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

Cells, Cultured ; Fibroblasts ; cytology ; drug effects ; metabolism ; Humans ; Lung ; cytology ; metabolism ; Macrophages, Alveolar ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Silicon Dioxide ; toxicity ; Silicosis ; metabolism

OBJECTIVETo investigate lipopolysaccharide (LPS)-induced changes of cytoskeletal filamentous actin in primary isolated pulmonary microvascular endothelial cells (PMVECs) from wild-type and RAGE knock-out mouse.

METHODSThe lungs of wild-type and RAGE knock-out mice were digested with collagenase type I to obtain endothelial cells purified by anti-CD31-coupled magnetic beads. The PMVEC identified by factor VIII labeling were stimulated with LPS at different concentrations and the changes of filamentous actin were observed by confocal microscopy.

RESULTSThe cultured primary cells showed typical endothelial cell phenotype as examined with factor VIII labeling. LPS stimulation caused rearrangement of the cytoskeletal filament F-actin in wild-type mouse PMVECs with stress fiber formation, but such changes were not obvious in RAGE knock-out mouse PMVECs.

CONCLUSIONMouse PMVECs of a high purity can be obtained by immune magnetic beads. RAGE is involved in LPS-induced destruction of mouse PMVEC cytoskeletons.

Actins ; metabolism ; Animals ; Cells, Cultured ; Cytoskeleton ; metabolism ; Endothelial Cells ; cytology ; Lipopolysaccharides ; Lung ; cytology ; Mice ; Mice, Knockout ; Microvessels ; cytology ; Phenotype ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; genetics ; metabolism

To determine whether lentinan could upregulate the expression of human-beta-defensin-2(HBD-2) in pulmonary epithelial cells (SPC-A-1), we stimulated pulmonary epithelial cells with lentinan and detected the expression of HBD-2mRNA by RT-PCR test. The results demonstrated that the expression of HBD-2mRNA in SPC-A-1 could be induced by lentinan in a concentration and time-dependent manner.
Adjuvants, Immunologic ; pharmacology ; Epithelial Cells ; metabolism ; Humans ; Lentinan ; pharmacology ; Lung ; cytology ; metabolism ; RNA, Messenger ; genetics ; metabolism ; beta-Defensins ; genetics ; metabolism

OBJECTIVETo study the influence of human cytomegalovirus (HCMV) infection on cell cycle and the expression of replication licensing factor Cdt1 in human embryonic lung fibroblastic (HEL) cells and to explore the pathogenesis of HCMV infection.

METHODSHEL cells were synchronized in the G0/G1 phase by the serum starvation method. The synchronized HEL cells were infected with HCMV, and those that were not subjected to HCMV infection were used as the control group. The HEL cells were harvested at 12, 24, 48, 72 and 96 hrs of HCMV infection. The cell cycle of HEL cells was detected by the flow cytometry. The expression of Cdt1 mRNA in HEL cells was determined by reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSThe cells in the G1 phase in the control group was significantly more than in the HCMV-infected group 12 and 24 hrs after infection (P < 0.01). The expression of Cdt1 mRNA in the HCMV-infected group was significantly lower 12 and 24 hrs after infection but increased significantly 48 hrs after infection compared with the control group (P < 0.05). The expression of Cdt1 mRNA reached a peak at 12 hrs of infection in the control group, but at 48 hrs of infection in the HCMV-infected group, which markedly lagged behind the control group.

CONCLUSIONSHCMV infection arrests the cell cycle of HEL cells at the G1 phase. HCMV infection makes Cdt1 expression delay. HCMV infection can interfere cell cycle of HEL cells possibly through affecting the expression of Cdt1.

Cell Cycle ; Cell Cycle Proteins ; genetics ; Cells, Cultured ; Cytomegalovirus ; pathogenicity ; Embryo, Mammalian ; cytology ; Fibroblasts ; cytology ; metabolism ; Humans ; Lung ; cytology ; metabolism ; RNA, Messenger ; analysis

OBJECTIVETo investigate the changes in human airway smooth muscle cell (HASMC) migration and related signaling pathway after interference with PTEN gene expression.

