OBJECTIVETo study the effects of conventional mechanical ventilation (CMV) with low tidal volume on developmental porcine lungs by examining the expression of growth factors and inflammatory mediators.

METHODSTwelve preterm piglets born at 99 days of gestational age, 12 term neonatal piglets and 11 young piglets (4-5-weeks old) were randomly placed on CMV or were not ventilated (control group). The ventilator settings were adjusted to provide a tidal volume of 6-8 mL/kg in order to maintain a normal blood-gas value. After 6 hrs (preterm piglets) or 24 hrs (neonatal and young piglets) of mechanical ventilation, the mRNA expression of growth factors PDGF-B, IGF-I, KGF, HGF, VEGF and TGF-beta1 and proinflammatory cytokines IL-1beta, IL-6, IL-8 and TNF-alpha in the lung tissue was measured using RT-PCR. Growth factor protein expression was measured with immunohistochemistry.

RESULTSIn preterm piglets, the CMV group had increased mRNA expression of PDGF-B (5.11+/-0.10 vs 4.88+/-0.01), IL-1beta (4.95+/-0.27 vs 4.08+/-0.37), IL-6 (4.76+/-0.27 vs 4.00+/-0.28) and IL-8 (5.31+/-0.57 vs 4.15+/-0.46), but decreased IGF-I mRNA expression (3.54+/-0.13 vs 3.80+/-0.11) compared with those in the control group (P<0.05 or 0.01). In term neonatal piglets and young piglets, there were no significant differences in the mRNA expression of growth factors and proinflammatory cytokines between the CMV and control groups.

CONCLUSIONSCMV caused inflammatory injury in immature lungs by increasing the expression of proinflammatory cytokines and PDGF-B and decreasing IGF-I expression. However, CMV had no effects on pulmonary expression of growth factors and inflammatory mediators in term neonatal piglets and young piglets.

Animals ; Bronchoalveolar Lavage Fluid ; chemistry ; Immunohistochemistry ; Inflammation Mediators ; analysis ; Intercellular Signaling Peptides and Proteins ; analysis ; genetics ; Lung ; metabolism ; Peroxidase ; analysis ; Phospholipids ; analysis ; RNA, Messenger ; analysis ; Respiration, Artificial ; Reverse Transcriptase Polymerase Chain Reaction ; Swine ; Tidal Volume ; Vascular Endothelial Growth Factor A ; analysis ; genetics

OBJECTIVETo investigate the differentially expressed genes in keloids in comparison with normal skin using cDNA microarray.

METHODSThe cDNA microarray consisting of 8064 clones of human genes was employed to detect and screen the differentially expressed genes in keloid and normal skin tissues. Semi-quantitative RT-PCR was applied to verify the results of gene microarray.

RESULTSTotally 277 differentially expressed genes were identified in keloids in comparison with normal skin tissue, including 163 up-regulated genes and 114 down-regulated ones according to the designed data filter criteria. These differentially expressed genes belonged to 26 different functional gene families involving different biological processes. RT-PCR yielded results were consistent with those of microarray study.

CONCLUSIONA variety of genes are involved in the formation of keloids. The 277 differentially expressed genes comprise the differential gene expression profile of keloids and describe the general changes in the gene expressions in keloid at transcriptional level. Further analysis of the identified genes might help reveal the molecular mechanism of abnormal scarring.

Connective Tissue Growth Factor ; Gene Expression Profiling ; Humans ; Immediate-Early Proteins ; genetics ; Intercellular Signaling Peptides and Proteins ; genetics ; Keloid ; genetics ; Oligonucleotide Array Sequence Analysis ; methods ; Reverse Transcriptase Polymerase Chain Reaction ; Skin ; metabolism ; pathology

OBJECTIVETo study the role of connective tissue growth factor (CTGF) in the pathogenesis of human keloid.

METHODSHuman keloid fibroblasts (HKF) were isolated from human keloid and cultured in vitro. The cells were then divided into 3 groups according to different processing, i.e. ASODN treatment (AT), in which phosphorothioate CTGF antisense oligonucleotides (ASODN) labeled by fluorescent isothiocyananate were transfected into the HKFs by liposome; liposome control (LC, with liposome only); control groups (without liposome or ASODN). The distribution of CTGF ASODN in all groups of cells was observed under fluorescent microscope. The CTGF mRNA index (RI) of HKF was assessed by reverse transcription polymerase chain reaction method (RT-PCR). The collagen synthesis of HKF was assessed by (3)H-proline incorporation method.

