BACKGROUNDRe-epithelialization has remained a major obstacle in both tracheal and lung transplantations. This study examines the realization of re-epithelialization by epithelial inoculation in a rat heterotopic tracheal transplantation model.

METHODSThe original epithelia of tracheas from donor Wistar rats were removed and the tracheas were then inoculated with 10(6)/ml in vitro cultured epithelial cells of the Spraque-Dawley (SD) rat phenotype. These allo-tracheas were then heterotopically transplanted into SD rats. After 28 days, the allo-trachea tissues were recovered and assessed for epithelial morphology and cellular differentiation using immunohistochemical analysis. An additional experimental group was used to compare the outcomes of re-epithelialization in immunosuppressed animals.

RESULTSHistological examination showed that allografts with epithelial inoculation maintained patent tracheal lumens, which were obliterated in controls. Recipient immunosuppression facilitated the formation of an integrated ciliated epithelial layer, further demonstrated by the presence of a dense cilia population, a well-developed plasma membrane, and readily recognizable intercellular junctions. Epithelial cellular differentiation markers such as cytokeratin 14 and 18, and cystic fibrosis transmembrane conductance regulator (CFTR) were all positive in allografts under immunosuppression.

CONCLUSIONConcurrent recipient-derived epithelial inoculation with immunosuppression can result in complete re-epithelialization with the recipient phenotype and suppress the luminal obliteration process in heterotopic transplantations.

Allografts ; cytology ; Animals ; Bronchiolitis Obliterans ; surgery ; Epithelial Cells ; cytology ; Female ; Male ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar ; Trachea ; cytology ; transplantation ; Transplantation, Heterotopic

Aim of the present study is to investigate activation effect of nobiletin on cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activity. CFTR-mediated iodide influx assay and patch-clamp tests were done on FRT cells stably co-transfected with human CFTR and EYFP/H148Q. Nobiletin potently activated CFTR chloride channel activity in a dose- and time-dependent manner. The CFTR blocker CFTR(inh)-172 could completely reverse the effect. Preliminary mechanism study indicated that nobiletin activated CFTR chloride channel through a direct binding way. In addition, ex vivo tests done on mice trachea showed that nobiletin time-dependently stimulated submucosal gland fluid secretion. Nobiletin may be a therapeutic lead compound in treating CFTR-related diseases including disseminated bronchiectasis.
Animals ; Benzoates ; pharmacology ; Cystic Fibrosis Transmembrane Conductance Regulator ; antagonists & inhibitors ; drug effects ; metabolism ; Dose-Response Relationship, Drug ; Epithelial Cells ; metabolism ; Exocrine Glands ; secretion ; Flavones ; administration & dosage ; pharmacology ; Humans ; Mice ; Patch-Clamp Techniques ; Rats ; Rats, Inbred F344 ; Thiazolidines ; pharmacology ; Thyroid Gland ; cytology ; Time Factors ; Trachea ; secretion