METHODHASMCs were infected with an adenovirus vector and RNA interference vector of human PTEN gene to establish the cell model with PTEN gene over-expression (Ad-GFP-PTEN-HASMC) and one with PTEN gene silencing (Ad-shPTEN-HASMC), using Ad-GFP-infected and a blank cells as the negative controls and LY294002 as the positive control. Fluorescence microscopy and flow cytometric analysis were used to evaluate the transfection efficiency, and Western blotting was performed to examine the expression of PTEN and the activation of AKT and ERK1/2 signal pathway. Transwell assay and wound healing assay were used to assess the migration of HASMCs.

RESULTSThe adenovirus over-expression vector and RNA interference vector significantly affected the expression of human PTEN gene. Up-regulation of PTEN gene resulted in a slow-down of the HASMC migration, an inhibition of PI3K/AKT signal pathway at the protein level but no changes in Ras-Raf-MEK1/2-ERK1/2 signal pathway. Down-regulated PTEN gene expression, however, was not associated with an enhancement of HASMC migration, but activated PI3K/AKT signal pathway and inhibited Ras-Raf-MEK1/2-ERK1/2 signal pathway.

CONCLUSIONUpregulation of PTEN gene can effectively inhibit airway smooth muscle cell migration, the effect of which is probably mediated by the PI3K/AKT pathway.

Adenoviridae ; genetics ; Bronchi ; cytology ; Cell Movement ; Cells, Cultured ; Gene Expression ; Genetic Vectors ; Humans ; Lung ; cytology ; Myocytes, Smooth Muscle ; cytology ; metabolism ; pathology ; PTEN Phosphohydrolase ; metabolism ; RNA Interference ; Transfection

OBJECTIVETo study the expression and significance of fragile histidine triad (FHIT) and Ki-67 in transformed epithelial cells induced by Yunnan tin mine dust.

METHODSEvery second generation of immortalized human bronchial epithelial cells (BEAS-2B) and human embryo lung fibroblasts (WI-38) were exposed to 100 µg/ml Yunnan tin mine dust for 72 h, until the ninth generation. The cells were subsequently co-cultured from the 11th generation. Experimental setup: B group, B (W) group, B (W 100) group, B100 group, B100 (W) group, B100 (W100) group. The expressions of FHIT and Ki-67 in epithelial cells were determined by the method of immunocytochemistry at the 16th, 26th and 36th generation. The percentage of Ki-67 positive cells was calculated as proliferation index.

RESULTSThe expression of FHIT was observed in BEAS-2B cells. The expression levels of FHIT among B group, B (W) group and B (W 100) group had not instinctive difference. At the 16th generation, the expression of FHIT in the B100 group was decreased compared with that in the B group and the expression of FHIT between B100 (W) group and B100 (W100) group was lower than that in the B100 group. At the 26th generation, the expression of FHIT was decreased compared with that at the 16th generation in the B100, B100 (W) and B100 (W100) groups. However, At the 36th generation, positive expression were observed again in the B100, B100 (W) and B100 (W100) groups and the expression levels were in incremental order. At the 16th, 26th and 36th generation, the proliferation indexes of B group, B (W) group and B (W 100) group were all < 3%. The proliferation indexes of B100, B100 (W) and B100 (W100) were increased step by step with the generation elongation.

CONCLUSIONSFHIT could be a target at which Yunnan tin mine dust induces transformation of BEAS-2B cells. The proliferation activation of BEAS-2B cells can be improved by Yunnan tin mine dust.

Acid Anhydride Hydrolases ; metabolism ; Cell Line ; Cell Transdifferentiation ; China ; Dust ; Epithelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Ki-67 Antigen ; metabolism ; Lung ; cytology ; Neoplasm Proteins ; metabolism ; Tin ; toxicity