RESULTSA large amount of fluorescence could be observed in the cytoplasm of HKFs in AT 12 hours after transfection, but not in LC and C groups. The CTGF mRNA index of HKF in AT group 48 hours after transfection was significantly lower than that in LC and C groups (0.12 +/- 0.62 vs 0.51 +/- 0.18 vs 0.54 +/- 0.35, P < 0.01). The (3)H-proline incorporation rate in AT group (108.96 +/- 79.05) was lower than that in LC and C groups (P < 0.01).

CONCLUSIONThe expression of CTGF gene and collagen synthesis of the cultured HKF could be inhibited by CTGF ASODN, implying that CTGF played a role in the development of excessive fibrosis of human keloid.

Collagen ; biosynthesis ; Connective Tissue Growth Factor ; Fibroblasts ; metabolism ; Humans ; Immediate-Early Proteins ; antagonists & inhibitors ; genetics ; physiology ; Intercellular Signaling Peptides and Proteins ; genetics ; physiology ; Keloid ; etiology ; metabolism ; Oligonucleotides, Antisense ; pharmacology ; RNA, Messenger ; analysis ; Transfection

Bioactive small molecules play a crucial role in maintaining structural and functional homeostasis of cardiovascular system. However, systemic research pattern is rare because of the multiple forms, diverse effects and complicated interaction of these molecules. Therefore firstly, they can be classified into different single 'families' according to their internal relationships. Secondly, studies may be focused on the network of members within each 'family' as well as network among different 'families', especially the physiological and pathological significance of each member in the cardiovascular diseases. Lastly, efforts should be made to establish a primary and simple regulation network as the strategy of researches on biology of cardiovascular systems, and to promote the transition to a network covering more complicated multiplex 'families', even the whole body.
Biological Products ; chemistry ; genetics ; metabolism ; Cardiovascular Diseases ; drug therapy ; Cardiovascular Physiological Phenomena ; Homeostasis ; drug effects ; Humans ; Intercellular Signaling Peptides and Proteins ; chemistry ; genetics ; metabolism ; Neuroimmunomodulation ; drug effects ; Protein Binding ; Proteins ; chemistry ; Receptors, Drug ; analysis

OBJECTIVETo observe the effect of high glucose, angiotensin II (AngII) and Losartan on the expression of connective tissue growth factor (CTGF) mRNA in cultured mesangial cells (MCs).

METHODSMCs of SD rats were isolated and cultured. High glucose (30 mmol/L) and AngII (10(-9), 10(-7), and 10(-5) mol/L) were added to the medium for 72 hours to observe the influence on CTGF mRNA expression. Losartan of 10(-5) mol/L and AngII of 10(-5) mol/L were added to the medium to observe the effects of Losartan on CTGF mRNA expression stimulated by AngII. The expressions of CTGF mRNA were detected by reverse transcriptase polymerase chain reaction (RT-PCR).

RESULTSRT-PCR showed that high glucose and AngII up-regulated the expression of CTGF mRNA, and AngII stimulated the expression in a dose-dependent manner. Expression of CTGF mRNA induced by AngIIwas partially suppressed by 10(-5) mol/L Losartan (P < 0.05).

CONCLUSIONSHigh glucose and AngII can enhance the expression of CTGF mRNA and thus be involved in the process of renal fibrosis. Losartan can have a partial fibrogenesis-inhibiting effect, with implications for the treatment of renal fibrosis.

Angiotensin II ; pharmacology ; Animals ; Cells, Cultured ; Connective Tissue Growth Factor ; Gene Expression ; drug effects ; Glomerular Mesangium ; metabolism ; Glucose ; pharmacology ; Immediate-Early Proteins ; genetics ; Intercellular Signaling Peptides and Proteins ; genetics ; Losartan ; pharmacology ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo explore the role of exogenous connective tissue growth factor (CTGF) in the collagen III synthesis of human renal tubular epithelial cell line HK2 in vitro.

