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+CD25-T细胞中FOXP3mRNA的表达,可能与TGF-β1有关。
AIM: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 peripheric blood CD4^+CD25^-T cells can be isolated in vitro by discontinuous density gradient centrfugal and dynal immunomagnetic beads.The expression of FOXP3 mRNA in E.coil LPS-induced human CD4^+CD25^-T cells may correlate with the expression of TGF-β1.
作者
苏苗赏
徐漫欢
陈福将
李昌崇
张维溪
陈小芳
陈慧
SU Miao-shang;XU Man-huan;CHEN Fu-jiang;LU Chang-chong;ZHANG Wei-xi;CHEN Xiao-fang;CHEN Hui(Department of Pediatries,Second Affiliated Hospital of WenzhouMedical College,Wenzhou 325027,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
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)
