OBJECTIVETo investigate the effect of cytomegalovirus (CMV) infection on actin and microfilament in human embryo fibroblast cells (HF) and its relationship with CMV replication.

METHODSCell morphology was observed after the infection of CMV. Western-blot was used to measure the expression levels of beta-actin, G-actin and F-actin proteins. CMV immediately early antigen (CMV IE) in HF cells was analyzed by indirect immunofluorescence assay. Microfilament alteration was determined by cytoskeleton fluorescence probe.

RESULTCMV IE was demonstrated in more than 95% of HF cells after infection, which was primarily located in nucleus. The shape of HF cells changed from thin shuttle like to round and thick ball like, even escaping from wall after infection by CMV. Compared with control group, the expression of G-actin protein increased at 24 h of CMV infection (0.941 +/-0.061 compared with 0.714 +/-0.119, P <0.05), then decreased at 72 h, 96 h respectively(0.218 +/-.035, 0.230 +/-0.055 compared with 0.714 +/-0.119, P <0.05). The levels of F-actin in infected cells gradually decreased at 24 h, 72 h and 96 h compared with control HF cells (0.256 +/-0.021, 0.127 +/-0.032, 0.026 +/-0.008 compared with 0.373 +/-0.050, P<0.05). In infected HF cells, microfilaments were found ruptured, arranged turbulently. Cells fused and fluorescence density of microfilament markedly reduced.

CONCLUSIONCytomegalovirus can induce alteration of actins and microfilament, which may be associated with its infection, replication and reactivity in host cells.

Actin Cytoskeleton ; metabolism ; Actins ; biosynthesis ; genetics ; Antigens, Viral ; analysis ; Cells, Cultured ; Cytomegalovirus ; Cytoskeleton ; metabolism ; Embryo, Mammalian ; Fibroblasts ; metabolism ; ultrastructure ; virology ; Humans ; Immediate-Early Proteins ; analysis

OBJECTIVE: To investigate the expression of c-fos in rats' skin during wound healing. METHODS: Immunohistochemistry was conducted on paraffin section from incised wounding model of rat skin. RESULTS: Fos protein improved from the time of 10 min after wounding in the wound edge, then it reached peak at 3 h. 24 h after injury, the quantity of Fos expression had no difference with that of normal skin. CONCLUSION Fos is sensitive after wound, but should be used with other criteria in wounding interval estimation as it's unstediness.
Animals ; Genes, Immediate-Early ; Immunohistochemistry ; Male ; Proto-Oncogene Proteins c-fos/biosynthesis* ; Rats ; Rats, Sprague-Dawley ; Skin/metabolism* ; Time Factors ; Wounds and Injuries/metabolism*

Co-infection of herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV) is not uncommon in immunocompromised hosts. Importantly, organ transplant recipients concurrently infected with HSV-1 and HCMV have a worse clinical outcome than recipients infected with a single virus. However, factors regulating the pathologic response in HSV-1, HCMV co-infected tissues are unclear. We investigated the potential biologic role of HCMV gene product immediate early 1 (IE1) protein in HSV-1-induced syncytial formation in U373MG cells. We utilized a co-infection model by infecting HSV-1 to U373MG cells constitutively expressing HCMV IE1 protein, UMG1-2. Syncytial formation was assessed by enumerating nuclei number per syncytium and number of syncytia. HSV-1-induced syncytial formation was enhanced after 24 hr in UMG1-2 cells compared with U373MG controls. The amplified phenotype in UMG1-2 cells was effectively suppressed by roscovitine in addition to inhibitors of viral replication. This is the first study to provide histological evidence of the contribution of HCMV IE1 protein to enhanced cytopathogenic responses in active HSV-1 infection.
Cell Line, Tumor ; Giant Cells/*virology ; Herpesvirus 1, Human/*growth & development ; Humans ; Immediate-Early Proteins/biosynthesis/*metabolism ; Protein Kinase Inhibitors/pharmacology ; Purines/pharmacology ; Transfection ; Virus Replication/drug effects

To investigate the mechanism and the suppressive effect of human cytomegalovirus (HCMV) on colony forming unit-megakaryocyte (CFU-MK), semi-solid culture system was used to observe the effect of HCMV AD169 strain on CFU-MK's growth of 18 cord blood samples. HCMV DNA and immediate early (IE) protein mRNA in CFU-MK was detected by PCR and reverse transcription-polymerase chain reaction (RT-PCR). Our results showed that HCMV AD169 significantly suppressed the formation of CFU-MK in vitro. Compared with the mock group, the CFU-MK colonies decreased by 21.6%, 33.8% and 46.3%, respectively, in all the 3 infected groups (P<0.05), suggesting the suppression and the titer of the virus was dose-dependent. Both HCMV DNA and the expression of HCMV IE protein mRNA were positively detected in the colony cells of viral infected group. It is concluded that HCMV AD169 strain could inhibit the differentiation and proliferation of CFU-MK by directly infecting their progenitors. There was early transcription of HCMV IE protein in CFU-MK infected by virus.
Antigens, CD34 ; metabolism ; Antigens, Viral ; biosynthesis ; genetics ; Cell Division ; drug effects ; Cells, Cultured ; Cytomegalovirus ; genetics ; physiology ; Cytomegalovirus Infections ; DNA, Viral ; biosynthesis ; genetics ; Humans ; Immediate-Early Proteins ; biosynthesis ; genetics ; Megakaryocytes ; cytology ; virology ; Molecular Sequence Data ; Stem Cells ; cytology ; virology

Animals ; Cells, Cultured ; Connective Tissue Growth Factor ; Hepatocytes ; cytology ; metabolism ; Immediate-Early Proteins ; biosynthesis ; genetics ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; genetics ; Male ; Plasmids ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Rats ; Transfection

OBJECTIVETo explore the effect of connective tissue growth factor on the pathogenesis of human hypertrophic scar.

METHODSNormal skin and hypertrophic scar fibroblasts were cultured in vitro. The collagen synthesis of fibroblasts were measured by H3-proline incorporation method. The expression of connective tissue growth factor protein and mRNA of fibroblasts were detected with immunocytochemistry staining and reverse transcription polymerase chain reaction methods.

RESULTSCompared with normal skin fibroblast, the collagen synthesis and the expression of connective tissue growth factor protein and mRNA in the hypertrophic scar fibroblast was higher (P < 0.01).

CONCLUSIONConnective tissue growth factor may play an important role in promoting the fibrotic process of hypertrophic scar.

Cells, Cultured ; Cicatrix, Hypertrophic ; genetics ; metabolism ; pathology ; Collagen ; biosynthesis ; Connective Tissue Growth Factor ; Fibroblasts ; metabolism ; pathology ; Gene Expression ; Humans ; Immediate-Early Proteins ; genetics ; Immunohistochemistry ; Intercellular Signaling Peptides and Proteins ; genetics ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo observe the expression of connective tissue growth factor (CTGF) in the tubulointerstitium in type 2 diabetic KKA(y) mice and the effect of rosiglitazone on it.

METHODSKKA(y) and C57 BL/6 mice aged 16 weeks ( n = 5 in each group) were sacrificed as controls before treatment. Another 20 KKA(y) mice were treated with rosiglitazone (30 mg x kg (-1) d (-1), n = 10) or placebo (n = 10). The mice were sacrificed at 20 and 24-week-age (n = 5 at each time point). Protein expression of transforming growth factor-beta1 (TGF-beta1 ), CTGF, peroxisome proliferator-activated receptor-gamma (PPARgamma) , and fibronectin were assayed by Western blot, while protein CTGF, PPARgamma, and alpha-smooth muscle actin ( alpha-SMA) were assayed by immunohistochemistry in kidney tissue sections.

RESULTSProteinuria was significantly decreased in mice aged 24 weeks treated by rosiglitazone than same-aged mice treated with placebo [ (44. 53+/-1. 96) vs (63. 66 +/-5. 57) microg/24 h, P < 0. 05 ]. The expressions of TGF-beta1, CTGF, and fibronectin in mice aged 20 weeks treated with rosiglitazone decreased by 37% , 21% , and 52% than same-aged control (P <0. 01) , and those were decreased by 61% , 50% , and 51% in mice aged 24 weeks treated with rosiglitazone compared with same-aged control mice (P < 0. 01). CTGF in the tubulointerstitium were respectively downregulated by 25% and 44. 9% in treated mice aged 20 weeks and 24 weeks compared with the same-aged control mice ( P < 0. 01). The PPARgamma appeared in diabetic mice and increased by 18. 1% in mice aged 24 weeks and treated with rosiglitazone than the same-aged control mice (P <0. 05).

CONCLUSIONHeterogeneous rosiglitazone may upregulate the expression of PPARgamma in renal cortex, and remarkably inhibit the expressions of CTGF in the tubulointerstitium and renal cortex in diabetic KKA(y) mice.

Animals ; Connective Tissue Growth Factor ; Diabetes Mellitus, Experimental ; drug therapy ; metabolism ; Diabetes Mellitus, Type 2 ; drug therapy ; metabolism ; Down-Regulation ; Female ; Fibronectins ; biosynthesis ; Immediate-Early Proteins ; biosynthesis ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; Kidney Cortex ; metabolism ; Kidney Tubules ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Mutant Strains ; PPAR gamma ; agonists ; biosynthesis ; Thiazolidinediones ; pharmacology ; Transforming Growth Factor beta1 ; biosynthesis ; Up-Regulation

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 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

Liver cirrhosis is one of the major complications of hepatitis C virus (HCV) infection, but the mechanisms underlying HCV-related fibrogenesis are still not clear. Although the roles of HCV core protein remain poorly understood, it is supposed to play an important role in the regulation of cellular growth and hepatocarcinogenesis. The aim of this study was to examine the role of HCV core protein on the hepatic fibrogenesis. We established an in vitro co-culture system with primary hepatic stellate cell (HSC) isolated from rats, and a stable HepG2-HCV core cell line which had been transfected with HCV core gene. The expressions of fibrosis-related molecules transforming growth factor beta1 (TGF-beta1), transforming growth factor b receptor II (TGF beta RII), alpha-smooth muscle actin (alpha-SMA) and connective tissue growth factor (CTGF) were analyzed via histological or molecular methods. In addition, the expression levels of matrix metaloprotinase-2 (MMP-2) and collagen type I (Col I) from the co-cultured media were measured by zymogram and ELISA, respectively. The expressions of alpha-SMA, TGF-beta1, Col I, TGF beta RII and MMP-2 were significantly increased in the co-culture of stable HepG2-HCV core with HSC. Moreover, the significant increases of CTGF and TGF-beta1 in the HCV core-expressing cells were observed by either Northern or Western blot analysis. These results suggest that HCV core protein may contribute to the hepatic fibrogenesis via up-regulation of CTGF and TGF-beta1.
Actins/metabolism ; Animals ; Cell Line, Tumor ; Cells, Cultured ; Coculture Techniques ; Collagen Type I/metabolism ; Gelatinase A/metabolism ; Immediate-Early Proteins/*biosynthesis ; Intercellular Signaling Peptides and Proteins/*biosynthesis ; Liver/metabolism/*pathology ; Liver Cirrhosis/*metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Receptors, Transforming Growth Factor beta/metabolism ; Research Support, Non-U.S. Gov't ; Transforming Growth Factor beta/*metabolism ; Up-Regulation ; Viral Core Proteins/genetics/*metabolism