Yi Shao-qiong, Peng Hong, Wang Hua, Wang Lan, Chen Jie, Zhao Guang-yu, Gao Jie-ying
Institute of Microbiology and Epidemiology, Academy of Military Medical Science, Beijing 100071, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2006 Sep;22(5):579-81.
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.
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.
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.
The mCD1.1 expressed on the membrane of CHO cells could stimulate the proliferation of NKT cells.
获得稳定表达小鼠CD1.1（mCD1.1）的细胞系，并研究mCD1.1对多种组织来源淋巴细胞的刺激作用。
分离肠上皮细胞并提取其总RNA。通过逆转录聚合酶链反应（RT-PCR）获得编码mCD1.1的基因，然后经BamH I和EcoR I酶切克隆至pcDNA3.1载体。将构建好的质粒pcDNA3.1-mCD1.1通过电穿孔法转染至中国仓鼠卵巢（CHO）细胞。挑选出在含G418培养基中生长良好的克隆，采用RT-PCR和流式细胞术（FCM）分析其mCD1.1表达情况。将表达mCD1.1的CHO细胞与从肝脏或肠系膜淋巴结新鲜分离的淋巴细胞共培养，同时设置有无脂多糖（LPS）刺激组。用丝裂霉素C处理CHO细胞以抑制其增殖。采用噻唑蓝（MTT）法检测淋巴细胞增殖情况。此外，对共培养体系中收集的淋巴细胞用荧光素标记的抗小鼠NK1.1和CD3单克隆抗体进行染色，通过FCM检测双阳性细胞。
通过RT-PCR获得了编码mCD1.1的基因，与部分作者报道的基因一致，但与Genbank报道的基因有一个碱基差异。获得了许多稳定表达mCD1.1的克隆。表达mCD1.1的CHO细胞在有或无LPS刺激时均能刺激淋巴细胞增殖，并提高NK1.1和CD3双阳性细胞的比例。
CHO细胞膜上表达的mCD1.1能刺激自然杀伤T（NKT）细胞增殖。