METHODSCultured HK2 cells were randomly assigned to three groups: placebo-control, low-dose CTGF-treated (2.5 ng/mL) and high-dose CTGF-treated groups (20 ng/mL). Cell morphological changes were observed under an inverted microscope. Collagen III alpha mRNA expression was detected using RT-PCR. Immunohistochemistry staining was used to assess the levels of intracellular collagen III alpha protein.

RESULTSAfter 48 hrs of low- or high- dose CTGF treatment, the appearances of HK2 cells were changed from oval to fusiform. High-dose CTGF treatment increased collagen III alpha mRNA expression (0.4461+/-0.0274 vs 0.2999+/-0.0115; P<0.05) as well as the protein expression of collagen III alpha (0.4075+/-0.0071 vs 0.3503+/-0.0136; P<0.05) compared with the placebo-control group.

CONCLUSIONSCTGF can induce morphological changes of human renal tubular epithelial cells in vitro. High concentration of CTGF may increase the synthesis of collagen III alpha.

Cells, Cultured ; Collagen Type III ; biosynthesis ; genetics ; Connective Tissue Growth Factor ; Epithelial Cells ; drug effects ; metabolism ; Humans ; Immediate-Early Proteins ; pharmacology ; Immunohistochemistry ; Intercellular Signaling Peptides and Proteins ; pharmacology ; Kidney Tubules ; drug effects ; metabolism ; RNA, Messenger ; analysis

OBJECTIVETo explore the relationship of airway remodeling with epidermal growth factor receptor (EGFR) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) levels in asthmatic mice and the effect of EGFR tyrosine kinase inhibitor (AG1478) on airway remodeling.

METHODSTwenty-four male BALB/c mice were randomly divided into three groups: normal control, asthma, AG1478-treated. Mice were sensitized and challenged with ovalbumin (OVA) and a mouse mode1 of asthma was prepared. Collagen deposition was determined in Masson-stained lung sections. Periodic acid Schiff (PAS) staining was used to observe the proliferation of goblet cells. Immunohistochemistry was used to determine the protein expression of HB-EGF. RT-PCR was used to determine the mRNA expression of HB-EGF and EGFR.

RESULTSThe characteristic changes of airway remodeling occurred in the asthma group. The expression of HB-EGF and EGFR in the epithelial cells of bronchi in the asthma group was significantly higher than that in the normal control group. Compared with the asthma group, the AG1478-treated group had decreased inflammation reactions, decreased collagen deposition and proliferation of goblet cells and lower expression of EGFR and HB-EGF.

CONCLUSIONSEGFR tyrosine kinase inhibitor (AG1478) ameliorates the progression of airway remodeling in mice with asthma by inhibitions of EGFR and HB-EGF expression and EGFR signal pathway.

Airway Remodeling ; Animals ; Asthma ; drug therapy ; pathology ; Heparin-binding EGF-like Growth Factor ; Immunohistochemistry ; Intercellular Signaling Peptides and Proteins ; analysis ; genetics ; Male ; Mice ; Quinazolines ; Receptor, Epidermal Growth Factor ; antagonists & inhibitors ; genetics ; physiology ; Tyrphostins ; therapeutic use

Animals ; Collagen Type I ; genetics ; Connective Tissue Growth Factor ; Corneal Injuries ; Fibronectins ; analysis ; Immediate-Early Proteins ; analysis ; genetics ; physiology ; Immunohistochemistry ; Intercellular Signaling Peptides and Proteins ; analysis ; genetics ; physiology ; RNA, Messenger ; analysis ; Rabbits ; Signal Transduction ; physiology ; Smad Proteins ; physiology ; Transforming Growth Factor beta ; analysis ; genetics ; physiology ; Transforming Growth Factor beta1 ; Wound Healing ; physiology

BACKGROUNDThe peritoneum response to peritoneal dialysis can lead to fibrosis. The transforming growth factor beta1 (TGF-beta1) plays a key role in regulating tissue repair and remodelling after injury. Connective tissue growth factor (CTGF), a downstream mediator of TGF-beta1 inducing fibrosis, has been implicated in peritoneal fibrosis. Vascular endothelial growth factor (VEGF) plays a key role in angiogenesis that can hasten peritoneal fibrosis. In this study, we investigated the effect of small interfering RNA (siRNA) of CTGF by pRETRO-SUPER (PRS) retrovirus vector on the expression of CTGF and VEGF in human peritoneal mesothelial cells.

