OBJECTIVE: To investigate the relationship between necroptosis and apoptosis in MCET3-E1 cell death induced by glucocorticoids. METHODS: MC3T3-E1 cells were incubated with 10-6 mol/L dexamethasone followed by treatment with the apoptosis inhibitor z-VAD-fmk (40 μmol/L) or the necroptosis inhibitor necrostatin-1 (40 μmol/L) for 2 h. At 72 h after incubation with dexamethasone, the cells were harvested to determine the cell viability using WST-1 assay and the rate of necrotic cells using annexin V/PI double staining; the percentage of apoptotic cells was determined using Hoechst staining. The mitochondrial membrane potential and the level of ATP in the cells were also evaluated. Transmission electron microscopy was used to observe the microstructural changes of the cells. The expressions of RIP-1 and RIP-3 in the cells were detected by Western blotting. RESULTS: At a concentration of 10-6 mol/L, dexamethasone induced both apoptosis and necroptosis in MC3T3- E1 cells. Annexin V/PI double staining showed that inhibition of cell apoptosis caused an increase in cell necrosis manifested by such changes as mitochondrial swelling and plasma membrane disruption, as shown by electron microscopy; Hoechst staining showed that the percentage of apoptotic cells was significantly reduced. When necroptosis was inhibited by necrostatin-1, MC3T3-E1 cells showed significantly increased apoptosis as shown by both AV/PI and Hoechst staining, and such changes were accompanied by changes in mitochondrial membrane potential and ATP level in the cells. CONCLUSIONS In the process of dexamethasone-induced cell death, necroptosis and apoptosis can transform reciprocally accompanied by functional changes of the mitochondria.
3T3 Cells ; Adenosine Triphosphate ; Animals ; Apoptosis ; Cell Death ; drug effects ; Dexamethasone ; Membrane Potential, Mitochondrial ; Mice ; Microscopy, Electron ; Mitochondria ; ultrastructure ; Necrosis

OBJECTIVETo culture rat prostate glandular epithelial cells and study their barrier functions in vitro.

METHODSRat prostate glandular epithelial cells were cultured in vitro. The expression of the tight junction protein claudin-1 was determined by immunohistochemistry, the structure and composition of the epithelial cells observed under the inverted microscope and transmission electron microscope. The transepithelial electrical resistances (TEERs) were monitored with the Millicell system. The permeability of the prostate glandular epithelial cells was assessed by the phenol red leakage test.

RESULTSCompact monolayer cell structures were formed in the prostate glandular epithelial cells cultured in vitro. Immunohistochemistry showed the expression of the tight junction protein claudin-1 and transmission electron microscopy confirmed the formation of tight junctions between the adjacent glandular epithelial cells. The TEERs in the cultured prostate glandular epithelial cells reached the peak of about (201.3 ± 3.5) Ω/cm2 on the 8th day. The phenol red leakage test manifested a decreased permeability of the cell layers with the increase of TEERs.

CONCLUSIONThe structure and function of rat prostate glandular epithelial cells are similar to those of brain capillary endothelial cells, retinal capillary endothelial cells, and intestinal epithelial cells. In vitro cultured prostate glandular epithelial cells have the barrier function and can be used as a model for the study of blood prostate barrier in vitro.

Animals ; Cell Membrane Permeability ; Cells, Cultured ; Claudin-1 ; metabolism ; Electric Impedance ; Epithelial Cells ; pathology ; physiology ; ultrastructure ; In Vitro Techniques ; Male ; Microscopy, Electron, Transmission ; Phenolsulfonphthalein ; pharmacokinetics ; Prostate ; metabolism ; pathology ; Rats ; Tight Junctions

