技术资料

BACKGROUND Recent studies have suggested that both poly(l-lactide-co-1,5-dioxepan-2-one) (or poly(LLA-co-DXO)) and poly(l-lactide-co-$\epsilon$-caprolactone) (or poly(LLA-co-CL)) porous scaffolds are good candidates for use as biodegradable scaffold materials in the field of tissue engineering; meanwhile,their surface properties,such as hydrophilicity,need to be further improved. METHODS We applied several different concentrations of the surfactant Tween 80 to tune the hydrophilicity of both materials. Moreover,the modification was applied not only in the form of solid scaffold as a film but also a porous scaffold. To investigate the potential application for tissue engineering,human bone marrow mesenchymal stem cells (hMSCs) were chosen to test the effect of hydrophilicity on cell attachment,proliferation,and differentiation. First,the cellular cytotoxicity of the extracted medium from modified scaffolds was investigated on HaCaT cells. Then,hMSCs were seeded on the scaffolds or films to evaluate cell attachment,proliferation,and osteogenic differentiation. The results indicated a significant increasing of wettability with the addition of Tween 80,and the hMSCs showed delayed attachment and spreading. PCR results indicated that the differentiation of hMSCs was stimulated,and several osteogenesis related genes were up-regulated in the 3{\%} Tween 80 group. Poly(LLA-co-CL) with 3{\%} Tween 80 showed an increased messenger Ribonucleic acid (mRNA) level of late-stage markers such as osteocalcin (OC) and key transcription factor as runt related gene 2 (Runx2). CONCLUSION A high hydrophilic scaffold may speed up the osteogenic differentiation for bone tissue engineering. View Publication

The proteasome inhibitor bortezomib is the most successfully applied chemotherapeutic drug for treating multiple myeloma. However,its clinical efficacy reduced due to resistance development. The underlying molecular mechanisms of bortezomib resistance are poorly understood. In this study,by combining in silico analysis and sgRNA library based drug resistance screening assay,we identified SENP2 (Sentrin/SUMO-specific proteases-2) as a bortezomib sensitive gene and found its expression highly downregulated in bortezomib resistant multiple myeloma patient's samples. Furthermore,down regulation of SENP2 in multiple myeloma cell line RPMI8226 alleviated bortezomib induced cell proliferation inhibition and apoptosis,whereas,overexpression of SENP2 sensitized these cells to bortezomib treatment. We further demonstrate that knockdown of SENP2 in RPMI8226 cells increased SUMO2 conjugated I$\kappa$B$\alpha$ that resulted in the activation of NF-$\kappa$B. Taken together,we report that silencing of SENP2 and consequent activation of NF-$\kappa$B through the modulation of I$\kappa$B$\alpha$ sumoylation as a novel mechanism inducing bortezomib resistance in multiple myeloma. View Publication

Background Nab-paclitaxel has been widely used in treating breast cancer and pancreatic patients for its low toxicity and high efficiency. However,its role in gastric cancer (GC) remains ambiguous. The aim of our study was to test the anti-tumor activity of nab-paclitaxel using GC patient-derived organoids. Methods By using the organoid culture system,we describe the establishment of human gastric cancer organoid lines from surgical samples of three patients with gastric cancer. The consistency of these organoids with original cancer tissues was evaluated by histopathological examination. The characteristics of the cancer organoids were tested using immunofluorescence (IF) staining. Using organoids,the anti-tumor efficiencies of nab-paclitaxel,5-Fu and epirubicin were compared by CCK8 assay and Annexin V-FITC/PI staining. Results Three organoids were successfully established and passaged. The morphology of the established GC organoids was consistent with original cancer tissues. The IC50 of nab-paclitaxel was 3.68 $\mu$mol/L in hGCO1,2.41 $\mu$mol/L in hGCO2 and 2.91 $\mu$mol/L in hGCO3,which was significantly lower than those of 5-FU (72.99 $\mu$mol/L in hGCO1,28.32 $\mu$mol/L in hGCO2 and 2.91 $\mu$mol/L in hGCO3) and epirubicin (25.85$\mu$mol/L in hGCO1,15.15 $\mu$mol/L in hGCO2 and 7.60 $\mu$mol/L in hGCO3). When each organoid lines were treated with nab-paclitaxel for increasing period of time,the percentage of the apoptotic cells in each organoid increased accordingly. Conclusion Nab-paclitaxel showed strong anti-tumor activity and had the potential to become front-line drug for treating GC patients. Gastric cancer organoid may be a good tool to predict in vivo response to drugs. View Publication

Emerging evidence indicates that in myelodysplastic syndromes (MDS),the bone marrow (BM) microenvironment may also contribute to the ineffective,malignant haematopoiesis in addition to the intrinsic abnormalities of haematopoietic stem precursor cells (HSPCs). The BM microenvironment influences malignant haematopoiesis through indirect mechanisms,but the processes by which the BM microenvironment directly contributes to MDS initiation and progression have not yet been elucidated. Our previous data showed that BM-derived stromal cells (BMSCs) from MDS patients have an abnormal expression of focal adhesion kinase (FAK). In this study,we characterise the morpho-phenotypic features and the functional alterations of BMSCs from MDS patients and in FAK knock-downed HS-5 cells. The decreased expression of FAK or its phosphorylated form in BMSCs from low-risk (LR) MDS directly correlates with BMSCs' functional deficiency and is associated with a reduced level of haemoglobin. The downregulation of FAK in HS-5 cells alters their morphology,proliferation,and differentiation capabilities and impairs the expression of several adhesion molecules. In addition,we examine the CD34+ healthy donor (HD)-derived HSPCs' properties when co-cultured with FAK-deficient BMSCs. Both abnormal proliferation and the impaired erythroid differentiation capacity of HD-HSPCs were observed. Together,these results demonstrate that stromal adhesion mechanisms mediated by FAK are crucial for regulating HSPCs' homeostasis. View Publication

Freshly isolated,uncultured,autologous adipose derived regenerative cells (ADRCs) have emerged as a promising tool for regenerative cell therapy. The Transpose RT system (InGeneron,Inc.,Houston,TX,USA) is a system for isolating ADRCs from adipose tissue,commercially available in Europe as a CE-marked medical device and under clinical evaluation in the United States. This system makes use of the proprietary,enzymatic Matrase Reagent for isolating cells. The present study addressed the question whether the use of Matrase Reagent influences cell yield,cell viability,live cell yield,biological characteristics,physiological functions or structural properties of the ADRCs in final cell suspension. Identical samples of subcutaneous adipose tissue from 12 subjects undergoing elective lipoplasty were processed either with or without the use of Matrase Reagent. Then,characteristics of the ADRCs in the respective final cell suspensions were evaluated. Compared to non-enzymatic isolation,enzymatic isolation resulted in approximately twelve times higher mean cell yield (i.e.,numbers of viable cells/ml lipoaspirate) and approximately 16 times more colony forming units. Despite these differences,cells isolated from lipoaspirate both with and without the use of Matrase Reagent were independently able to differentiate into cells of all three germ layers. This indicates that biological characteristics,physiological functions or structural properties relevant for the intended use were not altered or induced using Matrase Reagent. A comprehensive literature review demonstrated that isolation of ADRCs from lipoaspirate using the Transpose RT system and the Matrase Reagent results in the highest viable cell yield among published data regarding isolation of ADRCs from lipoaspirate. View Publication

H7 avian influenza viruses represent a major public health concern,and worldwide outbreaks raise the risk of a potential pandemic. Understanding the memory B cell response to avian (H7) influenza virus infection in humans could provide insights in the potential key to human infection risks. We investigated an epizootic of the highly pathogenic A(H7N7) in the Netherlands,which in 2003 led to infection of 89 persons and one fatal case. Subtype-specificity of antibodies were determined for confirmed H7N7 infected individuals (cases) (n = 19),contacts of these cases (n = 21) and a comparison group controls (n = 16),by microarray,using recombinant hemagglutinin (HA)1 proteins. The frequency and specificity of memory B cells was determined by detecting subtype-specific antibodies in the culture supernatants from in vitro stimulated oligoclonal B cell cultures,from peripheral blood of cases and controls. All cases (100{\%}) had high antibody titers specific for A(H7N7)2003 (GMT {\textgreater} 100),whereas H7-HA1 antigen binding was detected in 29{\%} of contacts and 31{\%} of controls,suggesting that some of the H7 reactivity stems from cross reactive antibodies. To unravel homotypic and heterotypic responses,the frequency and specificity of memory B cells were determined in 2 cases. Ten of 123 HA1 reactive clones isolated from the cases bound to only H7- HA1,whereas 5 bound both H7 and other HA1 antigens. We recovered at least four different epitopal reactivities,though none of the H7 reactive antibodies were able to neutralize H7 infections in vitro. Our study serologically confirms the infection with H7 avian influenza viruses,and shows that H7 infection triggers a mixture of strain -specific and cross-reactive antibodies. View Publication

Abdominal aortic aneurysm (AAA) is life-threatening,for which efficient nonsurgical treatment strategy has not been available so far. Several previous studies investigating the therapeutic effect of mesenchymal stem cells (MSCs) in AAA indicated that MSCs could inhibit aneurysmal inflammatory responses and extracellular matrix destruction,and suppress aneurysm occurrence and expansion. Vascular smooth muscle cell (VSMC) phenotypic plasticity is reported to be predisposed in AAA initiation and progression. However,little is known about the effect of MSCs on VSMC phenotypic modulation in AAA. In this study,we investigate the therapeutic efficacy of umbilical cord mesenchymal stem cells (UC-MSCs) in elastase-induced AAA model and evaluate the effect of UC-MSC on VSMC phenotypic regulation. We demonstrate that the intravenous injection of UC-MSC attenuates elastase-induced aneurysmal expansion,reduces elastin degradation and fragmentation,inhibits MMPs and TNF-$\alpha$ expression,and preserves and/or restores VSMC contractile phenotype in AAA. Taken together,these results highlight the therapeutic and VSMC phenotypic modulation effects of UC-MSC in AAA progression,which further indicates the potential of applying UC-MSC as an alternative treatment candidate for AAA. View Publication