METHODSRetrovirus producing CTGF siRNA were constructed from the inverted oligonucleotides and transferred into packaging cell line PT67 with lipofectamine, and the virus supernatant was used to infect human peritoneal mesothelial cell (HPMC). The cells were divided into seven groups: low glucose DMEM, low glucose DMEM + TGF-beta1 5 ng/ml, low glucose DMEM + TGF-beta1 5 ng/ml + PRS-CTGF-siRNA(1-4) and low glucose DMEM + TGF-beta1 5 ng/ml + PRS. The expression of CTGF and VEGF were measured by semiquantitative RT-PCR and Western blot.

RESULTSLow levels of CTGF and VEGF were detected in confluent HPMCs. Following stimulation with TGF-beta1, the levels of CTGF and VEGF were significantly upregulated (P < 0.01). Introduction of PRS-CTGF-siRNA(1-4) resulted in the significant reduction of CTGF mRNA and protein, and VEGF mRNA (P < 0.01), especially in groups PRS-CTGF-siRNA1 and PRS-CTGF-siRNA4. The introduction of PRS void vector did not have these effects (P > 0.05).

CONCLUSIONSThe expression of CTGF siRNA mediated by PRS retrovirus vector can effectively reduce the level of CTGF and VEGF induced by TGF-beta1 in cultured HPMCs. This study may provide potential therapeutic strategies to prevent the peritoneal fibrosis.

Animals ; Base Sequence ; Cells, Cultured ; Connective Tissue Growth Factor ; Epithelial Cells ; metabolism ; Humans ; Immediate-Early Proteins ; analysis ; antagonists & inhibitors ; genetics ; Intercellular Signaling Peptides and Proteins ; analysis ; genetics ; Mice ; Molecular Sequence Data ; NIH 3T3 Cells ; Peritoneum ; cytology ; metabolism ; RNA, Messenger ; analysis ; RNA, Small Interfering ; pharmacology ; Retroviridae ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transforming Growth Factor beta1 ; pharmacology ; Vascular Endothelial Growth Factor A ; analysis ; genetics

OBJECTIVETo explore the in vitro methods of isolation and culture of human fetal epidermal stem cells (HFESCs) and the feasibility of the cultured cells as the target cells for gene transfection.

METHODSThe HFESCs were isolated by means of type IV collagen rapid adhering method. The culture medium for HFESCs was prepared according to that for human fetal fibroblasts. The cultured cells were identified by immunohistochemistry staining of keratin-19 and integrin-beta1, cell cycle analysis and clone forming rate determination. Then the cultured cells were gene transfected in vitro by liposome mediating method in which eukaryon expression vector pcDNA3.1/VEGF165 containing vascular endothelial growth factor 165 (VEGF165) were transfected into cultured cells, or by virus vector mediating method in which recombinant adenovirus accompanied vector (raav) containing green fluorescent protein (GFP) (raav/GFP) were transfected into the cultured cells, respectively. The results of in vitro gene transfection of HFESCs were observed by immunohistochemisty staining and fluorescence microscope.

RESULTSHFESCs grew well and formed large clones with higher cloning efficiency and higher ratio of G1 cells than keratinocytes. The cultured cells were strongly positive with immunohistochemistry staining of keratin-19 and integrin-beta1. After being gene-transfected by pcDNA3.1/VEGF165, the VEGF165 of HFESCs showed positive immunohistochemistry staining property, while the HFESCs transfected by raav/GFP exhibited strong fluorescence.

CONCLUSIONHFESCs could be isolated and cultured in vitro by means of rapid adherence to type IV collagen. It seemed feasible that HFESCs were gene transfected with liposome or adeno-associated virus as the vector.

Cell Adhesion ; Cell Cycle ; physiology ; Cells, Cultured ; Endothelial Growth Factors ; genetics ; metabolism ; Epidermis ; Fetus ; G1 Phase ; Green Fluorescent Proteins ; Humans ; Immunohistochemistry ; Integrin beta1 ; analysis ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; Keratinocytes ; cytology ; Keratins ; analysis ; Luminescent Proteins ; genetics ; metabolism ; Lymphokines ; genetics ; metabolism ; Microscopy, Fluorescence ; Plasmids ; genetics ; Stem Cells ; chemistry ; cytology ; metabolism ; Transfection ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors