Objective To construct a morphine tolerance model in primarily cultured striatal neurons,and screen the differentially expressed genes in this model using suppression subtractive hybridization (SSH).Methods Sbtracted cDNA libraries were constructed using SSH from normal primarily cultured striatal neurons and long-term morphine treated striatal neurons (10-5 mol/L for 72 hours).To check reliability of the cell culture model,RT-PCR was performed to detect the cAMP-responsive element-binding protein (CREB) mRNA expression.The subtracted clones were prescreened by PCR.The clones containing inserted fragments from forward libraries were sequenced and submitted to GenBank for homology analysis.And the expression levels of genes of interest were confirmed by RT-PCR.Results CREB mRNA expression showed a significant increase in morphine treated striatal neurons (62.85±1.98) compared with normal striatal neurons (28.43±1.46,P<0.01).Thirty-six clones containing inserted fragments were randomly chosen for sequence analysis.And the 36 clones showed homology with 19 known genes and 2 novel genes.The expression of 2 novel genes,mitochondrial carrier homolog 1 (Mtch1;96.81±2.04 vs.44.20±1.31,P<0.01) and thymoma viral proto-oncogene 1 (Akt1;122.10±2.17 vs.50.11±2.01,P<0.01),showed a significant increase in morphine-treated striatal neurons compared with normal striatal neurons.Conclusions A reliable differential cDNA library of striatal neurons treated with long-term morphine is constructed.Mtch1 and Akt1 might be the candidate genes for the development of morphine tolerance.
Bo BaiHai-qing LiuJing ChenYa-lin LiHui DuHai LuPeng-li Yu
Microglia are considered to be potential anti- gen-presenting cells and have the ability to present antigen under pathological conditions. Nevertheless, whether and how microglia are involved in immune regulation are lar- gely unknown. Here, we investigated the suppressive activity of microglia during experimental autoimmune encephalomyelitis （EAE） induced by myelin oligodendro- cyte glycoprotein, with the goal of understanding their role in regulating the T cell reaction. Using flow cytometric analysis, we found that microglia were characterized by increased cell number and up-regulated programmed death ligand-1 （PD-L1） at the peak phase of EAE. Meanwhile, both the CD4＋ T cells and microglia that infiltrated the central nervous system expressed higher levels of PD1, the receptor for PD-L1, accompanied by a decline of Thl cells. In an ex vivo co-culture system, microglia from EAE mice inhibited the proliferation of antigen-specific CD4＋ T cells and the differentiation of Thl cells, and this was significantly inhibited by PD-L 1 blockade. Further, microglia suppressed Thl cells via nitric oxide （NO）, the production of which was dependent on PD-L1. Thus, these data suggest a scenario in which microglia are involved in the regulation of EAE by suppressing Thl-cell differenti- ation via the PD-L1-NO pathway.
