针灸实验中非麻醉状态下鼠科动物固定方法应用国内外进展

针灸实验研究多以鼠科动物为实验对象。为便于针灸操作,多采用在非麻醉状态下固定实验动物。目前针灸研究中采用的动物固定方法包括束缚固定、置高台固定和悬吊固定。因不同固定方法均会产生不同程度的应激反应,影响实验动物生理、病理指标,从而影响针灸疗效正确评估。该文通过梳理国内外针灸实验中不同固定方法对针灸效应干预的比较,为选择恰当的动物固定方法提供建议,以达到对针灸效应最小干预的目的。

PLP2, a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type I interferon production

Infections by coronaviruses such as severe acute respiratory syndrome （SARS） coronavirus （SCoV） and mouse hepatitis virus A59 （MHV-A59） result in very little type I interferon （IFN） production by host cells, which is potentially responsible for the rapid viral growth and severe immunopathology associated with SARS. However, the molecular mechanisms for the low IFN production in cells infected with coronaviruses remain unclear. Here, we provide evidence that Papain-like protease domain 2 （PLP2）, a catalytic domain of the nonstructural protein 3 （nsp3） of MHV-A59, can bind to IRF3, cause its deubiquitination and prevent its nuclear translocation. As a consequence, co-expression of PLP2 strongly inhibits CARDIF-, TBK1- and IRF3-mediated IFNp reporter activities. In addition, we show that wild-type PLP2 but not the mutant PLP2 lacking the deubiquitinase （DUB） activity can reduce IFN induction and promote viral growth in cells infected with VSV. Thus, our study uncovered a viral DUB which coronaviruses may use to escape from the host innate antiviral responses.

cAMP elevators inhibit LPS-induced IL-12 p40 expression by interfering with phosphorylation of p38 MAPK in Murine Peritoneal Macrophages

cAMP mediated signaling may play a suppressive role in immune response. We previously found that the cAMP-elevators (CTx and 8-Br-cAMP) inhibited IL-12, IL-la, IL-6 gene expression, but increased the transcriptional levels of IL-10 and IL-IRa in LPS-treated murine peritoneal macrophages. The present study examined a possible molecular mechanism involved in cAMP elevators-induced inhibition of IL-12 p40 expression in response to LPS. Our data demonstrated that cAMP elevators downregulated IL-12 p40 mRNA expression and IL-12 p70 production in murine peritoneal macrophages. Subsequent studies revealed that cAMP-elevators blocked phosphorylation of p38 MAPK, but did not affect the activity of NF-kB binding to IL-12 promoter (-136/-112). This is the first report that cAMP elevators inhibit LPS-induced IL-12 production by a mechanism that is associated, at least in part, with p38-dependent inhibition by cAMP signaling pathways.

Isolation and characterization of the murine Nanog gene promoter

Nanog protein is expressed in the interior cells of compacted morulae and maintained till epiblasts but downregulated by implantation stage. It is also expressed in embryonic stem cells, embryonic carcinoma cells and embryonic germ cells but disappeared in differentiated ES cells. In this study, we have isolated, sequenced, and performed the first characterization of the Nanog promoter. The transcription start sites were mapped by primer extension analysis. Two promoter regions were found upstream the transcription start sites and the expression of major Nanog promoter/ reporter gene construct is abolished in differentiated F9 EC cells as compared to the undifferentiated counterpart. We also showed that a putative octamer motif (ATGCAAAA) is necessary for the major promoter activity. Gel shift and supershift assays showed that Oct-1, Oct-4 and Oct-6 protein selectively bind to the octamer motif.

Induction of graft-versus-leukemia effect using a mixture of syngeneic plus G-CSF primed haploidentical bone marrow grafts in mice

Objective： To explore whether the graft-versus-leukemia （GVL） effects could be enhanced and acute graft-versus-host disease （aGVHD） could be relieved by syngeneic bone marrow mixed with G-CSF-primed H-2 haploidentical marrow grafting. Methods： Female L615 （H-2^k） mice were recipient mice and male （615×BALB/c） F1 （6BF1） （H-2^k×H-2^d） mice were donors respectively. Donor mice were injected subcutaneously with G-CSF daily at 0.01 μg/g for 6 days, and splenocytes were harvested on day 7. A total of 615 mice were loaded with L615 tumor cells and received 8.5 Gy （^60Co γ-ray） irradiation three days later, followed by a mixed bone marrow transplantation （MBMT）. The allo-grafts consisted of a mixture of syngenetic plus G-CSF-mobilized （control diluents） H-2 haploidetical marrow cells. GVL effects were monitored by survival time and survival rate of recipient mice. GVHD was assessed by clinical signs of weight loss, ruffled fur, diarrhea and histological changes of skin, liver and small intestines. AIIogeneic chimerism was detected using cytogenetic analysis. Enzyme-linked immunosorbent assay （ELISA） method was used to detect cytokines （IL-2, IL-4 and IFN-γ）. Fluorescence-activated cell sorting （FACS） analysis was used to detect T-cell phenotype. Results： （1） The mice merely received L615 leukemia cells were all died of leukemia. The L6t5 mice of 3：1 and 4：1 MBMT groups survived longer than those post syngeneic BMT （P 〈 0.01）. （2） The survival time of mice in the G-CSF-treated MBMT groups was longer than that of non-primed MBMT groups （P 〈 0.05）. Administration of G-CSF-treated 6BF1 mice could markedly increase the survival rate of 3：1 and 4：1 MBMT mice （P 〈 0.01） with little or a little GVHD. （3） As the post-transplanted time prolonged, the rates of allogeneic chimerism were decreased. The chimerism rates decreased to zero when the mice died of leukemia relapse. （4） L3T4^＋ cells and relative ratio in both subsets were significantly reduced in G-CSF-treated donor mice. After G-CSF-treated donors, splenocytes from recipients at day 14 post-MBMT showed an increased production of IL-4 and a decreased production of IL-2 and IFN-γ. Conclusion： Syngeneic bone marrow mixed with H-2 haploidentical marrow grafts had a potential way to increase GVL effects, and this GVL effects could be enhanced with little or a little GVHD by G-CSF pretreatment of donors. Improved survival in recipients of G-CSF-mobilized donors is associated with increased IL-4 production and decreased IL-2 and IFN-γ production.