OBJECTIVETo investigate the feasibility of transfection of recombinant human endothelial nitric oxide synthase (eNOS) into human hypertrophic scar fibroblasts (HSFbs), and to observe NO secretion and the synthesis of collagen I and III.

METHODSRecombinant human eNOS with karyocyte expressive vector was constructed in vitro, then was transfected into HSFbs which was isolated from hypertrophic scar tissues and cultured in vitro (T group). The HSFbs untransfected (normal culture) or transfected with empty-vector was used as control group and empty-vector group respectively. The mRNA expression of eNOS, collagen I and III was determined by Realtime PCR. The content of NO was determined by NO assay kit.

RESULTSThe expression of eNOS mRNA in T group was 5.92 +/- 0.21, which was obviously higher than that in empty-vector group (0.98 +/- 0.13, P < 0.05). The expression of collagen I mRNA (0.76 +/- 0.15), and collagen III (0.79 +/- 0.08) in T group was significantly lower than those in empty-vector group (0.98 +/- 0.15, 1.02 +/- 0.12, P < 0.05, respectively). The content of NO in T group (36.1 +/- 0.8 micromol/L) was obviously higher than that in empty-vector group (28.4 +/- 1.0 micromol/L, P < 0.01) and control group (27.7 +/- 1.3 micromol/L, P < 0.01).

CONCLUSIONHSFbs can be the target cells for eNOS gene transfection. The transfected cells can express eNOS and produce NO, which inhibit the synthesis of collagen.

Cicatrix, Hypertrophic ; metabolism ; Collagen Type I ; genetics ; metabolism ; Collagen Type III ; metabolism ; Fibroblasts ; metabolism ; Humans ; In Vitro Techniques ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type III ; genetics ; RNA, Messenger ; metabolism ; Transfection

OBJECTIVEThe aim of this study was to investigate the expression of collagen in the process of masseter muscle reattachment to the cortical and cancellous bones of mandible.

METHODSA total of nine adult goats were used in the study. One was the control. The other eight were treated with bilateral detachment of the masseter muscles. The biopsies of bone and muscle were taken at 2, 4, 8 and 12 weeks after the operation. The characteristics of the healing muscle-bone interfaces were examined using immunohistochemical techniques.

RESULTSImmunohistochemical analysis illustrated that the locations of collagen type I, II and III were different during the healing process, but similar in the cortical and cancellous bones.

CONCLUSIONThis study demonstrates that the distribution of the three types of collagens at the muscle-bone interfaces is associated with time, but not related with their locations.

Animals ; Collagen ; biosynthesis ; genetics ; Collagen Type I ; biosynthesis ; genetics ; Collagen Type II ; biosynthesis ; genetics ; Collagen Type III ; biosynthesis ; genetics ; Female ; Goats ; Male ; Mandible ; metabolism ; pathology ; surgery ; Masseter Muscle ; metabolism ; pathology ; surgery ; Wound Healing ; physiology

AIMTo observe the effects of hypoxia on DNA synthesis and the expression of collagen type I and III mRNA in cultured adult rat cardiac fibroblasts.

METHODSCardiac fibroblasts(CFs) were isolated from adult Wistar rat ventricule and cultured in vitro either in normoxic or hypoxic condition. Studies were conducted with the second passage of CFs. The changes of DNA synthesis was determined by measuring the incorporation of 3H-TdR into DNA and the changes of expression of pro-alpha1 (I) collagen, pro-alpha1(III) mRNA were measured by in situ hybridization respectively.

RESULTSThe 3H-TdR incorporation of CFs was increased by 34% (P < 0.05) and 36% (P < 0.01) after 6 h, 12 h hypoxia (2% O2) exposure respectively. The level of pro-alpha1(I) collagen mRNA expression was significantly elevated in the cells under hypoxia for 4 h, 8 h, and 12 h. The expression of pro-alpha1(III) mRNA increased when cells were cultured under hypoxia for 2 h.

CONCLUSIONThese results suggest that hypoxia alone can upregulate DNA synthesis and expression of collagen type I and III mRNA in adult rat cardiac fibroblasts. It may be one of the important mechanisms by which hypoxic myocardial fibrosis occur.

Animals ; Cell Hypoxia ; Cells, Cultured ; Collagen Type I ; metabolism ; Collagen Type III ; metabolism ; DNA ; biosynthesis ; Fibroblasts ; metabolism ; Male ; Myocytes, Cardiac ; cytology ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar

OBJECTIVETo investigate the effect of Qindan Capsule (QDC) on gene and protein expression of vascular adventitial collagen I and III (VAC1 and VAC3) in spontaneously hypertensive rats (SHR), for further research the possible mechanism of vascular adventitial fibroblasts remodeling.

METHODSThirty-two SHR, 40 weeks old, were equally randomized into the model group and the three treated groups treated respectively with high (750 mg/Kg x d) and low dosage QDC (150 mg/Kg x d), and losartan (30 mg/Kg x d), once a day for 12 weeks. Besides, a normal blank control group and a normal QDC (750 mg/Kg x d) medicated group were set up with same aged Wistar-Kyoto rats. Systolic blood pressures of rats were monitored, gene and protein expressions of VAC1 and VAC3 in rats' thoracic aortic adventitia were detected at the end of experiment using immune-histochemical staining and real-time quantitative fluorescent PCR respectively.

RESULTSCompared with the model group, blood pressure as well as the gene and protein expressions of VAC1 and VAC3 were all lower in the two QDC (high and low dosage) treated groups (P < 0.05).

CONCLUSIONQDC could not only effectively reduce the blood pressure in SHR, but also suppress and even reverse their thoracic aorta adventitial vascular remodeling, which is displayed by the obvious lowering of VAC1 and VAC2 expression levels.

Animals ; Aorta, Thoracic ; metabolism ; Capsules ; Collagen Type I ; genetics ; metabolism ; Collagen Type III ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Hypertension ; drug therapy ; metabolism ; Male ; Phytotherapy ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY

OBJECTIVESTo study the effects of Sanqi qisodium hyaluronate gel on collagen-I and collagen-III expression in the process of rabbits' epidural scar formation after operation.

METHODSNinety-six white rabbits with 6-month-old, half males and half females, weighted from 2 to 2.5 kg, which were randomly divided into normal saline group (A), Sanqi group (B), qisodium hyaluronate group (C) and Sanqi qisodium hyaluronate gel group (D). The laminectomy of rabbits were performed in group A, B, C, D, the duras were surrounded with normal saline, Sanqi liquid, qisodium hyaluronate and Sanqi qisodium hyaluronate gel respectively. Animals of each group were killed at 1, 2, 4, 8 weeks after operation. Use Masson staining for histological observation of collagen, and in situ hybridization staining for the analysis of collagen-I and collagen-III expression.

RESULTSIn the Masson staining, Sanqi qisodium hyaluronate gel group was more regular than the control group in the shape of collagen texture. As to the expression of collagen-I, and Sanqi qisodium hyaluronate gel group was lower than normal saline group, the Sanqi group and qisodium hyaluronate group at 4 weeks after using medicine (P < 0.01); while the Sanqi qisodium hyaluronate gel group was higher than normal saline group, Sanqi group and qisodium hyaluronate group in the collagen-III expression (P < 0.01).

CONCLUSIONSSanqi qisodium hyaluronate gel could improve collagen's arrangement of the rabbit's epidural scar after operation, reduce its rigidity and increase flexibility.

Animals ; Cicatrix ; etiology ; metabolism ; Collagen Type I ; genetics ; metabolism ; Collagen Type III ; genetics ; metabolism ; Epidural Space ; Female ; Gels ; Gene Expression Regulation ; drug effects ; Hyaluronic Acid ; chemistry ; Laminectomy ; adverse effects ; Male ; RNA, Messenger ; genetics ; metabolism ; Rabbits

OBJECTIVETo investigate the expression of PDGF receptor-beta and its correlation with extracellular matrix in hepatic tissue during hepatic fibrosis.

METHODSThe model of hepatic fibrosis in rats was induced by carbon tetrachloride. PDGF receptor-beta subunit, collagen I, collagen III and a-SMA in hepatic tissues of these rats were examined using immunohistochemistry. The correlation between PDGF receptor-beta subunit and collagen I, III was analyzed using SAS software after the results of immunohistochemistry were semi-quantified.

RESULTSPDGF receptor-beta subunit and a-SMA were not detected in normal controls. Collagen I and III were distributed in the portal tracts and beneath the endothelia of the central veins and of the Disse spaces. Two weeks after CCl4 injection, the PDGF receptor-beta and a-SMA were detected, and the expression of collagen I and III increased. At the end of 4 and 6 weeks, the above four proteins were further increased. Two weeks after CCl4 injection, PDGF receptor-beta had no apparent correlation with collagen I and III. However, PDGF receptor-beta had a significant correlation with collagen I and III 2 weeks later, and the correlation coefficient was 0.74 and 0.60 respectively at 4 weeks, and 0.83 and 0.67 respectively at 6 weeks. PDGF receptor-beta had a significant correlation with a-SMA during the whole process of hepatic fibrosis and the correlation coefficient was 0.62, 0.69 and 0.81, respectively at the time of 2, 4 and 6 weeks after CCl4 injection.

CONCLUSIONThe PDGF receptor-beta was overexpressed during the process of hepatic fibrosis development, and it significantly correlated with collagen I and collagen III.

Animals ; Carbon Tetrachloride ; Carbon Tetrachloride Poisoning ; Collagen Type I ; biosynthesis ; genetics ; Collagen Type III ; biosynthesis ; genetics ; Extracellular Matrix ; metabolism ; Liver ; metabolism ; Liver Cirrhosis, Experimental ; chemically induced ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Receptor, Platelet-Derived Growth Factor beta ; biosynthesis ; genetics

OBJECTIVETo investigate the effect of leptin on collagen systhesis in wounded rats.

METHODSThirty male Wistar rats, weight (180 +/- 20)g, were randomly divided into three groups (n = 10) by weight: normal depilation group, wound control group and leptin treatment group and ten rats were included in each group. A full-thickness defect measuring 2 x 2.5 cm was made in the back of rats in wound control group and leptin treatment group. Each wound in rats of leptin treatment group was applied topically with 0.1 ml leptin solution (2.0 microg leptin), daily for 7 days and that of wound control group with equivalent saline solution. All rats were killed and then granulation tissues samples and skin were collected to examine the synthesis of collagen.

RESULTSHydroxyproline content in granulation tissues of in leptin treatment group (33.92 +/- 3.09) mg/g were significantly increased than those in control group (29.55 +/- 3.59 mg/g, P < 0.05). The mRNA expressions of collagen I and III were significantly enhanced in leptin treatment group (0.96 +/- 0.09, 0.09 +/- 0.06) than those in control group (0.80 +/- 0.03, 0.08 +/- 0.03). The levels of type I and III collagen were significantly increased in leptin treatment group than those in control group.

CONCLUSIONLeptin applied topically can accelerate wound healing through enhancing gene expression of type I and III collagen and synthesis of collagen in wound tissue.

Administration, Cutaneous ; Animals ; Collagen Type I ; genetics ; metabolism ; Collagen Type III ; genetics ; metabolism ; Leptin ; administration & dosage ; therapeutic use ; Male ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Wound Healing ; drug effects ; Wounds and Injuries ; drug therapy

OBJECTIVETo examine the effect of pcDNA3.1-VEGF165 vector to the angiogoiesis, expression of collagen type I and type III mRNA in soft tissue injury model.

METHODSThirty two Sprague-Daulay rats,weighted (180 +/- 20) g, were made tissue injury in the bilateral of vertebral region. Round wound (diameter 12 mm) was made by perforex on the back, removed the skin and 2 mm muscle, one side was experimental group by random and the other as control. The wound was done with sodium chloride (0.2 ml) in the control group, with the recombinant VEGF165 vector (0.2 ml, 200 mg) in the experimental group. The wound healing and other general state of health was observed after the operation. The specimens were obtained at 3,5, 7,14 and 30 days after injury. Draw the materials from the rats at the same time, all samples were divided into two parts. one ( > 0.1 g) was conserved in refrigerator at - 80 degrees C, which was extracted total RNA by TRIZOL, design the primer of rat's collagen type I and type III, RT-PCR analysis indicated that collagen type I, III. The other was fixed by 10% formalin. Examine wound healing of local tissue and count it' s MVD by HE staining.

RESULTSAll the rabbits were well alive, no death or infection. Wound healing time was shorter than the control one (14.2, 17.4 d). Inflammatory cell infiltrate, cellula intersitialis, fibroblast, collagen and the density of angiogenesis were more in the experimental group than in the control one. The MVD was significant difference between the two groups at 1, 2 weeks are 63.38 +/- 9.20, 52.72 +/- 7.06 and 76.64 +/- 12.27, 66.84 +/- 9.82 (P < 0.05). The expression of collagen type I , III mRNA was found in the third day, the peak was in the second week and then degression. The collagen type I , III mRNA and beta-actin specificitic belt were found and its initial template volume different, the results was trend of RT-PCR obtained.

CONCLUSIONSThe local application of pcDNA3.1-VEGF165 can enhance the expression of collagen type I, III mRNA, enhance angiogenesis and extra cellular matrix, both of which can shorten healing time of tissue injury.

Animals ; Collagen Type I ; metabolism ; Collagen Type III ; metabolism ; Genetic Vectors ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Soft Tissue Injuries ; genetics ; metabolism ; Transfection ; Vascular Endothelial Growth Factor A ; genetics ; Wound Healing

OBJECTIVETo investigate the role of the PI3K/Akt signaling pathway in hydrogen sulfide-induced alterations in expression of collagen I and collagen III in hepatic stellate cells.

METHODSIn vitro cultured rat hepatic stellate cells (HSC-T6) were treated with hydrogen sulfide, or left untreated for use as controls, and divided into groups for treatment with different inhibitors for the various factors involved in the PI3K/Akt signaling pathway. Reverse transcription-PCR was used to measure Collagen I and collagen III mRNA expression. Western blotting was used to detect the protein expression of PI3K and p-Akt, which are upstream proteins of the PI3K/Akt pathway.

RESULTSCompared with the untreated control cells, the hydrogen sulfide treated cells showed elevated expression of collagen I mRNA (F =14.635, P less than 0.05), collagen III mRNA (F =14.620, P less than 0.05), PI3K protein (F =26.672, P less than 0.05), and p-Akt (F =23.522, P less than 0.05). Compared to the cells treated with hydrogen sulfide alone, the cells treated with the various inhibitors showed lower expression of collagen I mRNA (F =14.635, P less than 0.05), collagen III mRNA (F=14.620, P less than 0.05), PI3K protein (F =26.672, P less than 0.05), and p-Akt protein (F =23.522, P less than 0.05).

CONCLUSIONHydrogen sulfide can activate the PI3K/Akt pathway and elevate the expression of collagen I and collagen III in rat hepatic stellate cells.

Animals ; Cells, Cultured ; Collagen Type I ; metabolism ; Collagen Type III ; metabolism ; Hepatic Stellate Cells ; drug effects ; metabolism ; Hydrogen Sulfide ; pharmacology ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; RNA, Messenger ; genetics ; Rats ; Signal Transduction

OBJECTIVETo observe the in vitro effects of gamma-interferon (IFNgamma) on gene expression of collagen I (Col I), III (Col III) and tissue inhibitor of metalloprotenase 1 (TIMP1) of HSC-T6 cells.

METHODSCultured HSC-T6 cells were exposed to IFNgamma at concentrations of 0.1, 1, 10, 10(2), 10(3), 10(4), 2.5 x 10(5), 5 x10(5) U/ml for 48 hours. 4,5-simethylthiazaoly colormetric assay was used to evaluate the effect of IFNgamma on HSC-T6 cell proliferation. After incubating with IFNgamma (1 U/ml, 10(2) U/ml and 10(4) U/ml) for 48 hours, HSC-T6 cells were harvested to detect Col I, Col III and TIMP1 steady state mRNA levels by quantitative reverse-transcription polymerase chain reaction (RT-PCR).

RESULTSThe Col I, Col III and TIMP1 mRNA levels of the control group were 2.86+/-0.21, 2.00+/-0.23 and 3.90+/-0.81, respectively. Col I and Col III mRNA levels in HSC-T6 cells treated by different concentrations of IFNgamma were lower than that of the controls (P < 0.01). There was no significant difference in TIMP1 mRNA levels between IFNgamma groups and controls.

CONCLUSIONIFNgamma suppresses expression of Col I and Col III whereas it has no effect on TIMP1 mRNA expression. The antifibrotic mechanism of IFNgamma may be partly due to its down-regulation of Col I and Col III mRNA levels in HSC-T6 cells.

Animals ; Cell Line ; Collagen Type I ; biosynthesis ; genetics ; Collagen Type III ; biosynthesis ; genetics ; Hepatocytes ; cytology ; metabolism ; Interferon-gamma ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Tissue Inhibitor of Metalloproteinase-1 ; biosynthesis ; genetics