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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。
OBJECTIVE: To obtain a cell line stably expressing mCD1.1 and study its stimulatory effects on liver and mesenteric lymph node lymphocytes. Methods: The intestinal epithelial cells of C57 mice were isolated and the total RNA was prepared. The mCD1.1 coding region gene was amplified by RTPCR and cloned into eukaryotic expression vector pcDNA3.1. The resulting recombinant plasmid pcDNA3.1mCD1.1 transferred to CHO cells by electroporation, selection of resistant clones in G418-containing selective medium, the use of RTPCR and flow cytometry clones picked mCD1.1 Identification of the expression. The cells expressing mCD1.1 were co-cultured with newly isolated lymphocytes from the liver and mesenteric lymph nodes (MLN), and the proliferation of CHO cells was inhibited with mitomycin C, and then the proliferation of lymphocytes was detected by MTT assay. In addition, After co-cultured with lymphocytes, CHO cells expressing mCD1.1 were isolated, and then labeled with PEantiNK1.1 and CychromeantiCD3. The proportion of NK1.1 positive cells in CD3 cells was determined by flow cytometry. Results: The coding region of mCD1.1 gene was obtained by RTPCR and the sequence was in accordance with the expectation. After several times of identification, it was proved that the cloned cells could stably express mCD1.1. CHOmCD1.1 cells were co-cultured with newly isolated lymphocytes, In the presence or absence of LPS, both stimulated lymphocyte proliferation and increased the proportion of NK1.1-positive cells in CD3 + cells. Conclusion: CHO cells expressing mCD1.1 can stimulate NKT cell proliferation.