稳定高表达小鼠Tim-4巨噬细胞系RAW264.7-T4的建立

目的建立研究小鼠Tim-4功能的细胞模型。方法常规分离小鼠腹腔巨噬细胞，RT-PCR扩增Tim-4全长基因片段，构建含Tim-4全基因片段的真核表达载体pcDNA3.0-Tim-4, 通过BamHⅠ、HindⅢ双酶切及测序鉴定其正确性。利用脂质体将pcDNA3.0和pcDNA3.0-Tim-4分别转染小鼠巨噬细胞系RAW264.7，通过G418加压筛选，建立稳定高表达Tim-4的RAW264.7，并采用RT-PCR、流式细胞术和免疫细胞化学染色法检测Tim-4 mRNA和蛋白的表达水平。结果RT-PCR扩增产物经BamHⅠ、HindⅢ双酶切，与pcDNA3.0连接构建的重组体经酶切和测序，结果与GenBank报道序列一致。RT-PCR结果显示，RAW264.7 T4细胞表达Tim-4 mRNA的水平显著高于对照组，流式细胞术显示RAW264.7-T4高水平表达Tim-4蛋白，免疫细胞化学染色表明Tim-4主要表达于胞膜。结论成功构建了携载小鼠Tim-4全基因的表达载体，并用该重组体建立了稳定高表达Tim-4 mRNA和蛋白的小鼠巨噬细胞系，为进一步探讨Tim-1与Tim-4的相互作用及对免疫细胞功能的影响奠定基础。

Establishment of RAW264.7-T4 cell line with stable and high expression of murine Tim-4

To establish a new macrophage cell line of murine Tim-4. MethodsPeritoneal macrophages were regularly separated from mice. RT-PCR was used to amplify the full gene fragment of murine Tim-4,with which to construct the pcDNA3.0 Tim-4 expression vector. Restriction endonucluease digestion and sequencing were used to verify its correctness. Also, RAW264.7 cells were separately tansfected with pcDNA3.0 and pcDNA3.0-Tim-4 by liposomes. After screening with a high level of G418, a new cell line expressing stable and high Tim-4 was established. The mRNA and protein levels were further determined by RT-PCR, FCM and immunochemistry staining. ResultsProducts of RT-PCR amplification were digested with BamHⅠand HindⅢ，then Tim-4 was cloned into pcDNA3.0 and pcDNA3.0-Tim-4 recombinant was constructed. The result was consistent with the sequence of GenBank after digestion and sequencing. The mRNA level of RAW264.7-Tim-4 was significantly higher than that of the control group, as were proteins expressed in these cells, which was shown by FCM and immunochemistry staining. Also, Tim-4 expression was mainly found on the surface of the macrophage and cytoplasmic expression was also found. ConclusionAn expression vector with the full murine Tim-4 gene and a new line with stable and high expression of Tim-4 were successfully established, which will set a good basis for further study on the interaction of Tim-1 and Tim-4 and its impact on immunocytes.