The protein expression of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl(-) channel, in ovarian stimulated premature female rat ovary during a cycle of follicle development and corpus luteum formation was investigated. Animals were injected with 10 U pregnant Mare's serum gonadotropin (PMSG) and subsequently 10 U hCG 48 h later. Time-dependent immunohistochemistry and Western blotting experiments were performed before and 24, 48, 72 h after hCG treatment. The immunohistochemistry revealed that administration of PMSG stimulated the CFTR expression in thecal cell layer and granulosa cell layer of mature follicles 48 h post injection, coincident with the PMSG-induced peak in follicular estradiol. However, the expression of CFTR in the granulose lutein cell layer and thecal lutein cell layer was time-dependently reduced following hCG injection, in accordance with the gradually increased progestogen level during luteum corpus formation. Western blotting analysis demonstrated that rat ovarian tissue expressed the special CFTR band at 170 kD. It is concluded that cAMP-dependent Cl(-) channels are involved in regulation of follicle development and luteum formation.
Connective Tissue Growth Factor/genetics ; Connective Tissue Growth Factor/*metabolism ; Connective Tissue Growth Factor/*physiology ; Corpus Luteum/growth & development ; Ovarian Follicle/growth & development ; Ovary/*metabolism ; Rats, Wistar

OBJECTIVETo explore the effects of connective tissue growth factor (CTGF) on the pathogenesis of human keloid.

METHODSCTGF antisense oligonucleotides (ASODN) conjugated with isothiocyanate fluorescence was encapsulated by liposome, and then added into the human keloid fibroblast (HKF) culturing media. The intracellular distribution of CTGF ASODN was observed with fluorescence microscopy in the fixed HKF. The proliferation of HKF was measured by MIT test. The apoptosis of HKF was measured with a flow cytometer. The collagen synthesis of HKF was measured by using H3-proline incorporation method.

RESULTSThe CTGF ASODN inhibited the proliferation and collagen synthesis of the HKF, compared with the control, but it increased the apoptosis after the transfection (P < 0.01).

CONCLUSIONCTGF ASODN may has anti-fibrotic effects on human keloid in vitro, and the CTGF may play an important role in promoting the fibrosis of human keloid.

Apoptosis ; Cell Differentiation ; Cells, Cultured ; Connective Tissue Growth Factor ; genetics ; Fibroblasts ; cytology ; Humans ; Keloid ; metabolism ; pathology ; Oligonucleotides, Antisense ; genetics ; Transfection

OBJECTIVE: To study the safety and efficiency of the transfection of antisense oligonucletide into kidney mediated by lipid microbubbles, and to evaluate its potential clinical application. METHODS: The potential and conditions regarding the transfection self-made lipid microbubbles (CY5)-labeled-oligonucleotide (ODN) or CY5-labeled-ODN connective tissue growth factor (CTGF) into the rat kidney were evaluated. Th e safety was evaluated by HE staining, liver and renal function tests. The transfection efficiency was evaluated by fluorescence microscopy. Th e expression of CTGF was detected by RT-PCR and Western blot. RESULTS: Self-made lipid microbubble and/or ultrasound significantly enhanced the efficiency of gene transfer and expression in the kidney. Especially, 85%-90% of total glomerular could be transfected. CY5-labeled-ODN expression could be observed in glomerular, tubular and interstitial area. Th ere was no significant change in blood tests aft er gene transfer. Levels of LDH in 7 days were decreased compared with that at the fi rst day aft er the transfection (P<0.05). CTGF expression was successfully suppressed by transfection of CTGF-antisense-ODN into kidney. CONCLUSION The ultrasound-mediated gene transfer by self-made lipid microbubble could enhance the efficiency of ODN and expression in the rat kidney. Th is self-made lipid microbubbles supplement may be use for transfection of target genes.
Animals ; Connective Tissue Growth Factor ; genetics ; metabolism ; Kidney ; metabolism ; Lipids ; chemistry ; Microbubbles ; Oligonucleotides, Antisense ; genetics ; RNA, Messenger ; Rats ; Transfection ; Ultrasonics

OBJECTIVEEstrogen is closely associated with hypospadias. The present study was to explore the molecular mechanism of hypospadias caused by estradiol.

METHODSFibroblasts obtained from the prepuce of hypospadiac and normal children were cultured in vitro and treated with 17-beta ethinyl estradiol (17-EE) at the concentrations of 1 micromol/L to 0.1 nmol/L for 2 hours, or at 0.1 micromol/L for 0.5, 1, 2, 4, 8, 16 and 24 hours. MTT assay was used to evaluate the effect of 17-EE on the proliferation of the cells, and RT-PCR was employed to detect the expressions of the activating transcription factor-3 (ATF3) and connective tissue growth factor (CTGF) in the hypospadiac tissue. The results were compared with those obtained from the nonhypospadiac tissue.

