产品说明

The intestinal epithelium plays an essential role in nutrient absorption,hormone release,and barrier function. Maintenance of the epithelium is driven by continuous cell renewal by stem cells located in the intestinal crypts. The amount and type of diet influence this process and result in changes in the size and cellular make-up of the tissue. The mechanism underlying the nutrient-driven changes in proliferation is not known,but may involve a shift in intracellular metabolism that allows for more nutrients to be used to manufacture new cells. We hypothesized that nutrient availability drives changes in cellular energy metabolism of small intestinal epithelial crypts that could contribute to increases in crypt proliferation. We utilized primary small intestinal epithelial crypts from C57BL/6J mice to study (1) the effect of glucose on crypt proliferation and (2) the effect of glucose on crypt metabolism using an extracellular flux analyzer for real-time metabolic measurements. We found that glucose increased both crypt proliferation and glycolysis,and the glycolytic pathway inhibitor 2-deoxy-d-glucose (2-DG) attenuated glucose-induced crypt proliferation. Glucose did not enhance glucose oxidation,but did increase the maximum mitochondrial respiratory capacity,which may contribute to glucose-induced increases in proliferation. Glucose activated Akt/HIF-1alpha signaling pathway,which might be at least in part responsible for glucose-induced glycolysis and cell proliferation. These results suggest that high glucose availability induces an increase in crypt proliferation by inducing an increase in glycolysis with no change in glucose oxidation. View Publication

Human pluripotent stem cells (hPSCs) possess great value in the aspect of cellular therapies due to its self-renewal and potential to differentiate into all somatic cell types. A few defined synthetic surfaces such as polymers and adhesive biological materials conjugated substrata were established for the self-renewal of hPSCs. However,none of them was effective in the generation of human induced pluripotent stem cells (hiPSCs) and long-term maintenance of multiple hPSCs,and most of them required complicated manufacturing processes. Polydopamine has good biocompatibility,is able to form a stable film on nearly all solid substrates surface,and can immobilize adhesive biomolecules. In this manuscript,a polydopamine-mediated surface was developed,which not only supported the reprogramming of human somatic cells into hiPSCs under defined conditions,but also sustained the growth of hiPSCs on diverse substrates. Moreover,the proliferation and pluripotency of hPSCs cultured on the surface were comparable to Matrigel for more than 20 passages. Besides,hPSCs were able to differentiate to cardiomyocytes and neural cells on the surface. This polydopamine-based synthetic surface represents a chemically-defined surface extensively applicable both for fundamental research and cell therapies of hPSCs. View Publication

This study investigates the electrophysiological properties and functional integration of different phenotypes of transplanted human neural precursor cells (hNPCs) in immunodeficient NSG mice. Postnatal day 2 mice received unilateral injections of 100,000 GFP+ hNPCs into the right parietal cortex. Eight weeks after transplantation,1.21% of transplanted hNPCs survived. In these hNPCs,parvalbumin (PV)-,calretinin (CR)-,somatostatin (SS)-positive inhibitory interneurons and excitatory pyramidal neurons were confirmed electrophysiologically and histologically. All GFP+ hNPCs were immunoreactive with anti-human specific nuclear protein. The proportions of PV-,CR-,and SS-positive cells among GFP+ cells were 35.5%,15.7%,and 17.1%,respectively; around 15% of GFP+ cells were identified as pyramidal neurons. Those electrophysiologically and histological identified GFP+ hNPCs were shown to fire action potentials with the appropriate firing patterns for different classes of neurons and to display spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs). The amplitude,frequency and kinetic properties of sEPSCs and sIPSCs in different types of hNPCs were comparable to host cells of the same type. In conclusion,GFP+ hNPCs produce neurons that are competent to integrate functionally into host neocortical neuronal networks. This provides promising data on the potential for hNPCs to serve as therapeutic agents in neurological diseases with abnormal neuronal circuitry such as epilepsy. View Publication

Pituitary somatotroph adenomas result in dysregulated growth hormone (GH) hypersecretion and acromegaly; however,regulatory mechanisms that promote GH hypersecretion remain elusive. Here,we provide evidence that STAT3 directly induces somatotroph tumor cell GH. Evaluation of pituitary tumors revealed that STAT3 expression was enhanced in human GH-secreting adenomas compared with that in nonsecreting pituitary tumors. Moreover,STAT3 and GH expression were concordant in a somatotroph adenoma tissue array. Promoter and expression analysis in a GH-secreting rat cell line (GH3) revealed that STAT3 specifically binds the Gh promoter and induces transcription. Stable expression of STAT3 in GH3 cells induced expression of endogenous GH,and expression of a constitutively active STAT3 further enhanced GH production. Conversely,expression of dominant-negative STAT3 abrogated GH expression. In primary human somatotroph adenoma-derived cell cultures,STAT3 suppression with the specific inhibitor S3I-201 attenuated GH transcription and reduced GH secretion in the majority of derivative cultures. In addition,S3I-201 attenuated somatotroph tumor growth and GH secretion in a rat xenograft model. GH induced STAT3 phosphorylation and nuclear translocation,indicating a positive feedback loop between STAT3 and GH in somatotroph tumor cells. Together,these results indicate that adenoma GH hypersecretion is the result of STAT3-dependent GH induction,which in turn promotes STAT3 expression,and suggest STAT3 as a potential therapeutic target for pituitary somatotroph adenomas. View Publication

Pancreatic ductal adenocarcinoma (PDA) is characterized by an extremely poor prognosis. An inflammatory microenvironment triggers the pronounced desmoplasia,the selection of cancer stem-like cells (CSCs) and therapy resistance. The anti-inflammatory drug aspirin is suggested to lower the risk for PDA and to improve the treatment,although available results are conflicting and the effect of aspirin to CSC characteristics and desmoplasia in PDA has not yet been investigated. We characterized the influence of aspirin on CSC features,stromal reactions and gemcitabine resistance. Four established and 3 primary PDA cell lines,non-malignant cells,3 patient tumor-derived CSC-enriched spheroidal cultures and tissues from patients who did or did not receive aspirin before surgery were analyzed using MTT assays,flow cytometry,colony and spheroid formation assays,Western blot analysis,antibody protein arrays,electrophoretic mobility shift assays (EMSAs),immunohistochemistry and in vivo xenotransplantation. Aspirin significantly induced apoptosis and reduced the viability,self-renewal potential,and expression of proteins involved in inflammation and stem cell signaling. Aspirin also reduced the growth and invasion of tumors in vivo,and it significantly prolonged the survival of mice with orthotopic pancreatic xenografts in combination with gemcitabine. This was associated with a decreased expression of markers for progression,inflammation and desmoplasia. These findings were confirmed in tissue samples obtained from patients who had or had not taken aspirin before surgery. Importantly,aspirin sensitized cells that were resistant to gemcitabine and thereby enhanced the therapeutic efficacy. Aspirin showed no obvious toxic effects on normal cells,chick embryos or mice. These results highlight aspirin as an effective,inexpensive and well-tolerated co-treatment to target inflammation,desmoplasia and CSC features PDA. View Publication

The polarization and migration of neural progenitor cells (NPCs) are critical for embryonic brain development and neurogenesis after brain injury. Although stromal-derived factor-1α (SDF-1α,CXCL12) and its receptor CXCR4 are well-known to mediate the migration of NPCs in the developing brain,the dynamic cellular processes and structure-related molecular events remain elusive. Transwell and microfluidic-based assays are classical assays to effectively study cellular migration. However,both of them have limitations in the analysis of a single cell. In this study,we modified the stripe assay and extended its applications in the study of NPC polarization and intracellular molecular events associated with CXCL12-mediated migration. In response to localized CXCL12,NPCs formed lamellipodia in the stripe assay. Furthermore,CXCR4 and Rac1 quickly re-distributed to the area of lamellipodia,indicating their roles in NPC polarization upon CXCL12 stimulation. Although the chemokine stripes in the assay provided concentration gradients that can be best used to study cellular polarization and migration through immunocytochemistry,they can also generate live imaging data with comparable quality. In conclusion,stripe assay is a visual,dynamic and economical tool to study cellular mobility and its related molecule mechanisms. View Publication

H89 inhibits the dissociation-induced phosphorylation of PKA and two substrates of Rho-associated coiled-coil containing protein kinase (ROCK),myosin light chain (MLC2) and myosin phosphatase target subunit 1 (MYPT1),significantly increases cell survival and colony formation,and strongly depresses dissociation-induced cell death and cell blebbing without affecting the pluripotency of hESCs and their differentiation in vitro. View Publication

To better characterize biological responses to atmospheric organic aerosols,the efficient delivery of aerosol to in vitro lung cells is necessary. In this study,chamber generated secondary organic aerosol (SOA) entered the commercialized exposure chamber (CULTEX® Radial Flow System Compact) where it interfaced with an electrostatic precipitator (ESP) (CULTEX® Electrical Deposition Device) and then deposited on a particle collection plate. This plate contained human lung cells (BEAS-2B) that were cultured on a membrane insert to produce an air-liquid interface (ALI). To augment in vitro assessment using the ESP exposure device,the particle dose was predicted for various sampling parameters such as particle size,ESP deposition voltage,and sampling flowrate. The dose model was evaluated against the experimental measured mass of collected airborne particles. The high flowrate used in this study increased aerosol dose but failed to achieve cell stability. For example,RNA in the ALI BEAS-2B cells in vitro was stable at 0.15 L/minute but decayed at high flowrates. The ESP device and the resulting model were applied to in vitro studies (i.e.,viability and IL-8 expression) of toluene SOA using ALI BEAS-2B cells with a flowrate of 0.15 L/minute,and no cellular RNA decay occurred. View Publication

Eicosanoids are a group of bioactive lipids that are shown to be important mediators of neutrophilic inflammation; selective targeting of their function confers therapeutic benefit in a number of diseases. Neutrophilic airway diseases,including cystic fibrosis,are characterized by excessive neutrophil infiltration into the airspace. Understanding the role of eicosanoids in this process may reveal novel therapeutic targets. The eicosanoid hepoxilin A3 is a pathogen-elicited epithelial-produced neutrophil chemoattractant that directs transepithelial migration in response to infection. Following hepoxilin A3-driven transepithelial migration,neutrophil chemotaxis is amplified through neutrophil production of a second eicosanoid,leukotriene B4 (LTB4). The rate-limiting step of eicosanoid generation is the liberation of arachidonic acid by phospholipase A2,and the cytosolic phospholipase A2 (cPLA2)α isoform has been specifically shown to direct LTB4 synthesis in certain contexts. Whether cPLA2α is directly responsible for neutrophil synthesis of LTB4 in the context of Pseudomonas aeruginosa-induced neutrophil transepithelial migration has not been explored. Human and mouse neutrophil-epithelial cocultures were used to evaluate the role of neutrophil-derived cPLA2α in infection-induced transepithelial signaling by pharmacological and genetic approaches. Primary human airway basal stem cell-derived epithelial cultures and micro-optical coherence tomography,a new imaging modality that captures two- and three-dimensional real-time dynamics of neutrophil transepithelial migration,were applied. Evidence from these studies suggests that cPLA2α expressed by neutrophils,but not epithelial cells,plays a significant role in infection-induced neutrophil transepithelial migration by mediating LTB4 synthesis during migration,which serves to amplify the magnitude of neutrophil recruitment in response to epithelial infection. View Publication

