T细胞主要为小体积细胞,电镜下观察细胞核呈圆形,染色质致密。体外抗人CD3/CD28mAb刺激培养的CD4+CD25-T细胞体积逐渐增大,胞质较丰富,电镜下观察细胞核呈椭圆形或肾型,染色质较稀疏。(2)FCM检测CD4+CD25-T细胞纯度达91.5%～96%。台盼蓝试验检测分离前后活细胞数无统计学意义(P>0.05)。(3)FCM检测表明,B5d组为CD4+CD25+T细胞的阳性率为(55.99±1.42)%与A5d组相比较有统计学意义(P<0.01);D5d组CD4+CD25+T细胞的阳性率为(1.99±0.83)%与A5d组的阳性率(1.29±0.04)%相比较无统计学意义。(4)ELISA测定表明,培养液中TGF-β1的浓度,B3d组为(1.60±0.09)μg/L、B5d组为(1.83±0.14)μg/L,分别与A3d组为(0.35±0.04)μg/L、A5d组为(0.33±0.08)μg/L相比较,均有统计学意义(P<0.01);而D3d、D5d组与A3d、A5d组相比较均无统计学意义。(5)RT-PCR检测表明,FOXP3mRNA的表达:以β-actin的A值作为内参照,B3d组为(0.84±0.07)、B5d组为(1.85±0.24)分别与A3d组(0.05±0.02)、A5d组(0.04±0.02)相比较,均有统计学意义(P<0.01);而D组与A组的各个组间相比较均无统计学意义。(6)LPS诱导的人外周血中CD4+CD25-T细胞培养液上清中TGF-β1的水平与该细胞中FOXP3mRNA的表达呈显著的正相关(r=0.812,P<0.01)。结论:用Percoll不连续密度梯度离心与免疫磁珠法体外分离培养的人外周血CD4+CD25-T细胞的活力及纯度较理想;LPS诱导的CD4+CDAIM： To isolate peripheric CD4 ^＋ CD25 ^- T cells in vitro, and study their biological characteristics. METHODS： Human pedpheric blood CD4^＋ CD25^-T cells were isolated by discontinuous density gradient centrfugal and dynal immunomagnetic beads, and then divided into three groups： control group（A）, LPS group （B） and LPS ＋ TGF-β1 mAb group （D）, After they were prepared 4 h, 3 d and 5 d, the percentage of CD4^＋ CD25 ^＋ T cells were tested by FCM, the levels of TGF-β1 in cultured supernatants were detected by ELISA, and the expression of FOXP3 mRNA was examined by RT-PCR. RFSULTS： （I）LM showed CD4^ ＋ CD25^- T cells were of spherical cell nucleus, After CD4 ^＋ CD25^- T cells were cultured by anti-CD3/CD28 in vitro, their cellular volume increased and cytoplasm contained more particles. TEM showed CD4^ ＋ CD25^- T cells were of oval or kidney-shaped caryon. （2）FCM showed the purity of CD4^ ＋ CD25^ - T cells was 91.5% - 96%. The trypan blue experiment showed the energometry before and after detachment had no obvious difference（ P 〉 0. 05 ）. （3） FCM showd the percentage of CD4^＋ CD25 ^＋ T cells in B5d group （55.99 ± 1.42） % increased markedly compared with that in A5d group（1.29 ±0. 04）%. （4） ELISA showed the levels of TGF-β1 in B3d（1.60 ±0.09） lag/L, in B5d（1.83 ±0. 14） lag/L were significantly increased compared with those in A3d （0. 35 ±0. 04） lag/L and A5d（0. 33 ±0. 08） lag/L（ P〈0. 01 ） ; but the levels in group D and group A had no significantdifference; （5）RT-PCR showed FOXP3 mRNA in B3d group（0. 84 ± 0. 07 ） and BSd group （ 1.85± 0. 24） increased strikingly compared with those in A3d（0. 05 ±0. 02） and ASd （0. 04 ±0. 02） （ P〈0. 01 ）, but those in group A and goup D had no significant difference. （6）There was significantly positive correlation between the expression of TGF-β1 and FOXP3 mRNA in the induced CD4 ^＋ CD25^- T cells （ r = 0. 812, P〈0. 01）. CONCLUSION： Human pe

关 键 词：T淋巴细胞亚型 转化生长因子 叉状头/翼状螺旋转录因子 脂多糖 

分 类 号：R392.12[医药卫生—免疫学]