RESULTSThe expressions of ATF3 and CTGF were significantly upregulated in the hypospadiac tissue as compared with the nonhypospadiac group. At the concentration of 1 micromol/L, 17-EE significantly inhibited the proliferation of the cells. ATF3 mRNA was elevated at 1-2 hours, while CTGF mRNA showed no significant changes in 24 hours.

CONCLUSIONATF3 and CTGF are two candidate genes involved in the etiology of hypospadias. And estradiol may induce hypospadias by upregulating the expressions of ATF3 and CTGF.

Activating Transcription Factor 3 ; genetics ; metabolism ; Cells, Cultured ; Child ; Connective Tissue Growth Factor ; genetics ; metabolism ; Estradiol ; pharmacology ; Estrogens ; pharmacology ; Fibroblasts ; metabolism ; Foreskin ; metabolism ; Humans ; Hypospadias ; genetics ; metabolism ; Male

Animals ; Connective Tissue Growth Factor ; Genetic Therapy ; Hepatocyte Growth Factor ; biosynthesis ; genetics ; Humans ; Immediate-Early Proteins ; biosynthesis ; genetics ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; genetics ; Liver Cirrhosis ; therapy ; Transforming Growth Factor beta ; biosynthesis ; genetics

Connective tissue growth factor (CTGF) is involved in the differentiation of lung fibroblast into myofibroblast and is considered as an important mediator in the pathogenesis of pulmonary fibrosis. In the present study, a CTGF small interference RNA (siRNA) expressing plasmid (CTGF-siRNA) was constructed and stably transfected into human lung fibroblast cell line, MRC-5. Stable clones with CTGF gene silencing (CTGF-siRNA/MRC-5) were successfully established by G418 screening and further confirmed by real-time quantitative PCR and Western blot. Cell proliferation was investigated by growth curve analysis, and cell doubling time of the CTGF-siRNA/MRC-5 cells was markedly longer than that of the control cells (P < 0.05). Compared with control cells, the expression of alpha-smooth muscle actin (alpha-SMA), the marker of myofibroblast differentiation, was decreased in CTGF-siRNA/MRC-5 cells. Moreover, the deposition of extracellular matrix (ECM) proteins (such as collagen type I and fibronectin) in CTGF-siRNA/MRC-5 cells was also declined. Our data suggest that CTGF may play an important role in the differentiation of lung fibroblast into myofibroblast, and that siRNA targeting CTGF gene might provide a new strategy for gene therapy of pulmonary fibrosis.
Cell Differentiation ; genetics ; Cell Proliferation ; Cells, Cultured ; Connective Tissue Growth Factor ; genetics ; Fibroblasts ; cytology ; Humans ; Lung ; cytology ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo explore the expression of connective tissue growth factor (CTGF) in pancreatic cancer and its influence on the proliferation and migration of cancer cells.

METHODSThe expression of CTGF in pancreatic cell line PANC-1 cells was analyzed by real-time PCR and in pancreatic carcinoma (50 cases) tissues by immunohistochemistry. The ability of proliferation and migration in vitro of PANC-1 cells was tested by MTT assay, scratch test and Boyden chamber test after the CTGF gene was overexpressed by Ad5-CTGF or silenced with Ad5-siCTGF transfection.

RESULTSCTGF was overexpressed in both pancreatic cancer cells and tissues. Overxpression of CTGF leads to increased proliferation and migration of PANC-1 cells. The CTGF-transfected PANC-1 cells showed apparent stronger proliferation ability and scratch-repair ability than that of empty vector controls. The results of Boyden chamber test showed that there were 34 cells/field (200× magnificantion) of the CTGF-transfected overexpressing cells, much more than the 11 cells/field of the empty vector control cells; and 6 cells/microscopic field of the Ad5-siCTGF-transfected silenced cells, much less than the 15 cells/field of the control cells.

CONCLUSIONSCTGF is overexpressed in both pancreatic cancer cells in vitro and in vivo, indicating that it may play an important role in the cell proliferation and migration in pancreatic cancer.

Adenoviridae ; genetics ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Connective Tissue Growth Factor ; genetics ; metabolism ; Humans ; Pancreatic Neoplasms ; metabolism ; pathology ; Recombinant Proteins ; genetics ; metabolism ; Transfection

OBJECTIVETo construct and identify a adenovirus vector of the expression of connective tissue growth factor (CTGF) and to explore the role of CTGF in the metabolism of glucose and lipid.

METHODSThe over-expressed plasmid of CTGF was cloned, and then the CTGF sequences were cloned into pAdTrack-CMW vector. The reformed E. coli BJ5183-sensitive bacteria that contain pAdEasy-1 were transformed with lined vector cut by Pme I enzyme. The recombinant adenovirus vector was cut with Pac I enzyme and obtained, then transfected 293A cells to produce virus. Through three times of amplification, the adenovirus infected the primary hepatocytes to determine the infection efficiency and CTGF expression. The mice were starved for several time periods, and then the liver RNA was extracted for real-time PCR to detect the expressions of CTGF under different nutritional conditions.

