mCD1.1分子的真核表达及其对NKT细胞的刺激作用  

Expression of mouse CD1.1 in CHO cells and its stimulatory function on NKT cells

作　　者：易绍琼[1] 彭虹[1] 王华[1] 王岚[1] 陈洁[1] 赵光宇[1] 高杰英[1] 

机构地区：[1]军事医学科学院微生物流行病研究所病源微生物生物安全国家重点实验室,北京100071

出　　处：《细胞与分子免疫学杂志》2006年第5期579-581,共3页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家重点基础研究发展规划(973)资助项目(G1999054103)

摘　　要：目的:获得稳定表达mCD1.1分子的细胞系,研究其对肝脏和肠系膜淋巴结淋巴细胞的刺激作用。方法:分离C57小鼠的小肠上皮细胞,制备总RNA,通过RTPCR扩增mCD1.1编码区基因,将其克隆至真核表达载体pcDNA3.1。将所得重组质粒pcDNA3.1mCD1.1通过电穿孔法转至CHO细胞中,在含G418的的选择性培养基中筛选抗性克隆,利用RTPCR和流式细胞术对挑取的克隆进行mCD1.1表达的鉴定。将表达mCD1.1的细胞与新分离的肝脏和肠系膜淋巴结(MLN)的淋巴细胞进行共培养,并用丝裂霉素C抑制CHO细胞的增殖,然后利用MTT法检测淋巴细胞的增殖情况;另外将表达mCD1.1的CHO细胞与淋巴细胞共培养后,分离淋巴细胞,然后用PEantiNK1.1和CychromeantiCD3对其进行标记,利用流式细胞术检测CD3细胞中NK1.1阳性细胞的比例。结果:通过RTPCR,得到mCD1.1的编码区基因,序列与预期相符;通过多次鉴定,证明筛选得到的细胞克隆可稳定表达mCD1.1;CHOmCD1.1细胞与新分离的淋巴细胞共培养,在有无LPS存在的情况下,均能刺激淋巴细胞发生增殖,并使CD3+细胞中NK1.1阳性细胞的比例增加。结论:表达mCD1.1的CHO细胞能够刺激NKT细胞增殖。AIM： To get cell lines which express mouse CD1.1 （ mCD1. 1 ） stably and to study the stimulatory effects of mCD1. 1 on lymphocytes from many tissues. METHODS： The intestinal epithelial cells were isolated and their total RNA was prepared. By RT-PCR, the gene coding mCD1.1 was obtained and then cloned into the pcDNA3.1 vector through BamH I and EcoR I. The reconstructed plas- mid, named pcDNA3.1-mCD1.1, was transfected into CHO cells by electroporation. The clones that grew well in the medium containing G418 were selected and their mCDI. 1 expression was analyzed by RT-PCR and flow cytometry （FCM）. The mCD1. 1-expressing CHO cells were co-cultured with lymphocytes freshly isolated from the liver or mesenteric lymph nodes with or without LPS. The CHO cells were treated with mitomycin C to inhibit their proliferation. The lymphocyte proliferation was detected by MTT. In addition, the lymphocytes collected from the co-culture system were stained with fluorescein-labeled monoclonal antibodies against mouse NK1. 1 and CD3. The double positive cells were detected by FCM. RESULTS： By RT-PCR, the gene coding mCD1.1 was acquired, identical to that reported by some authors with one base different from that reported by Genbank. Many clones that express mCD1. 1 stably were obtained. MCD1. 1-expressing CHO cells could stimulate lymphocytes to proliferate in the presence or absence of LPS and elevate the percentage of NK1. 1 and CD3 double-positive cells. CONCLUSION： The mCD1. 1 expressed on the membrane of CHO cells could stimulate the proliferation of NKT cells.

关 键 词：mCD1.1 NKT细胞 细胞增殖 

分 类 号：R392.1[医药卫生—免疫学]