摘要: Objective We aimed to investigate the roles and underlying mechanisms of YAP in the proliferation of neuroblastoma cells. Methods The expression level of YAP was evaluated by Western blotting and immunocytochemistry. Cell viability, cell proliferation and growth were detected by CCK-8, PH3 and Ki67 immunostaining, and the real-time cell analyser system. The nuclear and cytoplasmic proteins of p27(Kip1) were dissociated by the nuclear-cytosol extraction kit and were detected by Western blotting and immunocytochemistry. mRNA levels of Akt, CDK5 and CRM1 were determined by qRT-PCR. Results YAP was enriched in SH-SY5Y cells (a human neuroblastoma cell line). Knock-down of YAP in SH-SY5Y cells or SK-N-SH cell line (another human neuroblastoma cell line) significantly decreased cell viability, inhibited cell proliferation and growth. Mechanistically, knock-down of YAP increased the nuclear location of p27(Kip1), whereas serum-induced YAP activation decreased the nuclear location of p27(Kip1) and was required for cell proliferation. Meanwhile, overexpression of YAP in these serum-starved SH-SY5Y cells decreased the nuclear location of p27(Kip1), promoted cell proliferation and overexpression of p27(Kip1) in YAP-activated cells inhibited cell proliferation. Furthermore, knock-down of YAP reduced Akt mRNA and protein levels. Overexpression of Akt in YAP-downregulated cells decreased the nuclear location of p27(Kip1) and accelerated the proliferation of SH-SY5Y cells. Conclusions Our studies suggest that YAP promotes the proliferation of neuroblastoma cells through negatively controlling the nuclear location of p27(Kip1) mediated by Akt.

摘要: Objectives Secondary bacterial pneumonia is common following influenza infection. However, it remains unclear about the underlying molecular mechanisms. Materials and methods We established a mouse model of post-influenza S aureus pneumonia using conditional Shp2 knockout mice (LysM(Cre/+):Shp2(flox/flox)). The survival, bacterial clearance, pulmonary histology, phenotype of macrophages, and expression of type I interferons and chemokines were assessed between SHP2 deletion and control mice (Shp2(flox/flox)). We infused additional KC and MIP-2 to examine the reconstitution of antibacterial immune response in LysM(Cre/+):Shp2(flox/flox) mice. The effect of SHP2 on signal molecules including MAPKs (JNK, p38 and Erk1/2), NF-kappa B p65 and IRF3 was further detected. Results LysM(Cre/+):Shp2(flox/flox) mice displayed impaired antibacterial immunity and high mortality compared with control mice in post-influenza S aureus pneumonia. The attenuated antibacterial ability was associated with the induction of type I interferon and suppression of chemo-attractants KC and MIP-2, which reduced the infiltration of neutrophils into the lung upon secondary bacterial invasion. In additional, Shp2 knockout mice displayed enhanced polarization to alternatively activated macrophages (M2 phenotype). Further in vitro analyses consistently demonstrated that SHP2-deficient macrophages were skewed towards an M2 phenotype and had a decreased antibacterial capacity. Moreover, SHP2 modulated the inflammatory response to secondary bacterial infection via interfering with NF-kappa B and IRF3 signalling in macrophages. Conclusions Our findings reveal that the SHP2 expression enhances the host immune response and prompts bacterial clearance in post-influenza S aureus pneumonia.

摘要: Objectives Silicate bioactive glass (BG) has been widely demonstrated to stimulate both of the hard and soft tissue regeneration, in which ion products released from BG play important roles. However, the mechanism by which ion products act on cells on cells is unclear. Materials and methods Human umbilical vein endothelial cells and human bone marrow stromal cells were used in this study. Fluorescence recovery after photobleaching and generalized polarization was used to characterize changes in cell membrane fluidity. Migration, differentiation and apoptosis experiments were carried out. RNA and protein chip were detected. The signal cascade is simulated to evaluate the effect of increased cell membrane fluidity on signal transduction. Results We have demonstrated that ion products released from BG could effectively enhance cell membrane fluidity in a direct and physical way, and Si ions may play a major role. Bioactivities of BG ion products on cells, such as migration and differentiation, were regulated by membrane fluidity. Furthermore, we have proved that BG ion products could promote apoptosis of injured cells based on our conclusion that BG ion products increased membrane fluidity. Conclusions This study proved that BG ion products could develop its bioactivity on cells by directly enhancing cell membrane fluidity and subsequently affected cell behaviours, which may provide an explanation for the general bioactivities of silicate material.

摘要: Objectives Genetic engineering of human-induced pluripotent stem cell-derived neural stem cells (hiPSC-NSC) may increase the risk of genomic aberrations. Therefore, we asked whether genetic modification of hiPSC-NSCs exacerbates chromosomal abnormalities that may occur during passaging and whether they may cause any functional perturbations in NSCs in vitro and in vivo. Materials and Methods The transgenic cassette was inserted into the AAVS1 locus, and the genetic integrity of zinc-finger nuclease (ZFN)-modified hiPSC-NSCs was assessed by the SNP-based karyotyping. The hiPSC-NSC proliferation was assessed in vitro by the EdU incorporation assay and in vivo by staining of brain slices with Ki-67 antibody at 2 and 8 weeks after transplantation of ZFN-NSCs with and without chromosomal aberration into the striatum of immunodeficient rats. Results During early passages, no chromosomal abnormalities were detected in unmodified or ZFN-modified hiPSC-NSCs. However, at higher passages both cell populations acquired duplication of the entire long arm of chromosome 1, dup(1)q. ZNF-NSCs carrying dup(1)q exhibited higher proliferation rate than karyotypically intact cells, which was partly mediated by increased expression ofAKT3located on Chr1q. Compared to karyotypically normal ZNF-NSCs, cells with dup(1)q also exhibited increased proliferation in vivo 2 weeks, but not 2 months, after transplantation. Conclusions These results demonstrate that, independently of ZFN-editing, hiPSC-NSCs have a propensity for acquiring dup(1)q and this aberration results in increased proliferation which might compromise downstream hiPSC-NSC applications.

摘要: Objective Adipose-derived mesenchymal stem cells (ADSCs) offer great promise as cell therapy for ischaemic diseases. Due to their poor survival in the ischaemic environment, the therapeutic efficacy of ADSCs is still relatively low. Interleukin-11 (IL-11) has been shown to play a key role in promoting cell proliferation and protecting cells from oxidative stress injury. The aim of this study was to determine whether IL-11 could improve therapeutic efficacy of ADSCs in ischaemic diseases. Methods and Results ADSCs were prepared from inguinal subcutaneous adipose tissue and exposed to hypoxic environment. The protein expression of IL-11 was decreased after hypoxic treatment. In addition, ADSCs viability was increased after IL-11 treatment under hypoxia. Moreover, IL-11 enhanced ADSCs viability in a dose-dependent manner under normoxia. Importantly, IL-11 promoted ADSCs proliferation and migration and protected ADSCs against hydrogen peroxide-induced cellular death. Notably, IL-11 enhanced ADSCs proliferation and migration, also promoted cell survival and apoptosis resistance by STAT3 signalling. In vivo, mice were subjected to limb ischaemia and treated with IL-11 overexpression ADSCs and control ADSCs. IL-11 overexpression ADSCs improved perfusion recovery in the ischaemic muscles. Conclusions We provide the evidence that IL-11 promoted ADSCs proliferation, stimulated ADSCs migration and attenuated ADSCs apoptosis by activation of STAT3 signalling. These results suggest that IL-11 facilitated ADSCs engraftment in ischaemic tissue, thereby enhanced ADSCs therapeutic efficacy.