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[Expression of mouse CD1.1 in CHO cells and its stimulatory function on NKT cells]
Shao-qiong Yi1, Hong Peng, Hua Wang
1Institute of Microbiology and Epidemiology, Academy of Military Medical Science, Beijing 100071, China.
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 plasmid, 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 mCD1.1 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.