RESULTSThe adenovirus of CTGF was successfully produced with an infection efficiency of 90%. The expressions of the CTGF were different under different nutritional conditions and showed a coincidence with the expression of peroxisome proliferators-activated receptor gamma coactivator 1 alpha. After the mice were starved for 24h, the expression of CTGF increased by (2.38 +/- 0.51) folds; after the mice were starved for 48 h, the expression of CTGF increased by (2.95 +/- 0.57) folds (P < 0.05).

CONCLUSIONCTGF is speculated to be involved in the metabolism of glucose and lipids.

Adenoviridae ; genetics ; Animals ; Cell Line ; Connective Tissue Growth Factor ; genetics ; Escherichia coli ; genetics ; Genetic Vectors ; Mice ; Mice, Inbred C57BL ; Plasmids ; Transfection

OBJECTIVETo investigate the anti-fibrogenesis property of intraportal vein small interfering RNA (siRNA) injection targeting connective tissue growth factor (CTGF) in a rat model of liver fibrosis induced by carbon tetrachloride (CCl4) and its effect on hepatic stellate cell (HSC) activation.

METHODS24 male rats were randomly divided into four group. rats received CCl4 by subcutaneous injections every three days for 6 consecutive weeks, and meantime they also obtained either siRNA targeting CTGF (as CTGF siRNA group), saline (as model group) or a control siRNA (as control siRNA group) by intraportal vein injection to rats liver at the same approach. Other rats received saline intraportal vein injection for 6 weeks (as normal control group). The expressions of CTGF and a-SMA protein were detected by Western blot. Hepatic histology was evaluated by HE staining and Sirius red staining. The collagen staining areas were measured quantitatively using a computer-aided manipulator with slight modifications. The number of active HSC were evaluated by immunohistochemistry.

RESULTSSix weeks after CCl4 injection, prominent upregulations were observed in the expressions of CTGF and a-SMA protein in saline or control siRNA-treated rats livers. In rats with CTGF siRNA treatment, the protein expressions of CTGF and a-SMA in liver decreased by 95%+/-2% and 86%+/-11% (F=21.234 and 12.473, P<0.01) respectively, the number of active HSC in liver decreased by 76%+/-9% (F=9.179, P<0.01) as compared to the model group. The attenuation of liver fibrosis was also observed in rats with CTGF siRNA treatment.

CONCLUSIONIntraportal vein siRNA injection targeting CTGF could significantly inhibit CTGF gene expression in rats, thereby attenuate liver fibrosis by decreasing the number of active HSCs.

Animals ; Connective Tissue Growth Factor ; genetics ; Gene Silencing ; Hepatic Stellate Cells ; metabolism ; Liver Cirrhosis ; genetics ; metabolism ; pathology ; therapy ; Male ; RNA, Small Interfering ; genetics ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effect of chemically synthetic small interfering RNA (siRNA) targeting connective tissue growth factor (CTGF) on the synthesis and secretion of extracellular matrix (ECM) in hepatic stellate cells (HSC).

METHODSChemically synthetic siRNA targeting CTGF was transfected into HSC T6 (an active HSC line in rats) by oligofectamine package, and untreated HSC T6 were used as control. Total RNA and protein of the cells, after their incubation with siRNA for 24, 48 and 72 hours, were extracted, and the supernatants were collected. The expressions of CTGF and type I and III collagen genes were detected by means of reverse transcription-polymerase chain reaction (RT-PCR) and/or Western blot. Contents of hyaluronic acid and type III pro-collagen in the supernatants were determined by radioimmunoassay.

RESULTSThe expression of CTGF at mRNA and protein level and type I and III collagen at mRNA levels were markedly down-regulated in siRNA-transfected HSCs. The contents of hyaluronic acid and type III pro-collagen in the supernatants decreased by 46%+/-7%, 52%+/-7%, 53%+/-7% and 29%+/-18%, 29%+/-7%, 27%+/-5%, compared with those of the blank control at 24, 48 and 72 hours.

CONCLUSIONSChemically synthetic anti-CTGF siRNA can significantly inhibit CTGF gene expression in HSC, and markedly reduce the synthesis and secretion of ECM including type I and III collagen and hyaluronic acid. The siRNA-directed suppression of CTGF gene in HSC was maintained for 72 hours. This suggests that chemically synthetic siRNA may be a potential in preventing and treating liver fibrosis and may have a promising future for development

Cell Line ; Connective Tissue Growth Factor ; Extracellular Matrix ; metabolism ; Gene Targeting ; Humans ; Immediate-Early Proteins ; genetics ; Intercellular Signaling Peptides and Proteins ; genetics ; Liver ; cytology ; metabolism ; RNA, Small Interfering ; genetics