目的克隆人CD45 c DNA并导入Hela细胞中表达,建立研究CD45功能的细胞模型。方法采用RT-PCR方法从人外周血单个核细胞中扩增CD45基因PTPRC的c DNA,将其克隆至p MD-18T载体。构建重组真核表达载体Pc DNA3.1-3xflag-CD45,经HindⅢ和XhoⅠ双酶切及测序验证。将其转染至Hela细胞,以流式细胞术(FCM)和免疫印迹(WB)分析CD45在Hela细胞中的表达情况,碱性磷酸酶试剂盒检测CD45的活性。结果分离到长约3900 bp的人PTPRC c DNA片段,将其插入p MD-18T载体获得了c DNA克隆。酶切和测序结果证实重组表达载体Pc DNA3.1-3xflag-CD45构建成功,FCM和WB分析表明CD45能在Hela细胞中有效表达,且表达的重组CD45蛋白具有生物学活性。结论成功获得人PTPRC c DNA克隆并在Hela细胞中有效表达,为进一步研究CD45功能奠定了基础。
Mannan-binding lectin(MBL)plays a key role in the lectin pathway of complement activation and can influence cytokine expression.Toll-like receptor 4(TLR4)is expressed extensively and has been demonstrated to be involved in lipopolysaccharide(LPS)-induced signaling.We first sought to determine whether MBL exposure could modulate LPS-induced inflammatory cytokine secretion and nuclear factor-kB(NF-kB)activity by using the monocytoid cell line THP-1.We then investigated the possible mechanisms underlying any observed regulatory effect.Using ELISA and reverse transcriptase polymerase chain reaction(RT-PCR)analysis,we found that at both the protein andmRNAlevels,treatment withMBLsuppresses LPS-induced tumor-necrosis factor(TNF)-a and IL-12 production in THP-1 cells.An electrophoretic mobility shift assay and western blot analysis revealed that MBL treatment can inhibit LPS-induced NF-kB DNA binding and translocation in THP-1 cells.While the binding of MBL to THP-1 cells was evident at physiological calcium concentrations,this binding occurred optimally in response to supraphysiological calcium concentrations.This binding can be partly inhibited by treatment with either a soluble form of recombinant TLR4 extracellular domain or anti-TLR4 monoclonal antibody(HTA125).Activation of THP-1 cells by LPS treatment resulted in increased MBL binding.We also observed that MBL could directly bind to the extracellular domain of TLR4 in a dose-dependent manner,and this interaction could attenuate the binding of LPS to cell surfaces.Taken together,these data suggest that MBL may affect cytokine expression through modulation of LPS-/TLR-signaling pathways.These findings suggest that MBL may play an important role in both immune regulation and the signaling pathways involved in cytokine networks.
