Animals ; Calpain ; metabolism ; Epithelial Cells ; metabolism ; Kidney ; cytology ; Kidney Tubules ; cytology ; metabolism ; Rats ; Reperfusion Injury ; metabolism

Phosphorylation of beta-catenin tyrosine residue 654 plays an important role in the epithelial to myofibroblast transition (EMT). Introducing mimic peptide of tyrosine residue 654 domain of beta-catenin into cells may influence phosphorylation of beta-catenin tyrosine residue 654. To deliver this mimic peptide into renal epithelial cells, we used penetratin as a vector, which is a novel cell permeable peptide, to deliver hydrophilic molecules into cells. A tyrosine 654 residue domain mimic peptide of beta-catenin (PM) with fused penetratin was constructed, purified and then detected for the penetration of the mimic peptide into human renal tubular epithelial cells (HK-2). The results showed that purified fusion mimic peptide could efficiently and rapidly translocate into human renal tubular epithelial cells. It is concluded that a cell-permeable peptides mimic of tyrosine residue 654 domain of beta-catenin was successfully obtained, which may provide a useful reagent for interfering the human renal tubular epithelial-mesenchymal transition.
Carrier Proteins/*metabolism ; Epithelial Cells/cytology ; Epithelial Cells/*metabolism ; Fibroblasts/cytology ; Fibroblasts/metabolism ; Kidney Tubules/*cytology ; Peptides/metabolism ; Permeability ; Phosphorylation ; Tyrosine/*metabolism ; beta Catenin/*metabolism

OBJECTIVETo establish and assess the Caco-2 cell in vitro absorption model.

METHODSCaco-2 cells were cultured on the millipore filters fixed in Snapwell transport chamber. The cell morphology, transepithelial electrical resistance, mannitol efflux rate and alkaline phosphatase activities were monitored during culture.

RESULTSAfter 21 days of in vitro culture, formation of tight junction was observed between the cells. The transepithelial electrical resistance reach a relatively stable value of 620-/+47 Omega.cm(2), the mannitol efflux rate was lower than 0.3%.h(-1).cm(-2), and the alkaline phosphatase activity in the apical side was significantly higher than that in the basolateral side.

CONCLUSIONThe established Caco-2 cell model shows similar morphology to intestinal epithelial cells with formation of polarity, and can be used as an in vitro model for absorption studies.

Caco-2 Cells ; Cell Culture Techniques ; methods ; Epithelial Cells ; cytology ; metabolism ; Humans ; Intestinal Absorption ; Intestines ; cytology

OBJECTIVETo investigate the possibility of differentiation of human mesenchymal stem cells (hMSC) into epidemic cells in vitro.

METHODShMSCs were segregated from normal adult human bone marrow by Percoll solution (1.073 g/ml) , and were cultured, purified, and amplified to 3th passage in vitro. Then the hMSCs were randomly divided into control group ( with treatment of normal L-DMEM medium) and experimental group (with treatment of L-DMEM medium containing epidermal growth factor,insulin,tretinoin, calcium chloride). After 7 days of culture, the morphologic changes of hMSCs in the 2 groups were observed with inverted phase contrast microscope. The expressions of P63 and PCK of hMSCs were assessed with immunohistochemical methods.

RESULTSThe shape of hMSCs in experimental group became irregular or oblong in shape, while that in control group were still in spindle shape. Immunohistochemical results showed that hMSCs were P63 and PCK positive in the experimental group, while those in control group were negative.

CONCLUSIONHuman mesenchymal stem cells can differentiate into epidemic cell in vitro.

Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; Cells, Cultured ; Epithelial Cells ; cytology ; Humans ; Keratins ; metabolism ; Membrane Proteins ; metabolism ; Mesenchymal Stromal Cells ; cytology

OBJECTIVETo study the effect of N-glycosylation on the modification of microvilli on the surface of rat liver epithelial cell WB-F344 and the growth of the cells in culture.

METHODSRecombinant adeno-associated virus (rAAV) expression vector pAGX (+) containing an antisense or a sense fragment of 6A8 cDNA encoding a human alpha-mannosidase was constructed. The recombinant vectors or the mock were transfected into WB-F344 cells by means of lipofectAmine. The transfected cells were selected in G418 medium and cloned by means of limiting dilution. Integration of the transfected DNA into host DNA was detected by neo PCR. Rat liver ER alpha-mannosidase activity in cell supernatant was measured by using P-nitrophenyl-alpha-D-mannopyranoside as a substrate. Microvilli on cell surface were observed upon a scan electron microscope. The growth curves of the cells in culture were drawn.

RESULTSThe cell clones transfected with antisense 6A8 showed reduction of ER alpha-mannosidase activity with various degrees. Clone AS1 and AS2 cell showed a pronounced reduction of the enzymatic activity. In the study on AS1 cells, Con A binding to the cells was found to be enhanced, cell growth in culture became slow from day 5. The microvilli on the cells were reduced and blunted.

CONCLUSIONSTransfection with antisense 6A8 resulted in reduction and blunting of microvilli on the surface of growing WB-F344 cells, which might be related to N-glycosylation modification.

Animals ; Cloning, Molecular ; Epithelial Cells ; cytology ; Glycosylation ; Liver ; cytology ; Microvilli ; Rats ; Transfection ; alpha-Mannosidase ; genetics ; metabolism

OBJECTIVE: To evaluate the feasibility of STR genotyping from trace epithelial cells on fountain pen and to discuss the impact of conservation time on DNA typing. METHODS: Seven fountain pens were separately used by each of the 17 volunteers 20 minutes per day for a month and then were preserved on day 1, 3, 5, 7, 14, 21, and 28. DNA was extracted from the epithelial cells on fountain pen by silicon bead and was genotyped by Identifier kit. The corresponding control samples were buccal swabs of the above volunteers. The detectable numbers of loci were counted for assessment. RESULTS: There were statistically significant differences in the DNA genotyping by detectable numbers of gene loci between buccal swabs and epithelial cells on fountain pen of different conservation times (P < 0.01). The differences of detectable numbers of loci between the epithelial cells on fountain pen preserved on day 1, 3, 5, 7, 14, 21, 28 and the corresponding oral swabs were also statistically significant (P < 0.01). More than 12 loci could be successfully genotyped in 41.2% samples from the epithelial cells on fountain pen if the tests were performed within 24 hours. CONCLUSION The trace epithelial cells on fountain pen can be used as biological samples for personal identification, but the conservation time would have influence on the results of DNA genotyping.
Epithelial Cells/metabolism* ; Forensic Medicine ; Genotype ; Humans ; Microsatellite Repeats/genetics* ; Mouth Mucosa/cytology* ; Skin/cytology*

No abstract available.
Animal ; Chloride Channels/metabolism ; Chloride Channels/biosynthesis* ; Choroid Plexus/metabolism* ; Choroid Plexus/cytology ; Epithelial Cells/metabolism*

OBJECTIVE: To detect cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) release in human nasal epithelia (HNE) induced by lipopolysaccharide (LPS) in different concentration gradient and time gradient, and to investigate their roles in nasal inflammatory pathogenesis. METHOD: Western Blot and fluorescent real time quantitative PCR were performed to detect the expression of COX-2 in HNE induced by LPS and blocked by selective inhibitor of COX-2. The concentrations of PGE2 were determined by enzyme immunoassay. RESULT: Low expressions of COX-2 and PGE2 were detected in normal HNE. COX-2 expression and PGE2 release increased in HNE induced by LPS in time-dependent or dose-dependent manner. The increased release of PGE2 was later than that of COX-2 expression. COX-2 expression and PGE2 release were dose-dependently attenuated by selective inhibitor of COX-2. CONCLUSION LPS effectively induces COX-2 expression and PGE2 release in HNE. And COX-2 is responsible for the synthesis of PGE2. These results indicate that the increased expression of COX-2 and PGE2 is involved in the inflammation of HNE induced by LPS in vitro.
Cells, Cultured ; metabolism ; Cyclooxygenase 2 ; metabolism ; Dinoprostone ; biosynthesis ; metabolism ; Epithelial Cells ; metabolism ; Humans ; Lipopolysaccharides ; Nasal Mucosa ; cytology ; metabolism

This study was purposed to optimize the culture conditions of the human amniotic epithelium cells (hAEC) in vitro, and detect the expression of hAEC pluripotent markers. Amnion tissues were separated from the underlying chorion through the spongy layer immediately after elective cesarean section of healthy pregnancy women at term. After the subsequent exposure to trypsin digestion, hAEC were cultured in DMEM with different supplements. The growth and proliferation potential of hAEC was evaluated, and the expression of cultured hAEC pluripotent markers was detected by using flow cytometry and immunohistochemistry methods. The results indicated that when being cultured in the mediums similar to that of embryonic stem cell culture supplemented with 10 ng/ml EGF, the hAEC grew better and the time for passage was shortened. In addition, compared to other culture conditions, under this condition, the cells could be passaged up to 5 times as much without obvious morphological changes, and the pluripotent marker SSEA-4 was detected in the cultured cells by flow cytometry. Meanwhile, the detection of immunofluorescence showed the expression of vimentin in cultured hAEC was strengthened as compared with primary cells. It is concluded that the culture condition similar to that for embryonic stem cells supplemented with EGF facilitates the proliferation and passage of hAEC in vitro.
Amnion ; cytology ; metabolism ; Cell Culture Techniques ; methods ; Cell Differentiation ; Cells, Cultured ; Epithelial Cells ; cytology ; metabolism ; Female ; Humans ; Pregnancy ; Stem Cells ; cytology ; metabolism

OBJECTIVE: To observe the expression profile of heat shock proteins (HSPs) including HSP70, inducible HSP90 (HSP86) and aB-crystallin in cells and tissues of lung adenocarcinoma. METHODS: Western blotting and reverse transcriptional-polymerase chain reaction (RT-PCR) were performed to detect the expression of HSP70, HSP86 and aB-crystallin both in the protein and mRNA level respectively. RESULTS: Compared with normal lung tissue and human bronchial epithelium (HBE) cells, RT-PCR and Western blotting showed that the expression of HSP70, HSP86 and alphaB crystallin increased significantly in both the mRNA and protein level in the cancer tissue and A549 human lung adenocarcinoma cells. Among the 3 sub-families of HSPs, the expression of HSP70 mRNA and protein increased most in both the lung tissue of cancer and A549 human adenocarcinoma cell lines. CONCLUSION The expression of HSPs is higher in the lung adenocarcinoma and A549 cells than that in the normal lung tissues and HBE cells. Among the HSP family, HSP70 is the most up-regulated member in the tissue and cells of lung adenocarcinoma.
Adenocarcinoma ; metabolism ; Adenocarcinoma of Lung ; Cells, Cultured ; Epithelial Cells ; cytology ; metabolism ; Heat-Shock Proteins ; metabolism ; Humans ; Lung ; cytology ; metabolism ; Lung Neoplasms ; metabolism ; Tumor Cells, Cultured