Neutrophil breach of the mucosal surface is a common pathological consequence of infection. We present an advanced co-culture model to explore neutrophil transepithelial migration utilizing airway mucosal barriers differentiated from primary human airway basal cells and examined by advanced imaging. Human airway basal cells were differentiated and cultured at air-liquid interface (ALI) on the underside of 3 μm pore-sized transwells,compatible with the study of transmigrating neutrophils. Inverted ALIs exhibit beating cilia and mucus production,consistent with conventional ALIs,as visualized by micro-optical coherence tomography (μOCT). μOCT is a recently developed imaging modality with the capacity for real time two- A nd three-dimensional analysis of cellular events in marked detail,including neutrophil transmigratory dynamics. Further,the newly devised and imaged primary co-culture model recapitulates key molecular mechanisms that underlie bacteria-induced neutrophil transepithelial migration previously characterized using cell line-based models. Neutrophils respond to imposed chemotactic gradients,and migrate in response to Pseudomonas aeruginosa infection of primary ALI barriers through a hepoxilin A3-directed mechanism. This primary cell-based co-culture system combined with μOCT imaging offers significant opportunity to probe,in great detail,micro-anatomical and mechanistic features of bacteria-induced neutrophil transepithelial migration and other important immunological and physiological processes at the mucosal surface. View Publication

Adult neural stem/precursor cells (NPCs) play a pivotal role in neuronal plasticity throughout life. Among ion channels identified in adult NPCs,voltage-gated delayed rectifier K(+) (KDR) channels are dominantly expressed. However,the KDR channel subtype and its physiological role are still undefined. We used real-time quantitative RT-PCR and gene knockdown techniques to identify a major functional KDR channel subtype in adult NPCs. Dominant mRNA expression of Kv3.1,a high voltage-gated KDR channel,was quantitatively confirmed. Kv3.1 gene knockdown with specific small interfering RNAs (siRNA) for Kv3.1 significantly inhibited Kv3.1 mRNA expression by 63.9% (P < 0.001) and KDR channel currents by 52.2% (P < 0.001). This indicates that Kv3.1 is the subtype responsible for producing KDR channel outward currents. Resting membrane properties,such as resting membrane potential,of NPCs were not affected by Kv3.1 expression. Kv3.1 knockdown with 300 nm siRNA inhibited NPC growth (increase in cell numbers) by 52.9% (P < 0.01). This inhibition was attributed to decreased cell proliferation,not increased cell apoptosis. We also established a convenient in vitro imaging assay system to evaluate NPC differentiation using NPCs from doublecortin-green fluorescent protein transgenic mice. Kv3.1 knockdown also significantly reduced neuronal differentiation by 31.4% (P < 0.01). We have demonstrated that Kv3.1 is a dominant functional KDR channel subtype expressed in adult NPCs and plays key roles in NPC proliferation and neuronal lineage commitment during differentiation. View Publication

Mobile sensing based on the integration of microfluidic device and smartphone,so-called MS2 technology,has enabled many applications over recent years,and continues to stimulate growing interest in both research communities and industries. In particular,it has been envisioned that MS2 technology can be developed for various cell functional assays to enable basic research and clinical applications. Toward this direction,in this paper,we describe the development of a MS2-based cell functional assay for testing cell migration (the Mkit). The system is constructed as an integrated test kit,which includes microfluidic chips,a smartphone-based imaging platform,the phone apps for image capturing and data analysis,and a set of reagent and accessories for performing the cell migration assay. We demonstrated that the Mkit can effectively measure purified neutrophil and cancer cell chemotaxis. Furthermore,neutrophil chemotaxis can be tested from a drop of whole blood using the Mkit with red blood cell (RBC) lysis. The effects of chemoattractant dose and gradient profile on neutrophil chemotaxis were also tested using the Mkit. In addition to research applications,we demonstrated the effective use of the Mkit for on-site test at the hospital and for testing clinical samples from chronic obstructive pulmonary disease patient. Thus,this developed Mkit provides an easy and integrated experimental platform for cell migration related research and potential medical diagnostic applications. View Publication