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构建人IL-34真核表达载体并将其转染到人骨髓间充质干细胞,观察高表达IL-34的骨髓间充质干细胞对THP-1细胞的影响。PCR扩增IL-34 DNA,并将其克隆到真核表达载体pIRES2-EGFP;将构建成功的重组体转染到骨髓间充质干细胞,Western blotting和ELISA分析IL-34在细胞中的表达;用高表达IL-34的骨髓间充质干细胞培养上清液来培养THP-1细胞,Real-time PCR分析THP-1细胞中IL-10和TNFα的表达变化。经双酶切和测序鉴定,成功构建了pIRES2-EGFP-IL-34重组体;转染至骨髓间充质干细胞的IL-34可以促进THP-1细胞表达IL-10和TNFα。结果表明,骨髓间充质干细胞表达分泌的IL-34对THP-1有调节作用。
The eukaryotic expression vector of human IL-34 was constructed and transfected into human bone marrow mesenchymal stem cells. The effect of high expression of IL-34 on bone marrow mesenchymal stem cells on THP-1 cells was observed. The IL-34 DNA was amplified by PCR and cloned into eukaryotic expression vector pIRES2-EGFP. The constructed recombinant was transfected into BMSCs, and the expression of IL-34 was analyzed by Western blotting and ELISA. THP-1 cells were cultured with supernatant of bone marrow-derived mesenchymal stem cells expressing IL-34, and the expression of IL-10 and TNFα in THP-1 cells was analyzed by Real-time PCR. The recombinant pIRES2-EGFP-IL-34 was successfully constructed by double enzyme digestion and sequencing. IL-34 transfected into bone marrow mesenchymal stem cells can promote the expression of IL-10 and TNFα in THP-1 cells. The results showed that IL-34 secreted by bone marrow mesenchymal stem cells can regulate THP-1.