OBJECTIVE: To construct phosphorylation sites domain (PSD) mutant of myristoylated alaninerich C kinase substrate (MARCKS) and explore the role of transient receptor potential melastatin 8 cation channels (TRPM8) and MARCKS in cold-induced synthesis and exocytosis of mucin (MUC) 5AC. METHODS: Human placental cDNA was used as a template to amplify the full coding region of MARCKS cDNA by PCR. Ser159, Ser 163, Ser 167, Ser 170 in the PSD were mutated to aspartic acids by an overlap PCR method. The resultant PSD mutant cDNA and the wild-type MARCKS cDNA were each subcloned into a mammalian expression vector pcDNA3.0. Recombinant constructs were confirmed by restriction enzyme digestion analysis and DNA sequencing. In intervention experiments, cells were pretreated with the TRPM8 channel antagonist BCTC and transfected with MARCKS-PSD mutant cDNA, and thereafter cold stimulation was applied. The levels of MUC5AC were measured by immunofluorescence and ELISA to clarify the roles of TRPM8 and PSD mutant on the synthesis and secretion of MUC5AC induced by cold, respectively. RESULTS: Restriction enzyme digestion analysis and DNA sequencing revealed that the pcDNA3.0- MARCKS and pcDNA3.0-MARCKS-PSD mutants were successfully constructed. The levels of intracellular and secreted MUC5AC of cold treated group were significantly higher than those of control group (P<0.05). BCTC attenuated the cold-induced synthesis and secretion of MUC5AC when compared with cold treated group (P<0.05). Transfection of 16HBE cells with the MARCKS-PSD mutant cDNA resulted in significant inhibition of mucin secretion in response to cold, and significantly higher level of intracellular MUC5AC than that of control group (P<0.01), whereas transfection with the vector DNA or the wild-type MARCKS cDNA had no effect on the mucin synthesis and secretion in response to cold (P>0.05). CONCLUSION TRPM8 and phosphorylation of MARCKS-PSD mediates the cold-induced exocytosis of MUC5AC by airway epithelial cells.
Base Sequence ; Cell Line ; Cold Temperature ; Epithelial Cells ; cytology ; metabolism ; Exocytosis ; physiology ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Molecular Sequence Data ; Mucin 5AC ; metabolism ; Mutation ; Myristoylated Alanine-Rich C Kinase Substrate ; Phosphorylation ; TRPM Cation Channels ; metabolism ; Trachea ; cytology ; metabolism

OBJECTIVETo assess the severity and reversibility of the chronic toxicity of a novel recombinant human granulocyte colony-stimulating factor (rhG-CSFa) in rats and the dose-effect relationship.

METHODSA total of 100 Sprague-Dawley rats (equal numbers of male and female) were randomly divided into five groups (20 rats in each group): four groups were treated with rhG-CSFa at 500, 100, 10, 1 µg/kg, respectively, and one group was treated with vehicle only to serve as the control. The rats were received subcutaneous injections of rhG-CSFa or vehicle daily for 13 weeks. During the course of the chronic toxicity study, the physical status, body weight, and food consumption were monitored. Half of the rats in each group (n = 10) were sacrificed after the last rhG-CSFa administration, and the other half were sacrificed at five weeks after the last rhG-CSFa administration. Urinalyses, blood biochemistry, hematological analysis, histopathological examination, and immunological tests were performed for each of the rats.

RESULTSThe hematological analyses revealed that the mean white blood cells count, neutrophils count, and neutrophils percentage were increased in male rats at the dose of 10 µg/kg or higher, and these were related with the biological activity of rhG-CSFa. Some small abnormalities were observed in the spleen of a few rats when used highest dose (500 µg/kg, a dosage of 200 folds higher than the normal clinical dosage), but these abnormalities were recovered within 5-week recovery period. No other rhG-CSFa-related abnormalities were observed in this chronic toxicity study.

CONCLUSIONNo significant toxicity and immunogenicity are observed with rhG-CSFa administration to rats in the chronic toxicity studies.

Animals ; Bilirubin ; urine ; Blood Chemical Analysis ; Dose-Response Relationship, Drug ; Female ; Granulocyte Colony-Stimulating Factor ; genetics ; toxicity ; Humans ; Lung ; cytology ; drug effects ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; Spleen ; cytology ; drug effects ; Trachea ; cytology ; drug effects

Since most of the respiratory epithelial cell lines are descended from neoplastic tissues or have been fused with neoplastic cells when being selected to a cell line, their biological behaviors are far different from normal respiratory epithelial cells. Aiming at better reflecting the biological properties of epithelial cells under respiratory pathological conditions, our study probed into a new isolation technique and culture method of mice respiratory epithelial cell. Pronase was applied for cell isolation from mouse trachea, and a modified medium with collagen-coated plate for primary culture. The ciliary movement can be observed under microscope, which demonstrates that the original biological functions of the tracheal epithelial cell have been reserved with our method. The research presents the efficacy, convenience and reliability of the separation technique and culture method established by this study, and laying preferable condition for cell models of respiratory diseases. Meanwhile, this method may be used for other animal models.
Animals ; Cell Separation ; methods ; Culture Media ; Epithelial Cells ; cytology ; Female ; Male ; Mice ; Mice, Inbred BALB C ; Primary Cell Culture ; methods ; Pronase ; pharmacology ; Trachea ; cytology

BACKGROUNDAn important physiological feature of asthma is the phenotypic change of airway smooth muscle cells (ASMCs), but the precise mechanisms behind the ASMCs' change remains unknown. Our study assessed whether p21Ras can directly modulate the phenotype of ASMCs.

METHODSRat ASMCs were treated with FTP III, a highly specific p21Ras inhibitor. ASMCs were identified via immunocytochemistry. The ultrastructure of cells was observed by electron microscopy, and the expression of α-actin was evaluated by Western blotting analysis. The levels of IL-6 and RANTES were measured by enzyme linked immunosorbent assay (ELISA).

RESULTSIt was observed that ASMCs in asthma exhibited a proliferative/secretory phenotype and were larger, denser and had many pseudopods, as well as increased signs of secretory organelles. Additionally, the level of α-actin, a marker of ASMCs, was reduced in asthmatic ASMCs and the secretion of IL-6 and RANTES was increased. When FTP III was added to asthmatic ASMCs it induced a contractile phenotype, with increased α-actin levels and reduced secretion of IL-6 and RANTES.

CONCLUSIONSIt appears that p21Ras induces asthmatic ASMCs to a proliferative/secretory phenotype, but its inhibitor FTP III, can significantly reverse this phenotype. The role of p21Ras in the ASMCs may be a new target for asthma treatment.

Actins ; metabolism ; Animals ; Asthma ; metabolism ; Blotting, Western ; Cells, Cultured ; Chemokine CCL5 ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Interleukin-6 ; metabolism ; Lung ; cytology ; Male ; Microscopy, Electron, Transmission ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; ultrastructure ; Organophosphonates ; pharmacology ; Proto-Oncogene Proteins p21(ras) ; antagonists & inhibitors ; metabolism ; Rats ; Rats, Sprague-Dawley ; Trachea ; cytology

OBJECTIVETo observe the homing and differentiation of marrow-derived mesenchymal stem cells (MSC) transplanted intravenously in smoke inhalation injured rabbits.

METHODSThirty-two New Zealand big ear rabbits were divided into normal control group (NC), inhalation injury group (II), normal control + MSC treatment group (NM), and MSC treatment group (MT) according to the random number table, with 8 rabbits in each group. Rabbits in NC group were injected with 10 mL phosphate buffered saline (PBS) via ear marginal vein. Rabbits in NM group were injected with 10 mL PBS containing the third generation MSC labeled by BrdU (1 × 10(7) per 10 mL PBS) via ear marginal vein. Severe smoke inhalation injury model was reproduced in the other two groups, among them rabbits in II group were treated as rabbits in NC group, rabbits in MT group treated as rabbits in NM group. On the 7th and 28th day post treatment (PTD), lung tissue and trachea tissue were harvested from four groups for observation on injury with HE staining. Homing of MSC in injured tissue was observed with immunohistochemistry staining. The differentiation of MSC into functional cells was observed with immunohistochemical double staining of combining nuclear marker BrdU with lung (trachea) membrane-specific marker aquaporin-5 (AQP-5), alkaline phosphatase (AKP), CD34, and cytokeratin respectively.

RESULTS(1) MSC homing in lung and trachea tissue was observed in MT group on PTD 7, which was not observed in NM group. (2) AQP-5, AKP, and CD34 positive MSC were observed in lung tissue in MT group on PTD 28, while cytokeratin positive MSC was not observed in trachea tissue. No positively marked MSC was observed in NM group. (3) Injury in lung and trachea was less severe in MT group than in II group; and the proliferation of fibroblasts was less in MT group.

CONCLUSIONSIntravenous injection of MSC to rabbits with smoke inhalation injury can migrate to lung and trachea tissue at obviously inflammatory site, and differentiate into alveolar epithelial cells typeI and II, and pulmonary vascular endothelial cells, which may participate in the process of tissue repair in smoke inhalation injury.

Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Cells, Cultured ; Endothelial Cells ; cytology ; Epithelial Cells ; cytology ; Lung ; cytology ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; Pulmonary Alveoli ; cytology ; Rabbits ; Smoke Inhalation Injury ; pathology ; Trachea ; cytology

OBJECTIVETo observe the effects of inhaled Chuankezhi injection (CKZ) on airway inflammation in a mouse model of asthma and dilation of isolated guinea-pig airway smooth muscle in vitro, which can provide pharmacodynamic evidence for CKZ treating acute attack of asthma.

METHODBALB/c mice were sensitized with ovalbumin (OVA) on Days 1, 15, and then were inhaled with OVA aerosol on Days 22-28. The sensitized mice were administered with inhalation of aerosolized CKZ injection (0.2, 0.4, 0.8 mL x kg(-1), bid), or intraperitoneal injection of CKZ (0.4 mL x kg(-1), bid), dexamethsone (0.5 mg x kg(-1) x d(-1)) and saline (control) on Days 22-28. Airway inflammation was evaluated by counting cells in bronchoalveolar lavage fluid (BALF) and by lung histology. The influences of CKZ on the dilation of tracheal smooth muscle in guinea-pig and the contraction induced by carbamylcholine (CCH)/histamine in vitro were also observed.

RESULTIn vivo, OVA-sensitized mice developed a significant airway inflammatory response that was significant inhibited by inhalation of CKZ (0.8 mL x kg(-1), bid), and intraperitoneal injection of CKZ (0.4 mL x kg(-1), bid) and dexamethasone (0.5 mg x kg(-1) x d(-1)). in vitro, CKZ did not dilate tracheal smooth muscles in guinea-pigs, and did not attenuate the contraction induced by carbamylcholine (CCH)/histamine.

CONCLUSIONCKZ can modulate airway inflammation in asthma, but has no dilation effect on the tracheal smooth muscle in guinea-pig in vitro. These results demonstrate that inhaled CKZ is not a preferred administration.

Animals ; Asthma ; drug therapy ; immunology ; Bronchoalveolar Lavage Fluid ; immunology ; Cells, Cultured ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Female ; Guinea Pigs ; Humans ; Injections ; Lung ; drug effects ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Muscle, Smooth ; drug effects ; immunology ; Respiratory System ; Trachea ; cytology ; drug effects ; immunology

Animals ; Aquaporin 3 ; analysis ; Cell Separation ; Fluorouracil ; pharmacology ; Humans ; Lung ; cytology ; Membrane Proteins ; analysis ; Octamer Transcription Factor-3 ; genetics ; Stem Cells ; cytology ; Trachea ; cytology

OBJECTIVEIn this study, we pretreated the mice ASMCs by dexamethasone (Dex) within 10 min, to test the peak of [Ca2+]i and phospho-PLCbeta (ser1105) in the cells by treated with Ach.

METHODSThe peak of [Ca2+]i was measured by Fura-2/AM methods and the phospho-PLCbeta-ser1105 was by Western blot, and compared with dexamethasone pretreated groups. Glucocorticoid receptor antagonist RU486 and the protein synthesis inhibitor cycloheximide groups were settled in our study.

RESULTSGlucocorticoids (GCs) significantly decreased the resting values and peak of [Ca2+]i elevation and elevated the intracellular levels of phospho-PLCbeta (ser1105) in 10 min. Neither the RU486 nor cycloheximide could alter the inhibitory effects of glucocorticoids stated above.

CONCLUSIONOur results demonstrate that glucocorticoids exert rapid inhibitory effects. The series of signal changes in this process that restrain the peak of [Ca2+]i may be responsible for the rapid nongenomic inhibitory effects of GCs by reducing the activity of PLC.

Animals ; Calcium ; metabolism ; Cells, Cultured ; Dexamethasone ; pharmacology ; Glucocorticoids ; pharmacology ; Guinea Pigs ; Male ; Mifepristone ; pharmacology ; Muscle, Smooth ; drug effects ; metabolism ; Phospholipase C beta ; metabolism ; Rats ; Rats, Sprague-Dawley ; Trachea ; cytology