To investigate the morphogenetic process of human adenovirus type 41 (HAdV-41), 293TE cells were infected with purified wild-type HAdV-41, and ultrathin sections of infected cells were prepared and observed under a transmission electron microscope. Results showed that HAdV-41 entered host cells mainly through three ways: non-clathrin-coated pit, clathrin-coated pit, and direct penetration of plasma membrane. In addition, cell microvilli might help HAdV-41 enter cells. After entering into cells, HAdV-41 virus particles could be found in vacuoles or lysosomes or be in a free state in cytoplasm. Only free virus particles could be found near nuclear pores (NP), suggesting that the virus needed to escape from lysosomes for effective infection and viral nucleoprotein entered the nucleus through NP. Progeny viruses were as-sembled in the nucleus. Three types of inclusion bodies, which were termed as fibrillous inclusion body, condense inclusion body, and stripped condense inclusion body, were involved in HAdV-41 morphogenesis. In the late phase of viral replication, the membrane integrity of the infected cells was lost and viral particles were released extracellularly. This study reveals the partial process of HAdV-41 morphogenesis and provides more biological information on HAdV-41.
Adenovirus Infections, Human ; virology ; Adenoviruses, Human ; genetics ; growth & development ; physiology ; ultrastructure ; Cell Membrane ; virology ; Cell Nucleus ; virology ; Humans ; Virus Release ; Virus Replication

Oval cells have a potential to differentiate into a variety of cell lineages including hepatocytes and biliary epithelia. Several models have been established to activate the oval cells by incorporating a variety of toxins and carcinogens, alone or combined with surgical treatment. Those models are obviously not suitable for the study on human hepatic oval cells. It is necessary to establish a new and efficient model to study the human hepatic oval cells. In this study, the hepatocyte growth factor (HGF) and epidermal growth factor (EGF) were used to induce differentiation of mouse embryonic stem (ES) cells into hepatic oval cells. We first confirmed that hepatic oval cells derived from ES cells, which are bipotential, do exist during the course of mouse ES cells' differentiation into hepatic parenchymal cells. RT-PCR and transmission electron microscopy were applied in this study. The ratio of Sca-1+/CD34+ cells sorted by FACS in the induction group was increased from day 4 and reached the maximum on the day 8, whereas that in the control group remained at a low level. The differentiation ratio of Sca-1+/CD34+ cells in the induction group was significantly higher than that in the control group. About 92.48% of the sorted Sca-1+/CD34+ cells on the day 8 were A6 positive. Highly purified A6+/Sca-1+/CD34+ hepatic oval cells derived from ES cells could be obtained by FACS. The differentiation ratio of hepatic oval cells in the induction group (up to 4.46%) was significantly higher than that in the control group. The number of hepatic oval cells could be increased significantly by HGF and EGF. The study also examined the ultrastructures of ES-derived hepatic oval cells' membrane surface by atomic force microscopy. The ES-derived hepatic oval cells cultured and sorted by our protocols may be available for the future clinical application.
Animals ; Antigens, CD34 ; genetics ; metabolism ; Antigens, Ly ; genetics ; metabolism ; Cell Differentiation ; drug effects ; genetics ; physiology ; Cell Line ; Embryonic Stem Cells ; cytology ; metabolism ; ultrastructure ; Epidermal Growth Factor ; pharmacology ; Flow Cytometry ; Gene Expression Regulation, Developmental ; drug effects ; Hepatocyte Growth Factor ; pharmacology ; Liver ; cytology ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Microfilament Proteins ; metabolism ; Microscopy, Atomic Force ; Microscopy, Electron, Transmission ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells ; cytology ; metabolism ; ultrastructure ; Time Factors

E-cadherin is a cell adhesion molecule that plays an important role in maintaining renal epithelial polarity and integrity. The purpose of this study was to determine the exact cellular localization of E-cadherin in pig kidney. Kidney tissues from pigs were processed for light and electron microscopy immunocytochemistry, and immunoblot analysis. E-cadhedrin bands of the same size were detected by immunoblot of samples from rat and pig kidneys. In pig kidney, strong E-cadherin expression was observed in the basolateral plasma membrane of the tubular epithelial cells. E-cadherin immunolabeling was not detected in glomeruli or blood vessels of pig kidney. Double-labeling results demonstrated that E-cadherin was expressed in the calbindin D28k-positive distal convoluted tubule and H(+)-ATPase-positive collecting duct, but not in the aquaporin 1-positive, N-cadherin-positive proximal tubule. In contrast to rat, E-cadherin immunoreactivity was not expressed at detectable levels in the Tamm-Horsfall protein-positive thick ascending limb of pig kidney. Immunoelectron microscopy confirmed that E-cadherin was localized in both the lateral membranes and basal infoldings of the collecting duct. These results suggest that E-cadherin may be a critical adhesion molecule in the distal convoluted tubule and collecting duct cells of pig kidney.
Animals ; Blotting, Western/veterinary ; Cadherins/*genetics/metabolism ; Cell Membrane/*metabolism/ultrastructure ; *Gene Expression Regulation ; Kidney/*metabolism ; Male ; Microscopy, Electron, Transmission/veterinary ; Sus scrofa/*genetics/metabolism

BACKGROUNDDendritic cells (DCs) are the most important professional antigen presenting cells that play a key role in initiating adaptive immune responses. The depletion and dysfunction of DCs contribute to the development of immunodeficiency or immunoparalysis in some lung diseases. In the present study, we investigated the effects of Fms-like tyrosine kinase-3 ligand (Flt3L) administration in vivo on lung DCs expansion to provide an experimental basis of Flt3L used as a potential therapeutic agent for the related lung disorders.

METHODSBalb/c mice were randomly divided into Flt3L group (n = 10) and control group (n = 10). Each mouse in the Flt3L group received subcutaneous administration of Flt3L at a dose of 10 µg once daily for nine consecutive days. Lung histology was observed, and CD11c and CD205 were immunologically labeled in lung tissue sections. Low-density lung cells were separated by density gradient centrifugation, and then subsets and MHC-II/I-A(d) expression of DCs were analyzed by flow cytometry.

RESULTSIn the Flt3L group the number and density of DC-like cells were markedly increased compared with the control group, mainly distributed in the alveolar septa. Immunological labeling in situ found that there were significantly higher numbers of CD11c(+) and CD205(+) DCs in lung mesenchymal tissue (P < 0.05), where they formed a denser reticular formation. Flow cytometry analysis demonstrated that the proportions of myeloid CD11c(+)CD11b(+) DCs and plasmacytoid CD11c(+)CD45R/B220(+) DCs in the low-density lung cells in the Flt3L group were significantly higher compared with the control group; showing 3.17- and 3.3-fold increase respectively (P < 0.05). The proportion of CD11c(+) DCs expressing MHC-II/I-A(d+) was significantly increased, with a 2.7-fold increase as compared with the control group (P < 0.05).

CONCLUSIONSFlt3L administration in vivo induces lung DCs expansion, favoring myeloid and plasmacytoid DC subsets, which are phenotypically more mature. Flt3L may be useful in the therapy to augment immune function of the lung.

Animals ; Antigens, CD ; metabolism ; Cells, Cultured ; Dendritic Cells ; drug effects ; metabolism ; ultrastructure ; Flow Cytometry ; Immunohistochemistry ; Lectins, C-Type ; metabolism ; Lung ; drug effects ; metabolism ; ultrastructure ; Male ; Membrane Proteins ; pharmacology ; Mice ; Mice, Inbred BALB C ; Microscopy, Electron, Transmission ; Minor Histocompatibility Antigens ; Random Allocation ; Receptors, Cell Surface ; metabolism

A sub-microsecond pulse generation applied for electroporation effects of tumor cell is presented in this paper. The principle of the generator is that the expected pulse waveform is intercepted from the RC discharge curve by controlling the on-off states of two IGBT modules with a synchronous controller. Experimental tests indicate that the generator can produce adjustable pulse waveform parameters with 0.5-3.5kV amplitude, 300-800 ns pulse duration, 1-400Hz repetition frequency rate, and it is suitable for the study of the electroporation effect experiments.
Animals ; Cell Line, Tumor ; Cell Membrane ; Electricity ; Electroporation ; instrumentation ; methods ; Equipment Design ; Microtechnology ; instrumentation ; Neoplasms ; ultrastructure

OBJECTIVETo observe the ultracytochemical localization of H(+)-adenosine triphosphatase (H(+)-ATPase) in the cell organelles.

METHODSThe localization of H(+)-ATPase in the cell organelles was observed in the hepatocytes and renal cells of Wistar rats using routine ultracytochemical methods.

RESULTSH(+)-ATPase activities were observed on the lysosomal membrane and nuclear envelope of the hepatocytes and proximal tubule epithelial cells of the nephron in Wistar rats.

CONCLUSIONThis finding supports the hypothesis that H(+)-ATPase (V-ATPase) is present on the plasma membrane and in the endomembrane system.

Animals ; Cell Membrane ; enzymology ; Hepatocytes ; cytology ; enzymology ; ultrastructure ; Histocytochemistry ; methods ; Kidney ; cytology ; enzymology ; ultrastructure ; Lysosomes ; enzymology ; Male ; Organelles ; enzymology ; Rats ; Rats, Wistar ; Vacuolar Proton-Translocating ATPases ; metabolism

The lower expression of CD20 antigen molecules on the B cell membrane is the primary characteristic of B-chronic lymphocytic leukemia (B-CLL). In this paper, we combined laser scanning confocal microscopy (LSCM) and quantum dots labeling to detect the expression and distribution of CD20 molecules on CD20+B lymphocyte surface. Simultaneously, we investigated the morphology and ultrastructure of the B lymphocytes that belonged to the normal persons and B-CLL patients through utilizing the atomic force microscope (AFM). In addition, we measured the force spectroscopy of CD20 antigen-antibody binding using the AFM tips modified with CD20 antibody. The fluorescent images indicated that the density of CD20 of normal CD20+B lymphocytes was much higher than that of B-CLL CD20+B cells. The AFM data show that ultrastructure of B-CLL CD20+B lymphocytes became more complicated. Moreover, the single molecular force spectroscopy data show that the special force of CD20 antigen-antibody was four times bigger than the nonspecific force between the naked AFM tip and cell surface. The force map showed that CD20 molecules distributed homogeneously on the normal CD20+B lymphocytes, whereas, the CD20 molecules distributed heterogenous on B-CLL CD20+B lymphocytes. Our data provide visualized evidence for the phenomenon of low-response to rituximab therapy on clinical. Meanwhile, AFM is possible to be a powerful tool for development and screening of drugs for pharmacology use.
Antigen-Antibody Reactions ; immunology ; Antigens, CD20 ; immunology ; B-Lymphocytes ; immunology ; ultrastructure ; Binding Sites, Antibody ; Cell Membrane ; immunology ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell ; immunology ; Microscopy, Atomic Force ; Microscopy, Confocal ; Quantum Dots

OBJECTIVETo determine the relationship of thick greasy tongue fur formation and permeability of vascular endothelial cells (ECs) with the protein expression of zonula occludens-1 (ZO-1).

METHODSSprague Dawley rats were randomly divided into a model group of severe acute pancreatitis (SAP) and a sham-operated (SO) group. The SAP rats were further divided into two subgroups on the basis of tongue-coating status: a thick greasy tongue fur group (SAP-TGF) and a normal tongue fur group (SAP-NF). Six rats were chosen randomly from every group mentioned above for an Evans blue assay 5 days after model establishment. For the histomorphology analysis, the expressions of ZO-1 protein and mRNA were studied by hematoxylin-eosin (H&E) staining, transmission electron microscope, Western blot, and Q-PCR using blood and tongue tissues, which were collected from 8 rats randomly chosen from each group.

RESULTSThe papillae density of the rat tongue surface and the caryocinesis frequency of the basal layer were significantly increased in the SAP-TGF group compared with the SO group (P<0.05). Evans blue levels in the tongue tissue of the SAP-TGF group were significantly higher than that of the SO and SAP-NF groups (P<0.05). Vascular ECs were wider and obviously fissured in the SAP-TGF group under transmission electron microscope observation. The protein and mRNA expression of ZO-1 in the SAP-TGF group were lower than those in the SAP-NF (P<0.05).

CONCLUSIONSReproductive activity enhancement of glossal epithelial cells was one of the main characteristics of thick greasy tongue fur formation. An increase in vasopermeability was closely associated with thick greasy tongue fur formation. Tight junction structural variation of vascular ECs might play an important role in the pathological and physiological process of thick greasy tongue fur formation.

Animals ; Blotting, Western ; Cell Membrane Permeability ; Endothelial Cells ; cytology ; Evans Blue ; metabolism ; Gene Expression Regulation ; Male ; Membrane Proteins ; genetics ; metabolism ; Phosphoproteins ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tongue ; pathology ; ultrastructure ; Zonula Occludens-1 Protein