OBJECTIVE: To investigate the role of AP-1 in the secretion of Type I collagen in TGF-beta1-stimulated human lung fibroblasts. METHODS: Human lung fibroblasts cell line (HLF-02) was cultured, and then stimulated with 10 microg/L TGF-beta1 at different time points. Curcumin was added into the culture medium to inhibit the AP-1 activity before incubating with TGF-beta1. AP-1 DNA binding activity was assayed by electrophoretic mobility shift assay (EMSA), and the expression of Type I collagen was detected by Western blot and RT-PCR. RESULTS: TGF-beta1 could induce the transcription and secretion of Type I collagen in HLF-02 cells(P<0.05). TGF-beta1 could upregulate the AP-1 DNA binding activity ( P<0.05). Curcumin ( 5, 10, 15, and 20 micromol/L) could inhibit the AP-1 DNA binding activity in TGF-beta1-stimulated cells (the inhibition ratio was 17.1%, 17.6%, 24.2%, and 31.3%; P<0.05). Curcumin (5, 10, 15, and 20 micromol/L) could also inhibit the secretion of Type I collagen significantly (the inhibition ratio was 62.1%, 58.8%, 62.1%, and 59.6%; P<0.05). CONCLUSION AP-1 is responsible for the secretion of TGF-beta1-induced Type I collagen in human lung fibroblasts.
Cell Line ; Collagen Type I ; metabolism ; Fibroblasts ; cytology ; drug effects ; metabolism ; Humans ; Lung ; cytology ; Signal Transduction ; Transcription Factor AP-1 ; metabolism ; Transforming Growth Factor beta1 ; pharmacology

OBJECTIVETo investigate the expression of extracellular matrix metalloproteinase inducer gene (EMMPRIN) regulated by the interactions between fibroblasts and colon cancer cells, and to study its role in the invasion and metastasis of colon cancer.

METHODSColon cancer cells (SW480) were co-cultured with fibroblasts (HELF) in RPMI 1640 media for 0, 12, 24 and 48 hours. The expression of EMMPRIN in SW480 cells and HELF cells was documented by RT-PCR and immunocytochemistry.

RESULTSThe mRNA and protein expressions of EMMPRIN in SW480 cells were remarkably increased by the co-culturing with HELF cells. Although without the endogenous expression, HELF cells began to express EMMPRIN in a time-dependent manner after being co-cultured with SW480 cells.

CONCLUSIONSIntercellular interactions between colon cancer cells and fibroblasts not only up-regulate the EMMPRIN expression in SW480 cells, but also induce its expression in HELF cells. Such interactions may play a crucial role in the invasion and metastasis of colon cancer.

Basigin ; biosynthesis ; genetics ; Cell Line, Tumor ; Cells, Cultured ; Coculture Techniques ; Colonic Neoplasms ; metabolism ; pathology ; Fibroblasts ; cytology ; metabolism ; Humans ; Lung ; cytology ; RNA, Messenger ; metabolism

Pigs are increasingly recognized as "natural" hosts of infection by human respiratory viruses because of their similarities to humans in terms of lung physiology, airway morphology, cell types, and distribution of cell receptors in the respiratory tract. We wished to explore the mechanisms of infection by respiratory viruses and screening of drug that could be used to treat respiratory-system diseases. Hence, we developed a model of well-differentiated porcine airway epithelial cells (PAECs) derived from pig-lung tissue and cultured them with serum-free medium under an air-liquid interface condition in vitro. We identified the PAEC model using scanning electron microscopy, electrophysiology, and immunohistology. To evaluate application of gene therapy of adeno-associated virus (AAV)6 on the PAEC model, we generated recombinant adeno-associated virus 6-green fluorescent protein (rAAV6-GFP) using the three-plasmid transfection method and infected PAECs from the apical surface with rAAV6-GFP. Results demonstrated that the PAEC model comprised a multilayer epithelial structure containing ciliated mucous secretory cells, with basal cells located directly beneath the multilayer. rAAV6-GFP could infect PAECs from the apical surface and efficiently transduce PAECs to mediate the long-term expression of the exogenous gene. Establishment of a model of well-differentiated PAECs in vitro could lay a solid foundation for the study of infection by respiratory pathogens, as well as the screening and gene therapy of agents used to treat diseases of the respiratory system.
Animals ; Cell Differentiation ; Dependovirus ; genetics ; Epithelial Cells ; cytology ; metabolism ; Green Fluorescent Proteins ; genetics ; HEK293 Cells ; Humans ; Lung ; cytology ; Membrane Potentials ; Mucins ; metabolism ; Swine ; Transduction, Genetic ; Tubulin ; metabolism