Mutational inactivation of ATRX ($\alpha$-thalassemia mental retardation X-linked) represents a defining molecular alteration in large subsets of malignant glioma. Yet the pathogenic consequences of ATRX deficiency remain unclear,as do tractable mechanisms for its therapeutic targeting. Here we report that ATRX loss in isogenic glioma model systems induces replication stress and DNA damage by way of G-quadruplex (G4) DNA secondary structure. Moreover,these effects are associated with the acquisition of disease-relevant copy number alterations over time. We then demonstrate,both in vitro and in vivo,that ATRX deficiency selectively enhances DNA damage and cell death following chemical G4 stabilization. Finally,we show that G4 stabilization synergizes with other DNA-damaging therapies,including ionizing radiation,in the ATRX-deficient context. Our findings reveal novel pathogenic mechanisms driven by ATRX deficiency in glioma,while also pointing to tangible strategies for drug development. View Publication

Our understanding of the signalling pathways regulating early human development is limited,despite their fundamental biological importance. Here,we mine transcriptomics datasets to investigate signalling in the human embryo and identify expression for the insulin and insulin growth factor 1 (IGF1) receptors,along with IGF1 ligand. Consequently,we generate a minimal chemically-defined culture medium in which IGF1 together with Activin maintain self-renewal in the absence of fibroblast growth factor (FGF) signalling. Under these conditions,we derive several pluripotent stem cell lines that express pluripotency-associated genes,retain high viability and a normal karyotype,and can be genetically modified or differentiated into multiple cell lineages. We also identify active phosphoinositide 3-kinase (PI3K)/AKT/mTOR signalling in early human embryos,and in both primed and na{\{i}}ve pluripotent culture conditions. This demonstrates that signalling insights from human blastocysts can be used to define culture conditions that more closely recapitulate the embryonic niche." View Publication

Self-assembling virus-like particles represent highly attractive tools for developing next-generation vaccines and protein therapeutics. We created ADDomer,an adenovirus-derived multimeric protein-based self-assembling nanoparticle scaffold engineered to facilitate plug-and-play display of multiple immunogenic epitopes from pathogens. We used cryo–electron microscopy at near-atomic resolution and implemented novel,cost-effective,high-performance cloud computing to reveal architectural features in unprecedented detail. We analyzed ADDomer interaction with components of the immune system and developed a promising first-in-kind ADDomer-based vaccine candidate to combat emerging Chikungunya infectious disease,exemplifying the potential of our approach. View Publication

High-grade gliomas are lethal brain cancers whose progression is robustly regulated by neuronal activity. Activity-regulated release of growth factors promotes glioma growth,but this alone is insufficient to explain the effect that neuronal activity exerts on glioma progression. Here we show that neuron and glioma interactions include electrochemical communication through bona fide AMPA receptor-dependent neuron-glioma synapses. Neuronal activity also evokes non-synaptic activity-dependent potassium currents that are amplified by gap junction-mediated tumour interconnections,forming an electrically coupled network. Depolarization of glioma membranes assessed by in vivo optogenetics promotes proliferation,whereas pharmacologically or genetically blocking electrochemical signalling inhibits the growth of glioma xenografts and extends mouse survival. Emphasizing the positive feedback mechanisms by which gliomas increase neuronal excitability and thus activity-regulated glioma growth,human intraoperative electrocorticography demonstrates increased cortical excitability in the glioma-infiltrated brain. Together,these findings indicate that synaptic and electrical integration into neural circuits promotes glioma progression. View Publication

Vaccine efficacy can be increased by arraying immunogens in multivalent form on virus-like nanoparticles to enhance B-cell activation. However,the effects of antigen copy number,spacing and affinity,as well as the dimensionality and rigidity of scaffold presentation on B-cell activation remain poorly understood. Here,we display the clinical vaccine immunogen eOD-GT8,an engineered outer domain of the HIV-1 glycoprotein-120,on DNA origami nanoparticles to systematically interrogate the impact of these nanoscale parameters on B-cell activation in vitro. We find that B-cell signalling is maximized by as few as five antigens maximally spaced on the surface of a 40-nm viral-like nanoparticle. Increasing antigen spacing up to {\~{}}25-30 nm monotonically increases B-cell receptor activation. Moreover,scaffold rigidity is essential for robust B-cell triggering. These results reveal molecular vaccine design principles that may be used to drive functional B-cell responses. View Publication